WO2018085379A2

WO2018085379A2 - Dual-activity nicotinamide phosphoribosyltransferase inhibitors

Info

Publication number: WO2018085379A2
Application number: PCT/US2017/059508
Authority: WO
Inventors: Stephen J. Gardell; Mauro Antonio DISPAGNA
Original assignee: Sanford Burnham Prebys Medical Discovery Institute
Current assignee: Sanford Burnham Prebys Medical Discovery Institute
Priority date: 2016-11-02
Filing date: 2017-11-01
Publication date: 2018-05-11
Anticipated expiration: 2019-05-02
Also published as: WO2018085379A3

Abstract

The present disclosure describes NAMPT modulatory compounds, and methods of identifying NAMPT modulatory compounds. The present disclosure also describes methods of testing NAMPT modulatory compounds for NTPase activity, cell mobility modulatory activity, and cell metastasis modulatory activity.

Description

DUAL-ACTIVITY NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE

INHIBITORS

CROSS-REFERENCE

[0001] This application claims the benefit of U. S. Provisional Application No. 62/416,508, filed November 2, 2016, which is entirely incorporated herein by reference for all purposes.

BACKGROUND

[0002] Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting step in the nicotinamide (NAM) salvage pathway which culminates in NAD⁺ biosynthesis. NAMPT synthesizes nicotinamide mononucleotide (NMN) and pyrophosphate from NAM and a-D-5- phosphoribosyl-l-pyrophosphate. NAMPT also catalyzes ATP hydrolysis which promotes NMN production via a phosphoenzyme intermediate (His247). Cancer cells are especially dependent on robust NAD⁺ biosynthesis due to heightened flux of NAD⁺ consuming pathways. Although small molecule NAMPT inhibitors have been tested for treatment of cancer, they have yielded lackluster results for reasons not fully understood.

SUMMARY

[0003] In one aspect, the present disclosure provides a method for preparing a pharmaceutical composition comprising a nicotinamide phosphoribosyltransferase (NAMPT) modulatory compound having a nucleoside triphosphatase (NTPase) modulatory activity, said method comprising: a) administering the NAMPT modulatory compound to a subject; and b) determining a modulation of NTPase activity of NAMPT, wherein the ability of the NAMPT modulatory compound to modulate NTPase activity of NAMPT is indicative of the NAMPT modulatory compound having an NTPase modulatory activity; and c) formulating the NAMPT modulatory compound having an NTPase modulatory activity with a pharmaceutically acceptable carrier.

[0004] In some embodiments, the modulation of NTPase activity is an increase in NTPase activity. In some embodiments, the modulation of NTPase activity is a decrease in NTPase activity. In some embodiments, determining the modulation of NTPase activity comprises using mass spectrometry. In some embodiments, the mass spectrometry is used to detect nucleoside diphosphate production. In some embodiments, the nucleoside diphosphate is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP. [0005] In some embodiments, determining the modulation of NTPase activity comprises using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0006] In one aspect, the present disclosure provides a method for preparing a pharmaceutical composition comprising a AMPT modulatory compound having an NTPase modulatory activity, the NAMPT modulatory compound having been administered to a subject and determined to modulate NTPase activity of NAMPT in a biological sample obtained from the subject, wherein the ability of the NAMPT modulatory compound to modulate NTPase activity of NAMPT in the biological sample is indicative of the NAMPT modulatory compound having an NTPase modulatory activity, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

[0007] In some embodiments, the NTPase modulatory activity is an increase in NTPase activity. In some embodiments, the NTPase modulatory activity is a decrease in NTPase activity. In some embodiments, the NTPase modulatory activity was determined using mass spectrometry. In some embodiments, the mass spectrometry was used to detect NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0008] In some embodiments, the NTPase modulatory activity was determined using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0009] In one aspect, the present disclosure provides a method for optimizing an NTPase modulatory activity of a NAMPT modulatory compound, wherein said method comprises determining the NTPase modulatory activity of the NAMPT modulatory compound. In some embodiments, the method further comprises modifying the NAMPT modulatory compound to optimize the NTPase modulatory activity. In some embodiments, optimizing the NTPase modulatory activity comprises increasing the NTPase modulatory activity. In some embodiments, optimizing the NTPase modulatory activity comprises decreasing the NTPase modulatory activity.

[0010] In some embodiments, determining the NTPase modulatory activity comprises using mass spectrometry. In some embodiments, the mass spectrometry is used to detect NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP. In some embodiments, determining the modulation of NTPase activity comprises using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP In some embodiments, the NDP is UDP.

[0011] In one aspect, the present disclosure provides a method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound, wherein an NTPase modulatory activity of the NAMPT modulatory compound has been identified, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier. In some embodiments, the NTPase modulatory activity is an increase in NTPase activity. In some embodiments, the NTPase modulatory activity is a decrease in NTPase activity. In some embodiments, the NTPase modulatory activity was determined using mass spectrometry. In some embodiments, the mass spectrometry was used to detect NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0012] In some embodiments, the NTPase modulatory activity was determined using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0013] A method for identifying a NAMPT modulatory compound, said method comprising: a) treating NAMPT with a test compound; b) determining a modulation of NMN formation by the NAMPT; and c) determining a modulation of NTPase activity of NAMPT; wherein the modulation of NMN formation of b) and the modulation of NTPase activity of c) identifies the test compound as an NAMPT modulatory compound. [0014] In some embodiments, the determining a modulation of NMN formation by the NAMPT comprises comparing: i) NMN formation by the NAMPT that has been treated with the test compound; and ii) NMN formation by NAMPT that has not been treated with the test compound. In some embodiments, the determining a modulation of NMN formation by the NAMPT comprises comparing: i) NMN formation by the NAMPT before it has been treated with the test compound; and ii) NMN formation by the NAMPT after it has been treated with the test compound. In some embodiments, the determining a modulation of NTPase activity of the NAMPT comprises comparing: i) NTPase activity of the NAMPT that has been treated with the test compound; and ii) NTPase activity of NAMPT that has not been treated with the test compound. In some embodiments, the determining a modulation of NTPase activity of the NAMPT comprises comparing: i) NTPase activity of the NAMPT before it has been treated with the test compound; and ii) NTPase activity of the NAMPT after it has been treated with the test compound.

[0015] In some embodiments, the modulation of NMN formation is a decrease in NMN formation. In some embodiments, the modulation of NMN formation is no change in NMN formation. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient. In some embodiments, the modulation of NMN formation is an increase in NMN formation. In some embodiments, the modulation of NTPase activity is an increase in NTPase activity. In some embodiments, the modulation of NTPase activity is no change in NTPase activity. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient. In some embodiments, the modulation of NTPase activity is a decrease in NTPase activity.

[0016] In some embodiments, determining the modulation of NTPase activity comprises using mass spectrometry. In some embodiments, the mass spectrometry is used to detect NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP. In some embodiments, screening the test compound for modulation of NTPase activity comprises using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0017] In one aspect, the invention provides a method for determining an NTPase modulatory activity of an NAMPT modulatory compound, said method comprising: a) treating NAMPT with the NAMPT modulatory compound; and b) determining a modulation of NTPase activity of NAMPT. In some embodiments, the determining an NTPase modulatory activity of an NAMPT modulatory compound comprises comparing: i) NTPase activity of the NAMPT that has been treated with the test compound; and ii) NTPase activity of NAMPT that has not been treated with the test compound. In some embodiments, the determining a modulation of NTPase activity of the NAMPT comprises comparing: i) NTPase activity of the NAMPT before it has been treated with the test compound; and ii) NTPase activity of the NAMPT after it has been treated with the test compound

[0018] In some embodiments, the modulation of NTPase activity is an increase in NTPase activity. In some embodiments, the modulation of NTPase activity is no change in NTPase activity. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient. In some embodiments, the modulation of NTPase activity is a decrease in NTPase activity.

[0019] In some embodiments, determining the modulation of NTPase activity comprises using mass spectrometry. In some embodiments, the mass spectrometry is used to detect NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0020] In some embodiments, determining the modulation of NTPase activity comprises using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during NDP production. In some embodiments, the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP. In some embodiments, the NDP is ADP. In some embodiments, the NDP is GDP. In some embodiments, the NDP is CDP. In some embodiments, the NDP is UDP.

[0021] In one aspect, the invention provides a method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising: a) determining the mobility of one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound; b) contacting the cells with the NAMPT modulatory compound; c) determining the mobility of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and d) comparing the mobility of the one or more cells before and after contacting the cells with the NAMPT modulatory compound; wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound.

[0022] In some embodiments, determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

[0023] In one aspect, the present disclosure provides a method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising: a) contacting one or more cells expressing NAMPT with the NAMPT modulatory compound; b) determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound; c) determining the mobility of one or more cells that have not been contacted with the NAMPT modulatory compound; and d) comparing the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound; wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound.

[0024] In some embodiments, determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay. In some embodiments, the cell mobility modulatory activity is a decrease in cell mobility. In some embodiments, the cell mobility modulatory activity is no change in cell mobility. In some embodiments, the cell mobility modulatory activity is an increase in cell mobility. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient.

[0025] In one aspect, the present disclosure provides a method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising: a) determining the metastatic state of one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound; b) contacting the cells with the NAMPT modulatory compound; c) determining the metastatic state of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and d) comparing the metastatic state of the one or more cells before and after contacting the cells with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound.

[0026] In some embodiments, determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

[0027] In one aspect, the invention provides a method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising: a) contacting one or more cells expressing NAMPT with the NAMPT modulatory compound; b) determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound; c) determining the metastatic state of one or more cells that have not been contacted with the NAMPT modulatory compound; and d) comparing the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound.

[0028] In some embodiments, determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay In some embodiments, determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

[0029] In some embodiments, the cell metastasis modulatory activity is a decrease in metastatic activity. In some embodiments, the cell metastasis modulatory activity is no change in metastatic activity. In some embodiments, the cell metastasis modulatory activity is an increase in metastatic activity. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof In some embodiments, the patient is a cancer patient.

[0030] In any one of the above embodiments, NTPase activity can be selected from the group consisting of ATPase activity, GTPase activity, CTPase activity, UTPase activity, or a combination thereof. In some embodiments, the NTPase activity is ATPase activity. In some embodiments, the NTPase activity is GTPase activity. In some embodiments, the NTPase activity is CTPase activity. In some embodiments, the NTPase activity is UTPase activity.

[0031] In any one of the above embodiments, the NAMPT modulatory compound can be a NAMPT inhibitor. In some embodiments, the NAMPT inhibitor is selected from the group consisting of FK-866, GNI-50, and CHS-828. In some embodiments, the NAMPT inhibitor is FK-866. In some embodiments, the NAMPT inhibitor is GNI-50. In some embodiments, the NAMPT inhibitor is CHS-828.

[0032] In any one of the above embodiments, the method can further comprise modifying the NAMPT modulatory compound to change its NTPase activity. In some embodiments, the change to the NTPase activity is an increase in NTPase activity. In some embodiments, the change to the NTPase activity is a decrease in NTPase activity.

[0033] In one aspect, the invention provides a method for treating cancer, said method comprising administering an NAMPT modulatory compound of any one of the above embodiments. In one aspect, the present disclosure provides a composition comprising an NAMPT modulatory compound as described herein. In some embodiments, the NAMPT modulatory compound excludes FK-866, CHS-828, GNI-50, GPP-78, GNE-618, STF-118804, STF-31, and KPT9274. In one aspect, the present disclosure provides a method for preparing the above composition, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

[0034] In one aspect, the present disclosure provides a method for identifying a NAMPT modulatory compound, said method comprising: a) treating NAMPT with a test compound; b) determining a modulation of NMN formation by the NAMPT; and c) determining a modulation of guanosine triphosphatase (GTPase) activity of NAMPT; wherein the modulation of NMN formation of b) and the modulation of GTPase activity of c) identifies the test compound as an NAMPT modulatory compound. In some embodiments, the determining a modulation of NMN formation by the NAMPT comprises comparing: i) NMN formation by the NAMPT that has been treated with the test compound; and ii) NMN formation by NAMPT that has not been treated with the test compound. In some embodiments, the determining a modulation of NMN formation by the NAMPT comprises comparing: i) NMN formation by the NAMPT before it has been treated with the test compound; and ii) NMN formation by the NAMPT after it has been treated with the test compound. In some embodiments, the determining a modulation of GTPase activity of the NAMPT comprises comparing: i) GTPase activity of the NAMPT that has been treated with the test compound; and ii) GTPase activity of NAMPT that has not been treated with the test compound. In some embodiments, the determining a modulation of GTPase activity of the NAMPT comprises comparing: i) GTPase activity of the NAMPT before it has been treated with the test compound; and ii) GTPase activity of the NAMPT after it has been treated with the test compound.

[0035] In some embodiments, the modulation of NMN formation is a decrease in NMN formation. In some embodiments, the modulation of NMN formation is no change in NMN formation. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient.

[0036] In some embodiments, the modulation of NMN formation is an increase in NMN formation. In some embodiments, the modulation of GTPase activity is an increase in GTPase activity. In some embodiments, the modulation of GTPase activity is no change in GTPase activity. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient. In some embodiments, the modulation of GTPase activity is a decrease in GTPase activity.

[0037] In some embodiments, determining the modulation of GTPase activity comprises using mass spectrometry. In some embodiments, the mass spectrometry is used to detect GDP production. In some embodiments, screening the test compound for modulation of GTPase activity comprises using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during GDP production.

[0038] In one aspect, the invention provides a method for determining a GTPase modulatory activity of an NAMPT modulatory compound, said method comprising: a) treating NAMPT with the NAMPT modulatory compound; and b) determining a modulation of GTPase activity of NAMPT. In some embodiments, the determining a GTPase modulatory activity of an NAMPT modulatory compound comprises comparing: i) GTPase activity of the NAMPT that has been treated with the test compound; and ii) GTPase activity of NAMPT that has not been treated with the test compound. In some embodiments, the determining a modulation of GTPase activity of the NAMPT comprises comparing: i) GTPase activity of the NAMPT before it has been treated with the test compound; and ii) GTPase activity of the NAMPT after it has been treated with the test compound

[0039] In some embodiments, the modulation of GTPase activity is an increase in GTPase activity. In some embodiments, the modulation of GTPase activity is no change in GTPase activity. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient. In some embodiments, the modulation of GTPase activity is a decrease in GTPase activity.

[0040] In some embodiments, determining the modulation of GTPase activity comprises using mass spectrometry. In some embodiments, the mass spectrometry is used to detect GDP production. In some embodiments, determining the modulation of GTPase activity comprises using a colorimetric assay. In some embodiments, the colorimetric assay detects inorganic phosphate produced during GDP production.

[0041] In one aspect, the present disclosure provides a method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates NAMPT GTPase activity, said method comprising: a) determining the mobility of one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound; b) contacting the cells with the NAMPT modulatory compound; c) determining the mobility of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and d) comparing the mobility of the one or more cells before and after contacting the cells with the NAMPT modulatory compound; wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound.

[0042] In some embodiments, determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

[0043] In one aspect, the invention provides a method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound with GTPase activity, said method comprising: a) contacting one or more cells expressing NAMPT with the NAMPT modulatory compound; b) determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound; c) determining the mobility of one or more cells that have not been contacted with the NAMPT modulatory compound; and d) comparing the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound; wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound.

[0044] In some embodiments, determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

[0045] In some embodiments, the cell mobility modulatory activity is a decrease in cell mobility. In some embodiments, the cell mobility modulatory activity is no change in cell mobility. In some embodiments, the cell mobility modulatory activity is an increase in cell mobility. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient.

[0046] In one aspect, the present disclosure provides a method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound with GTPase activity, said method comprising: a) determining the metastatic state of one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound; b) contacting the cells with the NAMPT modulatory compound; c) determining the metastatic state of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and d) comparing the metastatic state of the one or more cells before and after contacting the cells with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound.

[0047] In some embodiments, determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

[0048] In one aspect, the present disclosure provides a method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound with GTPase activity, said method comprising: a) contacting one or more cells expressing NAMPT with the NAMPT modulatory compound; b) determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound; c) determining the metastatic state of one or more cells that have not been contacted with the NAMPT modulatory compound; and d) comparing the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound.

[0049] In some embodiments, determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay. In some embodiments, determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay. In some embodiments, determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

[0050] In some embodiments, the cell metastasis modulatory activity is a decrease in metastatic activity. In some embodiments, the cell metastasis modulatory activity is no change in metastatic activity. In some embodiments, the cell metastasis modulatory activity is an increase in metastatic activity. In some embodiments, the method further comprises administering the NAMPT modulatory compound to a patient in need thereof. In some embodiments, the patient is a cancer patient.

[0051] In some embodiments, the NAMPT modulatory compound is an NAMPT inhibitor. In some embodiments, the NAMPT inhibitor is selected from the group consisting of FK-866, GNI- 50, CHS-828, and GNI-50. In some embodiments, the NAMPT inhibitor is FK-866. In some embodiments, the NAMPT inhibitor is GNI-50. In some embodiments, the NAMPT inhibitor is CHS-828. In some embodiments, the NAMPT inhibitor is GNI-50. In some embodiments, the NAMPT inhibitor is not FK-866, GNI-50, CHS-828, or GNI-50. In some embodiments, said method further comprising modifying the NAMPT modulatory compound to change its GTPase activity. In some embodiments, said change to the GTPase activity is an increase in GTPase activity. In some embodiments, said change to the GTPase activity is a decrease in GTPase activity. [0052] In one aspect, the present disclosure provides a method for treating cancer, the method comprising administering an NAMPT modulatory compound of any one of the above

embodiments.

[0053] In one aspect, the present disclosure provides a composition comprising an NAMPT modulatory compound of any one of the above embodiments.

[0054] In one aspect, the present disclosure provides a composition comprising an NAMPT modulatory compound of any one of the above embodiments.

[0055] In one aspect, the present disclosure provides a method for preparing the composition of the above embodiments, said method comprising combining the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1 A depicts a time course of NAMPT-mediated NTPase activity. NAMPT (200 nM) was incubated at 37 °C in the presence of ATP (·), GTP (A), UTP (o) or CTP (Δ) (2 mM). Samples were quenched at the indicated times and NDPs were quantified by LC/MS/MS. For each NTP, a "No NAMPT" sample was run to correct for non-enzymatic NTP hydrolysis. Data show NDP production (μηιοΐ NDP / μιηοΐ NAMPT); mean (sd) N=4.

[0057] FIG. IB depicts the Michaelis-Menten kinetics for NAMPT-mediated ATPase and GTPase activity. The reactions were performed with various ATP or GTP concentrations in the absence (·) or presence (o) of 1 μΜ FK-866. The ADP or GDP products were assayed by LC/MS/MS. Curve fitting of the data points was performed with GraphPad Prism software. Each data point shows mean (sd) N=4.

[0058] FIG. 1C depicts the impact of the H247A mutation on the ATPase and GTPase reactions catalyzed by NAMPT. NAMPT (200 nM) and NAMPT-H247A (1 uM) were incubated at 37 °C for 4 h in the presence of ATP or GTP (2 mM). ADP and GDP products were measured using LC MS MS. A "No NAMPT" sample was run to correct for non -enzymatic ATP/GTP hydrolysis. Data show NDP production (μΜ NDP / μΜ NAMPT / h); mean (sd) N=3.

[0059] FIG. 2A depicts enhanced synthesis of NMN by NAMPT in the presence of ATP, but not in the presence of other NTPs. NAMPT (20 nM) was incubated at 37 °C for 1 h in the presence of NAM and PRPP without ("No NTP") or with 2 mM of ATP, GTP, CTP or UTP. Samples were assayed for NMN production using the NMN fluorescent assay (see Methods). Data show mean (sd); N=3. *, p value < 0.05 compared to the "No NTP" sample.

[0060] FIG. 2B depicts the inhibition of NMN synthesis catalyzed by NAMPT in the presence of NTPs other than ATP. NAMPT (20 nM) was incubated at 37oC for 1 h with NAM and PRPP in the presence of ATP (1 mM; "ATP only"). Where indicated, 4 mM of CTP, GTP or UTP were also added. Samples were assayed for NMN production using the NMN fluorescent assay (see Methods). Data show mean, sd; N=3. *, p value < 0.05 compared to "ATP only" sample.

[0061] FIG. 3A depicts the high potency of FK-866 for stimulating the ATPase and GTPase activities of NAMPT. NAMPT (100 nM) was incubated at 37 °C for 6 h in the presence of ATP (·) or GTP (o) along with FK-866 (0, 0.25, 0.5, 1.0, and 2.0 μΜ). Samples were assayed for ADP and GDP by LC/MS/MS. "No NAMPT" samples were run in parallel to correct for non-enzymatic formation of ADP/GDP. Data show mean (sd); N=4.

[0062] FIG. 3B depicts the ability of FK-866 to stimulate NAMPT' s ATPase activity being blocked by CHS-828. NAMPT (100 nM) was incubated at 37 °C for 6 h in the presence of ATP (2 mM) and FK-866 (0.25 μΜ), CHS-828 (2.5 μΜ) or combined FK-866 and CHS-828 (at aforementioned concentrations). Samples were assayed for ADP by LC/MS/MS and a "No NAMPT" sample was used to correct for non- enzymatic hydrolysis of ATP. Data show mean (sd); N=4. *, p value < 0.05.

[0063] FIGS. 4A, 4B, and 4C depict the effect of FK-866, CHS-828, or GNI-50 on adenosine 5'- tetraphosphate (Ap4), ADP, and Pj formation. NAMPT (200 nM) was incubated with ATP (2mM) and FK-866 ( 1 μΜ), CHS-828 (1 μΜ), or GNI-50 ( 1 pM), and ADP, Pj, and AP4 formation were measured over time FIG 4 A depicts ADP formation over time. FIG. 4B depicts Pi formation over time FIG. 4C depicts Ap4 formation over time.

DETAILED DESCRIPTION

[0064] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology,

immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art. Chemistry terms used herein are used according to conventional usage in the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0065] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0066] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0067] The singular forms "a," "an," and "the" include the plurals unless the context clearly dictates otherwise. [0068] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0069] A "patient", "subject", or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).

[0070] Each embodiment described herein may be combined with any other embodiment described herein.

Methods of the present disclosure

[0071] The present disclosure described herein takes advantage of the new and surprising discovery of a novel enzymatic activity catalyzed by NAMPT. More particularly, NAMPT is promiscuous with respect to its ability to hydrolyze nucleoside triphosphates (NTPs). Hence, the known ability of NAMPT to consume ATP can be extended to CTP, GTP and UTP, although only ATP promotes NMN biosynthesis. Perhaps most surprisingly, applicant discovered that highly-potent inhibitors of NAMPT-mediated NMN formation (such as FK-866, CHS-828, and GNI-50) generally stimulate NTP hydrolysis by NAMPT. Remarkably, each of these NAMPT inhibitors was unique with respect to their impact on NTP hydrolysis. Thus, the ability of the NAMPT inhibitors to inhibit NMN formation was not inextricably linked to their ability to stimulate NTPase activity. The present disclosure capitalizes on this discovery by providing methods useful for, inter alia, identifying NAMPT modulatory compounds and determining the effects of NAMPT modulatory compounds on NAMPT-mediated NTPase activity, cell mobility, and cell metastasis. Applicant submits that the disparate effects of FK-866, CHS-828, and GNI- 50 on the NTPase activity of NAMPT produce different pharmacologic profiles that transcend the common ability of these molecules to inhibit NAMPT-mediated NMN formation and NAD⁺ biosynthesis.

[0072] The NAMPT reaction scheme as proposed involves the following sequential steps: (1) ATP binding, phosphorylation of residue His247 in NAMPT, followed by ADP release, (2) PRPP binding to the phosphorylated enzyme, followed by NAM binding to the (phospho- His247)-PRPP complex and (3) chemical reaction culminating in the release of products (NMN and PPi) and hydrolysis of the phosphorylated enzyme. The phosphoenzyme intermediate provides thermodynamic drive for NMN formation due to a striking increase in the affinity of the enzyme for its substrates PRPP and NAM. In the absence of NAM and PRPP substrates, NAMPT-catalyzed ATP hydrolysis is an apparent futile ATPase activity. Although the ATPase activity of NAMPT is stimulated by sundry NAMPT substrates and products, the known impact of modulators of NAMPT-mediated NMN formation on the constitutive ATPase activity was unknown.

Methods for identifying NAMPT modulatory compounds

[0073] In some embodiments, the present disclosure provides a method for identifying a NAMPT modulatory compound, the method comprising a) contacting NAMPT with a test compound (e.g., including treating NAMPT with a test compound); b) determining a modulation of NMN formation by NAMPT; and c) determining a modulation of nucleoside triphosphatase (NTPase) activity of NAMPT; wherein the modulation of NMN formation of b) and the modulation of NTPase activity of c) identifies the test compound as an NAMPT modulatory compound. A test compound may be any compound that the skilled worker wishes to test for NAMPT modulatory activity. The skilled worker may be guided by structural or functional analyses of NAMPT or NAMPT modulatory compounds, or by any other means known in the art.

[0074] In certain embodiments, determining a modulation of NMN formation by NAMPT comprises comparing: i) NMN formation by NAMPT that has been treated (e.g., including contacted) with a test compound; and ii) NMN formation by NAMPT that has not been treated (e.g., contacted) with the test compound. Thus, NMN formation can be determined (e.g., measured) by assaying NAMPT activity before and after NAMPT is treated (e.g., including contacted) with a test compound, or by comparing NMN formation by NAMPT that has been treated (e.g., including contacted) with the test compound and NMN formation by NAMPT that has not been treated (e.g., including contacted) with the test compound.

[0075] Likewise, NTPase activity can be determined (e.g., measured) by testing before and after NAMPT is treated (e.g., including contacted) with a test compound, or by comparing the NTPase activity of NAMPT that has been treated (e.g., including contacted) with the test compound and the NTPase activity of NAMPT that has not been treated (e.g., including contacted) with the test compound. Thus, in certain embodiments, determining a modulation of NTPase activity of the NAMPT comprises comparing: i) NTPase activity of the NAMPT that has been treated with the test compound; and ii) NTPase activity of NAMPT that has not been treated (e.g., including contacted) with the test compound. In certain embodiments, determining a modulation of NTPase activity of NAMPT comprises comparing: i) NTPase activity of the NAMPT before it has been treated (e.g., including contacted) with the test compound; and ii) NTPase activity of the NAMPT after it has been treated (e.g., including contacted) with the test compound.

[0076] The skilled worker will appreciate that the modulation of NMN formation refers to an increase in NMN formation or a decrease in NMN formation. However, in certain embodiments, the modulation of NMN formation refers to no change in NMN formation. The skilled worker also will appreciate that the modulation of NTPase activity refers to an increase in NTPase activity or a decrease in NTPase activity. However, in certain embodiments, the modulation of NTPase activity refers to no change in NTPase activity.

[0077] In certain embodiments, a method of identifying an NAMPT modulatory compound may further comprise administering the NAMPT modulatory compound to a patient in need thereof (e.g., a cancer patient). In certain embodiments, a method of identifying an NAMPT modulatory compound may be followed by administering the NAMPT modulatory compound to a patient in need thereof (e.g., a cancer patient). Without wishing to be bound by theory, it may be particularly beneficial to treat a patient (e.g., a cancer patient) with a NAMPT modulatory compound that decreases NMN formation. A NAMPT modulatory compound that decreases NMN formation may also be called a NAMPT inhibitor. Examples of NAMPT inhibitors include, but are not limited to, FK-866, GNI-50, and CHS-828. Without wishing to be bound by theory, it may be particularly beneficial to treat a patient (e.g., a cancer patient) with a NAMPT modulatory compound that increases the NTPase activity of NAMPT.

[0078] The skilled worker will appreciate that determining the modulation of NAMPT' s NTPase activity may be achieved by any method known in the art. For example, the modulation of NTPase activity may be measured directly or indirectly, e.g., by measuring nucleoside diphosphate (NDP) formation The skilled worker will appreciate that the choice of NDP to be measured is dependent upon the NTPase activity being determined. For example, where GPTase activity is being determined, GDP can be measured. Likewise, ADP can be measured for ATPase activity, CDP can be measured for CTPase activity, and UDP can be measured for UTPase activity. In certain embodiments, determining the modulation of NTPase activity comprises using mass spectrometry, for example, to measure NDP (e.g., GDP, ADP, CDP, or UDP) formation. In certain embodiments, the method comprises determining the modulation of NTPase activity by using a colorimetric assay, for example, to detect inorganic phosphate produced by an NTP to NDP conversion (e.g., GTP to GDP, ATP to ADP, CTP to CDP, or UTP to UDP).

Methods for determining an NTPase modulatory activity

[0079] In some embodiments, a method for determining an NTPase modulatory activity of an NAMPT modulatory compound comprises treating NAMPT (e.g., including contacting NAMPT) with the NAMPT modulatory compound and determining a modulation of NTPase activity of NAMPT. For example, in certain embodiments, determining an NTPase modulatory activity of an NAMPT modulatory compound comprises comparing: NTPase activity of NAMPT that has been treated (e.g., including contacted) with the test compound; and NTPase activity of NAMPT that has not been treated (e.g., including contacted) with the test compound. In certain embodiments, determining a modulation of NTPase activity of the NAMPT comprises comparing: NTPase activity of NAMPT before it has been treated (e.g., including contacted) with the test compound; and NTPase activity of NAMPT after it has been treated (e.g., including contacted) with the test compound. Thus, the skilled worker will appreciate that determining a modulation of NTPase activity of NAMPT can be achieved in the same preparation of NAMPT protein or in different preparations of NAMPT protein. For example, a preparation of NAMPT protein can be divided to provide two populations of NAMPT proteins, one of which is treated with a test compound and one of which is not treated with a test compound. Likewise, the skilled worker will appreciate that determining a modulation of NTPase activity of NAMPT in cells can be achieved in the same population of cells expressing NAMPT or in different populations of cells expressing NAMPT.

[0080] In certain embodiments, the modulation of NTPase activity is an increase in NTPase activity or a decrease in NTPase activity. However, in certain embodiments, the modulation of NTPase activity is no change in NTPase activity. In certain embodiments, a method for determining an NTPase modulatory activity of an NAMPT modulatory compound may further comprise administering the NAMPT modulatory compound to a patient in need thereof (e g , a cancer patient). In certain embodiments, a method for determining an NTPase modulatory activity of an NAMPT modulatory compound may be followed by administering the NAMPT modulatory compound to a patient in need thereof (e.g., a cancer patient). Without wishing to be bound by theory, it may be particularly beneficial to treat a patient (e.g., a cancer patient) with a NAMPT modulatory compound that increases NTPase activity of NAMPT.

[0081] In some embodiments, determining the modulation of NTPase activity comprises using mass spectrometry, for example, to detect NDP production. Depending on the NTPase activity being assayed for, the skilled worker can choose the appropriate NDP to detect (e.g., ADP, GDP, CDP, or UDP). In some embodiments, determining the modulation of NTPase activity comprises using a colorimetric assay, for example, to detect inorganic phosphate produced concomitantly with NDP production. Again, the skilled worker will appreciate that the NDP (e.g., ADP, GDP, CDP, or UDP) will depend on the NTPase activity being assayed for. Methods for determining a modulation of Ap4 formation

[0082] Modulators of the NTPase activity of NAMPT (e.g., NAMPT inhibitors, such as those that modulate NMN formation) also can have a disparate effect on the formation of a by-product of the NAMPT ATPase reaction: adenosine 5 ' -tetraphosphate (Ap4). Ap4 can be produced indirectly by NAMPT when the inorganic phosphate (Pi) produced by NAMPT 's ATPase activity combines with ATP to produce Ap4. Thus, the skilled worker will appreciate that Ap4 formation, as described herein, also can be replaced with Cp4 formation, Up4 formation, and Gp4 formation stemming from the CTPase activity, UTPase activity, or GTPase activity of NAMPT, respectively.

[0083] Intriguing biological activities have been attributed to Ap4. For instance, Ap4 has been shown to be a highly potent purinergic vasoconstrictor in rats that exerts its effect via activation of the P2X1 receptor. It also has been shown to produce a dose-dependent reduction of intraocular pressure in rabbits. The discovery that NAMPT modulatory compounds can affect Ap4 formation expands the pharmacological differences possible for NAMPT modulatory compounds.

[0084] In some embodiments, a method for determining a modulation of Ap4 formation by an NAMPT modulatory compound comprises treating NAMPT (e.g., including contacting NAMPT) with the NAMPT modulatory compound and determining a modulation of Ap4 formation by NAMPT. For example, in certain embodiments, determining a modulation of Ap4 formation by an NAMPT modulatory compound comprises comparing: Ap4 formation by NAMPT that has been treated (e.g., including contacted) with the test compound; and Ap4 formation by NAMPT that has not been treated (e.g., including contacted) with the test compound. In certain embodiments, determining a modulation of Ap4 formation by the NAMPT comprises comparing: Ap4 formation by NAMPT before it has been treated (e.g., including contacted) with the test compound; and Ap4 formation by NAMPT after it has been treated (e.g., including contacted) with the test compound. Thus, the skilled worker will appreciate that determining a modulation of Ap4 formation by NAMPT can be achieved in the same preparation of NAMPT protein or in different preparations of NAMPT protein. For example, a preparation of NAMPT protein can be divided to provide two populations of NAMPT proteins, one of which is treated with a test compound and one of which is not treated with a test compound. Likewise, the skilled worker will appreciate that determining a modulation of Ap4 formation by NAMPT in cells can be achieved in the same population of cells expressing NAMPT or in different populations of cells expressing NAMPT.

[0085] In certain embodiments, the modulation of Ap4 formation is an increase in Ap4 formation or a decrease in Ap4 formation. However, in certain embodiments, the modulation of NTPase activity is no change in Ap4 formation. In some embodiments, a modulation of Ap4 formation is directly correlated with an NTPase modulatory activity such as ATPase activity. For example, inhibition of ATPase activity can result in less (ADP + P_t) formation and thus, less Ap4 formation. By contrast, in some embodiments, a modulation of Ap4 formation is inversely correlated with an NTPase modulatory activity such as ATPase activity. In some embodiments, the effect of a NAMPT modulatory compound on NTPase activity can be independent of its effect on Ap4 formation. In certain embodiments, a method for determining a modulation of Ap4 formation may further comprise administering a NAMPT modulatory compound to a patient in need thereof (e.g., a hypertension patient). In certain embodiments, a method for determining a modulation of Ap4 formation may be followed by administering a NAMPT modulatory compound to a patient in need thereof (e.g., a hypertension patient). In some embodiments, determining the modulation of Ap4 formation comprises using mass spectrometry, for example, to detect Ap4 production.

Methods for identifying cell mobility and metastasis modulatory activity

[0086] In some embodiments, the present disclosure provides a method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates the NTPase activity of NAMPT, the method comprising: determining the mobility of one or more cells(e.g., including determining the mobility of one or more cell lines) expressing NAMPT before treating (e.g., including contacting) the one or more cells (e.g., the one or more cell lines) with the NAMPT modulatory compound; treating (e.g., including contacting) the cells (e.g., the one or more cell lines) with the NAMPT modulatory compound; determining the mobility of the one or more cells (e.g., the one or more cell lines) after treating (e.g., including contacting) the one or more cells (e.g., including the one or more cell lines) with the NAMPT modulatory compound; and comparing the mobility of the one or more cells (e.g., including the one or more cell lines) before and after treating (e.g., including contacting) the cells (e.g., the cell lines) with the NAMPT modulatory compound; wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound. Without wishing to be bound by theory, it may be beneficial to identify a cell mobility modulatory activity in the context of testing a NAMPT modulatory compound for anti -cancer properties, especially in view of the surprising discovery provided herein that NAMPT modulatory compounds have differing modulatory effects on the NTPase activity of NAMPT, including the surprising discover that NAMPT modulatory compounds can affect each NTPase activity differently. For example, the skilled worker might test a NAMPT modulatory compound that stimulates the GTPase activity of NAMPT for its effects on cell mobility, due to the established link between GTP and cytoskeletal

organization/dynamics. The skilled worker will appreciate that, when comparing mobility between cells, or populations of cells, that the same or similar type of cells, or populations of cells, can be used to facilitate the comparison. For example, two populations of primary cells, or cell lines, can be cultured under the same conditions. One of these populations can be treated with a NAMPT modulatory compound, while the other is not, thereby providing two populations of cells that are substantially similar except for the treatment with the NAMPT modulatory compound.

[0087] In certain embodiments, determining the mobility of the one or more cells (e.g., including determining the mobility of one or more cell lines) expressing NAMPT before treating (e.g., including contacting) the one or more cells (e.g., including the one or more cell lines) with the NAMPT modulatory compound and determining the mobility of the one or more cells (e.g., including the one or more cell lines) expressing NAMPT after treating (e.g., including contacting) the one or more cells (e.g., including the one or more cell lines) with the NAMPT modulatory compound comprises performing an in situ assay, an in vitro assay, or an in vivo assay.

[0088] In some embodiments, the present disclosure provides a method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates the NTPase activity of NAMPT, said method comprising: treating one or more cells (e.g., including one or more cell lines) expressing NAMPT with the NAMPT modulatory compound; determining the mobility of the one or more cell s (e.g., including one or more cell lines) that have been treated (e.g., including contacted) with the NAMPT modulatory compound; determining the mobility of one or more cells (e.g., including one or more cell lines) that have not been treated (e.g., including contacted) with the NAMPT modulatory compound; and comparing the mobility of the one or more cells (e.g., including one or more cell lines) that have been treated (e.g., including contacted) with the NAMPT modulatory compound and the mobility of the one or more cells (e.g., including one or more cell lines) that have not been treated (e.g., including contacted) with the NAMPT modulatory compound; wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound.

[0089] In certain embodiments, determining the mobility of the one or more cells (e.g., including one or more cell lines) that have been treated (e.g., including contacted) with the NAMPT modulatory compound and determining the mobility of the one or more cell lines that have not been treated (e.g., including contacted) with the NAMPT modulatory compound comprises performing an in situ assay, an in vitro assay or an in vivo assay. [0090] In certain embodiments, the cell mobility modulatory activity is a decrease in cell mobility or an increase in cell mobility. However, the skilled worker will appreciate that in some embodiments, the cell mobility modulatory activity is no change in cell mobility. In certain embodiments, the method of determining cell mobility modulatory activity further comprises administering the NAMPT modulatory compound to a patient in need thereof (e.g., a cancer patient). For example, a NAMPT modulatory compound that decreases cell mobility may be used to treat a cancer patient.

[0091] In some embodiments, the present disclosure provides a method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound that modulates the NTPase activity of NAMPT, said method comprising: determining the metastatic state of one or more cells expressing NAMPT before treating (e.g., including contacting) the one or more cells (e.g., including one or more cell lines) with the NAMPT modulatory compound; treating (e.g., including contacting) the cells with the NAMPT modulatory compound; determining the metastatic state of the one or more cells (e.g., including one or more cell lines) after treating (e.g., including contacting) the one or more cells (e.g., including one or more cell lines) with the NAMPT modulatory compound; and comparing the metastatic state of the one or more cells (e.g., including one or more cell lines) before and after treating (e.g., including contacting) the cells with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound. Without wishing to be bound by theory, it may be beneficial to identify a cell metastasis modulatory activity in the context of testing a NAMPT modulatory compound for anti-cancer properties, especially in view of the surprising discovery provided herein that NAMPT modulatory compounds exhibit differing abilities to modulate the NTPase activity of NAMPT, including the surprising discover that NAMPT modulatory compounds can affect each NTPase activity differently. For example, the skilled worker might test a NAMPT modulatory compound that stimulates the GTPase activity of NAMPT for its effects on cell metastasis, due to the established link between GTP and cytoskeletal organization. The skilled worker will appreciate that, when comparing metastatic activity between cells, or populations of cells, that the same or similar type of cells, or populations of cells, can be used to facilitate the comparison. For example, two populations of primary cells, or cell lines, can be cultured under the same conditions. One of these populations can be treated with a NAMPT modulatory compound, while the other is not, thereby providing two populations of cells that are substantially similar except for the treatment with the NAMPT modulatory compound.

[0092] In certain embodiments, determining the metastatic state of the one or more cells (e.g., including one or more cell lines) expressing NAMPT before treating (e.g., including contacting) the one or more cells (e.g., including one or more cell lines) with the NAMPT modulatory compound and determining the metastatic state of the one or more cells (e.g., including one or more cell lines) expressing NAMPT after treating (e.g., including contacting) the one or more cells (e.g., including one or more cell lines) with the NAMPT modulatory compound comprises performing an in situ assay, an in vitro assay, or an in vivo assay.

[0093] In some embodiments, the present disclosure provides a method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound that modulates the NTPase activity of NAMPT, said method comprising: treating one or more cells (e.g., including one or more cell lines expressing NAMPT with the NAMPT modulatory compound; determining the metastatic state of the one or more cells (e.g., including one or more cell lines) that have been treated (e.g., including contacted) with the NAMPT modulatory compound; determining the metastatic state of one or more cells (e.g., including one or more cell lines) that have not been treated (e.g., including contacted) with the NAMPT modulatory compound; and comparing the metastatic state of the one or more cells (e.g., including one or more cell lines) that have been treated (e.g., including contacted) with the NAMPT modulatory compound and the metastatic state of the one or more cells (e.g., including one or more cell lines) that have not been treated (e.g., including contacted) with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound. In certain embodiments, determining the metastatic state of the one or more cells (e.g., including one or more cell lines) that have been treated (e.g., including contacted) with the NAMPT modulatory compound and determining the metastatic state of the one or more cells (e.g., including one or more cell lines) that have not been treated (e.g., including contacted) with the NAMPT modulatory compound comprises performing an in situ assay, an in vitro assay, or an in vivo assay.

[0094] In certain embodiments, the cell metastasis modulatory activity is a decrease in metastatic activity or an increase in metastatic activity. However, the skilled worker will appreciate that in some embodiments, the cell metastasis modulatory activity is no change in metastatic activity. In certain embodiments, the method of determining cell metastasis modulatory activity further comprises administering the NAMPT modulatory compound to a patient in need thereof (e.g., a cancer patient). For example, a NAMPT modulatory compound that decreases cell metastatic activity may be used to treat a cancer patient.

[0095] The skilled worker will appreciate that the methods described herein also may be used to screen an NAMPT modulatory compound for its effect on cell mobility and/or metastasis, even if the NAMPT modulatory compound does not modulate NTPase activity. NTPase activity

[0096] Without limitation, NTPase activity can be ATPase activity, GTPase activity, CTPase activity, UTPase activity, or any combination thereof. For example, a NAMPT modulatory compound may be screened to fit a particular NTPase "profile" wherein certain levels of NTPase activity of NAMPT provide desired biological effects (e.g., decrease cell mobility or metastasis, or otherwise treat cancer). Likewise, the skilled worker may modify a NAMPT modulatory compound to change its ability to modulate the NTPase activity of NAMPT, for example, increasing or decreasing one or more NTPase activity. Thus, a NAMPT modulatory compound can be modified to provide particular therapeutic benefits.

Compositions and methods of treatment

[0097] The skilled worker will appreciate that identifying an NAMPT modulatory compound, or determining an NTPase modulatory activity, or a cell mobility modulatory activity, or a cell metastasis modulatory activity, of a NAMPT modulatory compound, is useful in the development of treatments for diseases and conditions in which NAMPT can regulate the size of a biologically-important NTP pool. NAMPT catalyzes the reversible formation of NMN and pyrophosphate (PPi) from NAM and a-D-5-phosphoribosyl-l-pyrophosphate (PRPP). NMN is subsequently converted to NAD⁺ in the presence of ATP by NMN-adenylyl transferase

(NMNAT), and cells need ongoing NAD⁺ biosynthesis due to its brisk consumption by sirtuins (SIRTs), poly(ADP ribose) polymerases (PARPs), mono ADP-ribosyltransferases (ARTs), and ADP-ribosyl cyclases. Cancer cells, for example, are especially dependent on robust NAD⁺ biosynthesis due to heightened flux of these NAD⁺ consuming pathways. Thus, modulating NAMPT activity provides a way to reduce or eliminate cancer cell populations, or to reduce or eliminate their mobility and/or metastatic capacity.

[0098] In certain embodiments, a method of identifying an NAMPT modulatory compound, or determining an NTPase modulatory activity, or a cell mobility modulatory activity, or a cell metastasis modulatory activity, of a NAMPT modulatory compound, may be followed by administering the NAMPT modulatory compound to a patient in need thereof (e.g., a cancer patient). In certain embodiments, the present disclosure provides a method for treating an NAMPT-mediated disease or condition (e.g., cancer), comprising administering an NAMPT modulatory compound as described herein. In particular embodiments, the present disclosure provides a method for treating a disease or condition (e.g., cancer) that is influenced by NAMPT activity, comprising administering an NAMPT modulatory compound identified by the methods of the present disclosure described herein Without wishing to be bound by theory, it may be particularly beneficial to treat a patient (e.g., a cancer patient) with a NAMPT modulatory compound that decreases NMN formation and/or increases NTPase activity. A NAMPT modulatory compound that decreases NMN formation may also be called a NAMPT inhibitor.

[0099] As used herein, "treating" a disease or condition refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms associated with diseases or conditions.

[00100] As used herein, "administering" or "administration of a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow, or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some aspects, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self- administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient. In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion, or intravenously, e.g., to a subject by injection. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[00101] As used herein, a "NAMPT" inhibitor refers to a molecule (including, without limitation, small molecules, macromolecules, and biological molecules) that inhibits NAMPT' s ability to form NMN. Examples of known NAMPT inhibitors include FK-866, CHS-828, GNI-50, GPP- 78, GNE-618, STF-118804, STF-31, and KPT9274. Structures of the above-mentioned NAMPT inhibitors are as follows.

[00102] As described above, NAMPT inhibitors that increase the NTPase activity of NAMPT can be particularly beneficial in the treatment of NAMPT-modulated diseases and conditions. In particular, the reduction of the intracellular NTP pools by NAMPT-mediated hydrolysis of GTP, CTP and UTP can have impactful biological consequences. For example, GTP exerts a multitude of cellular effects in part due to its fundamental role as an obligatory effector for signal transduction by GTPases. Even small alterations of the intracellular GTP pools in melanoma cells have been shown to regulate the activity of several small Rho-GTPases involved in cell invasion, suppressing the ability of melanoma cells to form invadopodia, degrade the extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Thus, NAMPT- mediated reduction of GTP can impact the activity of Rho GTPases, thereby regulating the structure/function of specialized actin-based cytoskeleton structures. This mechanistic link also is supported by evidence that NAMPT is localized to lamellipodia, cytoskeleton regions that mediate cellular motility and adhesion between cells and extracellular matrix; that FK-866 treatment reduces the activity of Cdc42, a member of the Rho-GTPase subfamily; and that immunoprecipitation of GTP-Cdc42 captures endogenous NAMPT. Thus, NAMPT modulatory compounds (e.g., NAMPT inhibitors) that increase GTPase activity (thereby depleting the local GTP pool) will affect cancer cells by reducing cell mobility and metastasis. CTP plays a central role in phospholipid synthesis by producing key intermediates such as CDP-diacylglycerol, CDP- choline and CDP-ethanolamine. This role is supported by ^LH-decoupled ³¹P magnetic resonance spectroscopy (³¹P MRS) evidence that FK-866 has a marked impact on the phospholipid pool. UTP is a precursor for activated forms of monosaccharides that serve as glycosyl donors in glycosylation reactions.

[00103] As used herein, NAMPT-modulated diseases and conditions are those in which the disease pathway involves NAMPT. NAMPT-mediated diseases and conditions include, but are not limited to, cancer, obesity, diabetes (e.g., Type 1 diabetes, Type 2 diabetes, and gestational diabetes), inflammatory diseases and disorders, and neurodegenerative diseases. In some embodiments, a NAMPT-modulated disease or condition is one in which the disease or condition has direct causality from NAMPT. As described herein, NAMPT modulatory compounds can have an effect on Ap4 formation. In some embodiments, modulating Ap4 formation can be used to treat primary or secondary hypertension, abnormal vasoconstriction, Raynaud's disease, occlusive diseases associated with inflammation, post-traumatic dystrophy, migraines, Buerger's disease, or diseases associated with increased intraocular pressure (e.g., glaucoma).

[00104] As used herein, the term "cancer" refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites (see, for example, PDR Medical Dictionary, 1 st edition (1995), incorporated herein by reference in its entirety for all purposes). The terms "neoplasm" and "tumor" refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated proliferation is removed. Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (such as a benign tumor) or malignant (such as a malignant tumor). Examples of general categories of cancer include, but are not limited to, carcinomas (malignant tumors derived from epithelial cells such as, for example, common forms of breast, prostate, lung and colon cancer), sarcomas (malignant tumors derived from connective tissue or mesenchymal cells), lymphomas (malignancies derived from hematopoietic cells), leukemias (malignancies derived from hematopoietic cells), germ cell tumors (tumors derived from totipotent cells, which in adults are most often found in the testicle or ovary and in fetuses, babies and young children, are most often found on the body midline, particularly at the tip of the tailbone), blastic tumors (a typically malignant tumor which resembles an immature or embryonic tissue) and the like. Examples of the types of neoplasms intended to be encompassed by the present disclosure include, but are not limited to, those neoplasms associated with cancers of neural tissue, blood forming tissue, breast, skin, bone, prostate, ovaries, uterus, cervix, liver, lung, brain, larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal gland, immune system, head and neck, colon, stomach, bronchi, and/or kidneys.

[00105] As used herein, the term "inflammatory diseases" refers to pathological states resulting in inflammation, typically caused by neutrophil chemotaxis. Examples of such disorders include inflammatory skin diseases including psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (IBD) (such as Crohn's disease and ulcerative colitis); ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and constriction after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma, hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute-lung injury; Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis;

encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoid arthritis (RA), Sjorgen's syndrome, vasculitis; diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder, multiple organ injury syndrome secondary to septicemia or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex mediated diseases including glomerulonephritis; sepsis; sarcoidosis; immunopathologic responses to tissue/organ transplantation; inflammations of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis,

hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis; chronic inflammation, autoimmune diabetes, rheumatoid spondylitis, gouty arthritis and other arthritic conditions, asthma, systemic lupus erythematosus, chronic pulmonary inflammatory disease, graft versus host reaction, Alzheimer's disease, and pyresis, along with any disease or disorder that relates to inflammation and related disorders.

[00106] As used herein, the term "diabetes" refers to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin and is characterized by hyperglycemia and glycosuria. Diabetes includes, but is not limited to, Type I diabetes, Type II diabetes, insulin-resistant diabetes (e.g., Mendenhall syndrome), gestational diabetes, and lipoatrophic diabetes.

[00107] As used herein, the term "neurodegenerative disease" refers to a disease or disorder in which neurons lose their structure or function, including diseases or disorders resulting in neuronal death. Neurodegenerative diseases include, but are not limited to, prion diseases (e.g., Creutzfeldt-Jakob disease), Alzheimer's disease, amyotrophic lateral sclerosis, corticobasal degeneration, frontotemporal dementia, HIV-related cognitive impairment, Huntington's disease, Lewy body dementias (e.g., dementia with Lewy bodies and Parkinson's disease with dementia), mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy, and vascular dementia.

[00108] In some embodiments, the present disclosure provides a composition comprising an NAMPT modulatory compound (e.g., a AMPT inhibitor) as described herein. In certain embodiments, the present disclosure provides a composition comprising an NAMPT modulatory compound (e.g., a NAMPT inhibitor) identified according to the methods of the present disclosure. In certain embodiments, the present disclosure provides a composition comprising an NAMPT modulatory compound (e.g., a NAMPT inhibitor) whose NTPase modulatory activity, or cell mobility modulatory activity, or cell metastasis modulatory activity, has been determined according to the methods of the present disclosure. In some embodiments, a composition of the present disclosure is useful as a medicament. In some embodiments, the present disclosure provides for the use of a composition of the present disclosure in the manufacture of a medicament. In some embodiments, it may be beneficial to include one or more excipients in the compositions of the present disclosure. One of skill in the art would appreciate that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipient because the combination of excipients would produce undesirable effects. One of skill in the art would be able to empirically determine which additional excipients, if any, to include in the formulations of the present disclosure. For example, a NAMPT modulatory compound can be combined with at least one pharmaceutically acceptable carrier such as a solvent, bulking agents, binder, humectant, disintegrating agent, solution retarder, disintegrant, glidant, absorption accelerator, wetting agent, solubilizing agent, lubricant, sweetening agent, or flavorant agent. A

"pharmaceutically acceptable carrier" refers to any diluent or excipient that is compatible with the other ingredients of the formulation, and which is not deleterious to the recipient. A pharmaceutically acceptable carrier can be selected on the basis of the desired route of administration, in accordance with standard pharmaceutical practices.

[00109] In some embodiments, the present disclosure provides a method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound having a NTPase modulatory activity, the method comprising administering the NAMPT modulatory compound to a subject; and determining a modulation of NTPase activity of NAMPT, wherein the ability of the NAMPT modulatory compound to modulate NTPase activity of NAMPT is indicative of the NAMPT modulatory compound having an NTPase modulatory activity; and formulating the NAMPT modulatory compound having an NTPase modulatory activity with a pharmaceutically acceptable carrier. In certain embodiments, the modulation of NTPase activity is an increase in NTPase activity or a decrease in NTPase activity.

[00110] In certain embodiments, determining the modulation of NTPase activity comprises using mass spectrometry, for example, to detect NDP (e.g., ADP, GDP, CDP, or UDP) production. In certain embodiments, determining the modulation of NTPase activity comprises using a colorimetric assay, for example, to detect inorganic phosphate produced during NDP (e.g., ADP, GDP, CDP, or UDP) production.

[00111] In some embodiments, the present disclosure provides a method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound having an NTPase modulatory activity, the NAMPT modulatory compound having been administered to a subject and determined to modulate NTPase activity of NAMPT in a biological sample obtained from the subject, wherein the ability of the NAMPT modulatory compound to modulate NTPase activity of NAMPT in the biological sample is indicative of the NAMPT modulatory compound having an NTPase modulatory activity, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier. As described above, an NTPase modulatory activity can be an increase in NTPase activity or a decrease in NTPase activity, and can be determined using mass spectrometry or a colorimetric assay.

[00112] In some embodiments, the present disclosure provides a method for optimizing an NTPase modulatory activity of a NAMPT modulatory compound, wherein said method comprises determining the NTPase modulatory activity of the NAMPT modulatory compound. Thus, the present disclosure provides a method of crafting an NAMPT modulatory compound to suit a particular need, such as a biological activity (e.g., for treating cancer). In certain embodiments, the method further comprises modifying the NAMPT modulatory compound to optimize the NTPase modulatory activity (e.g., by increasing the NTPase modulatory activity or decreasing the NTPase modulatory activity). As described above, NTPase activity can be determined using mass spectrometry or a colorimetric assay.

[00113] In some embodiments, the present disclosure provides a method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound, wherein an NTPase modulatory activity of the NAMPT modulatory compound has been identified, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier. In certain embodiments, the NTPase modulatory activity is an increase in NTPase activity. In certain embodiments, the NTPase modulatory activity is a decrease in NTPase activity. As described above, NTPase activity can be determined using mass spectrometry or a colorimetric assay.

[00114] In some embodiments, it can be beneficial to include a bulking agent in the compositions of the present disclosure. Bulking agents are commonly used in pharmaceutical compositions to provide added volume to the composition. Bulking agents are well known in the art. Accordingly, the bulking agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary bulking agents that may be used in the compositions and methods of the present disclosure.

[00115] Exemplary bulking agents can include carbohydrates, sugar alcohols, amino acids, and sugar acids. Bulking agents include, but are not limited to, mono-, di-, or poly-, carbohydrates, starches, aldoses, ketoses, amino sugars, glyceraldehyde, arabinose, lyxose, pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose, talose, heptose, glucose, fructose, methyl a- D-glucopyranoside, maltose, lactone, sorbose, erythrose, threose, arabinose, allose, altrose, gulose, idose, talose, erythrulose, ribulose, xylulose, psicose, tagatose, glucosamine,

galactosamine, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galactocarolose, pectins, amylose, pullulan, glycogen, amylopectin, cellulose, microcrystalline cellulose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, xanthin gum, sucrose, trehalose, dextran, lactose, alditols, inositols, sorbitol, mannitol, glycine, aldonic acids, uronic acids, aldaric acids, gluconic acid, isoascorbic acid, ascorbic acid, glucaric acid, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, neuraminic acid, pectic acids, maize starch, and alginic acid.

[00116] In some embodiments, it can be beneficial to include a disintegrant in the compositions of the present disclosure. Disintegrants aid in the breakup of solid compositions, facilitating delivery of an active pharmaceutical composition. Disintegrants are well known in the art.

Accordingly, the disintegrants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary disintegrants that may be used in the compositions and methods of the present disclosure. Exemplary disintegrants include crospovidone,

microcrystalline cellulose, sodium carboxymethyl cellulose, methyl cellulose, sodium starch glycolate, calcium carboxymethyl croscarmellose sodium, polyvinylpyrrolidone, lower alkyl- substituted hydroxypropyl cellulose, Indion 414, starch, pre-gelatinized starch, calcium carbonate, gums, sodium alginate, and Pearlitol Flash®.

[00117] In some embodiments, it can be beneficial to include a glidant in the compositions of the present disclosure. Glidants aid in the ability of a powder to flow freely. Glidants are well known in the art. Accordingly, the glidants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary glidants that may be used in the compositions and methods of the present disclosure. Exemplary glidants include colloidal silica (silicon dioxide), magnesium stearate, starch, talc, glycerol behenate, DL-leucine, sodium lauryl sulfate, calcium stearate, and sodium stearate.

[00118] In some embodiments, it can be beneficial to include a lubricant in the compositions of the present disclosure. Lubricants help keep the components of a composition from clumping. Lubricants are well known in the art. Accordingly, the lubricants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary lubricants that may be used in the compositions and methods of the present disclosure. Exemplary lubricants include calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumarate, vegetable based fatty acids, talc, mineral oil, light mineral oil, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, safflower oil, canola oil, coconut oil and soybean oil), silica, zinc stearate, ethyl oleate, and ethyl laurate.

[00119] In some embodiments, it can be beneficial to include a solubilizing agent in the formulations or compositions of the present disclosure. Solubilizing agents may be useful for increasing the solubility of a NAMPT modulatory compound or an excipient. The solubilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary solubilizing agents that may be used in the formulations or compositions of the present disclosure. In certain embodiments, solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceutically acceptable salts and/or combinations thereof.

[00120] The pH of the compositions of the present disclosure may be any pH that provides desirable properties for the formulation or composition. Desirable properties may include, for example, NAMPT modulatory compound stability and improved filtration efficiency.

[00121] In some embodiments, it can be beneficial to buffer the pH by including one or more buffers in the compositions. One of skill in the art would appreciate that an appropriate buffer may be chosen for inclusion in compositions of the present disclosure based on its pKa and other properties. Buffers are well known in the art. Accordingly, the buffers described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary buffers that may be used in the formulations or compositions of the present disclosure. In certain embodiments, a buffer includes, but is not limited to, Tris, Tris HC1, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium and potassium phosphate, Tris/Tris HC1, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof.

[00122] In some embodiments, it can be beneficial to include a surfactant in the compositions of the present disclosure. Surfactants, in general, reduce the surface tension of a liquid composition. This may provide beneficial properties such as improved ease of filtration. Surfactants also may act as emulsifying agents and/or solubilizing agents. Surfactants are well known in the art.

Accordingly, the surfactants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary surfactants that may be used in the formulations or compositions of the present disclosure. Surfactants that may be included include, but are not limited to, sorbitan esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

[00123] In some embodiments, it can be beneficial to include a toni city-adjusting agent in the reconstituted formulations and injectable compositions of the present disclosure. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration. Toni city-adjusting agents, thus, may be used to help make a formulation or composition suitable for administration. Tonicity-adjusting agents are well known in the art. Accordingly, the tonicity-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary tonicity-adjusting agents that may be used in the formulations or compositions of the present disclosure. Tonicity- adjusting agents may be ionic or non-ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

[00124] In some embodiments, it can be beneficial to include a stabilizing agent in the compositions of the present disclosure. Stabilizing agents help increase the stability of a

NAMPT modulatory compound in the compositions of the present disclosure. This may occur by, for example, reducing degradation or preventing aggregation of an anthracycline compound. Stabilizing agents are well known in the art. Accordingly, the stabilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary stabilizing agents that may be used in the formulations or compositions of the present disclosure.

Stabilizing agents can include, but are not limited to, emulsifiers and surfactants.

[00125] As described above, NAMPT modulatory compounds can be used to treat NAMPT- mediated diseases and conditions (e.g., cancer). A NAMPT modulatory compound also may be combined with another therapeutic compound or method to improve the outcome of the treatment. In some embodiments, a NAMPT modulatory compound is combined with a drug which may further treat cancer.

EXAMPLES

Example 1: Identification of NAMPT modulatory compounds

Experimental procedures

Chemicals

[00126] NAM, NMN, NAD, AMP, ADP, ATP, GDP, GTP, CDP, CTP, UDP, UTP,

imidodiphosphate (PNP), and acetophenone were purchased from Sigma Aldrich (St. Louis, MO), Tocris Biosciences (Avonmouth, Bristol, UK), Thermo Scientific (Waltham, MA) or MP Biomedicals (Santa Ana, CA) Isotopically labeled nucleotides, ^L80₂-NMN and ¹⁸0₂-NAD, were synthesized. Other internal standards, D₄-NAM, ¹³C¹⁰-¹⁵N₅-ATP, ¹³C₉-¹⁵N₃-CTP, ¹³C₁₀-¹⁵N₅- GTP, and ¹³C₉-¹⁵N₂-UTP, were purchased from Cambridge Isotope Laboratories, Inc.

(Cambridge, MA) and Sigma Aldrich. FK-866 (2-(£ N-/ -(l-benzoyl-4-piperidinyl)butyl]-3-(3- pyridinyl)-2-propenamide) and tris(hydroxy-propyl)-phosphine (THP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CHS-828 (N-/6-(4-chlorophenoxy)hexyl-N'-cyano- N"-4-pyridinyl-guanidine) was purchased from Cayman Chemicals (Ann Arbor, MI). GNI-50 was synthesized by Sanford Burnham Prebys Medical Discovery Institute.

Recombinant NAMPT

[00127] N-terminal His-tagged human NAMPT was expressed in E.coli using the BL21(DE3)- pLys plasmid (pBAD DEST 49) containing a cDNA insert encoding human NAMPT (6). The BL21 cells were grown in LB broth (EMD Millipore) containing 100 μg mL.i ampicillin and 30 μg mL.i chloramphenicol at 37 °C until turbidity reached an OD₆₀o of 0.8-0.9. NAMPT expression was induced by adding IPTG (0.8 mM). Cells were then grown overnight at 20 °C, harvested by centrifugation, resuspended in 20 mM Tris, 0.5 M NaCl, 5 mM imidazole (pH 7.9), 1 mM 2-mercaptoethanol (ME), and protease inhibitor cocktail and disrupted by sonication. Debris was removed by centrifugation at 20,000 rpm for 20 min. The supernatant (150 ml from 50 g of cells) was loaded onto a Ni-NTA column (50 ml of resin pre-equilibrated with disruption buffer). The column was washed with 20 mM Tris, 500 mM NaCl, 50 mM imidazole (pH 7.9), 1 mM ME and eluted with the same buffer containing 0.3 M imidazole. NAMPT was concentrated and then further purified using a HiLoad Superdex 200pg 16/600 column (GE Healthcare) run in the presence of 100 mM Hepes (pH 7.5), 100 mM NaCl, and 10 mM ME. Purified NAMPT was flash frozen and stored at -80°C. The NAMPT-H247A mutant expression constructs were derived from the pBAD-DEST49 expression after site-directed mutagenesis to change the nucleotide sequence using the QuikChange® XL Site-Directed Mutagenesis kit (Agilent Technologies) and custom oligonucleotide primers. The NAMPT mutant was expressed and purified as described above for WT NAMPT.

NAMPT Enzymatic Reactions

[00128] For the NTPase assays, NAMPT or NAMPT-H247A was incubated at 37 °C with an NTP in TMT buffer (50 mM Tris-HCl, 10 mM MgC12, 1 mM THP, pH 7.5). The routinely-used NTP concentration was 2 mM. However, for the kinetic analysis ATP and GTP hydrolysis, the NTP concentration ranged from 0.25 mM to 4 mM. Other agents (FK-866, CHS-828, GNI-50, PNP, NMN) were included where indicated. At the indicated times, an aliquot of the sample was quenched by the addition of an equal volume of 1 M perchloric acid (PCA). Samples were stored at -80 °C until processed for LC/MS/MS. The values for Vmax and Km (ATP and GTP hydrolysis) were deduced using the on-line Michaelis-Menten kinetics tool at

http://www.graphpad.com/quickcalcs/ttestl/?Format=SEM For the NMN production assay, NAMPT (20 nM) was incubated in the presence of NAM (10 μΜ), PRPP (50 μΜ) in TMT buffer. Where indicated, NTPs (2 mM) were also included. NMN was assayed using a chemical method which converts NMN into a fluorescent derivative. Briefly, an aliquot (37.5 μΐ) of the NMN- containing sample was sequentially mixed with 15 μΐ of 20% acetophenone (in DMSO) and 15 μΐ of 2M KOH. The mixture was placed on ice for approximately 10 min. Next, 67.5 μΐ of 100% formic acid was added to each sample, vortexed, and then incubated at 37 °C for 20 min. Samples (100 μΐ) were transferred to a 96-well opaque bottom plate and fluorescence (Ex/Em = 382/445 nm) was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA).

Preparation of Standards for LC/MS/MS

[00129] Individual stock solutions of standards were prepared by dissolving each nucleotide in 0.1% formic acid at concentrations ranging from 125 μΜ to 50 mM. Aliquots of these stock solutions were combined to prepare a calibration stock mixture with concentrations ranging from 20 μΜ to 50 mM. The working calibration curve was prepared by spiking 10 μΐ, of each calibration stock mixture into 90 μΐ, of 0.5 M PCA and 100

of 1 M ammonium formate. The working calibration curve concentrations were 0.25, 0.5, 1 , 2.5, 5, 10, 25, 50, 100, and 200 μΜ for NAM, NAD, GDP, GTP, CDP, CTP, UDP, and UTP. NMN working solutions were 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, and 2 μΜ. The AMP, ADP, and ATP working solutions were 0.625, 1.25, 2.5, 6.25, 12.5, 25, 62.5, 125, 250, and 500 μΜ. Individual stock solutions of internal standards were prepared by dissolving each isotopic labelled nucleotide in 0.1% formic acid resulting in concentrations of 0.5 mM to 50 mM. An internal standard mixture of all isotopic labeled nucleotide stock solutions was prepared with 0.1% formic acid to make internal standard concentrations ranging from 1.25 μΜ to 1.25 mM. A 10 μΐ, aliquot of the internal standard mixture was spiked into each working calibration mixture resulting in concentrations ranging from 0.125 μΜ to 125 μΜ.

Extraction of Nucleotides

[00130] A 100 μΙ_ aliquot of NAMPT assay sample was quenched with equal volume 1 M PCA and spiked with a 10 yh mixture of isotopic labeled internal standards. A 100 μΙ_. aliquot of 1 M ammonium formate was added to adjust the sample pH to ~4. Samples were vortexed and centrifuged at 18,000 x g for 5 min at 10 °C. The samples were passed through an AcroPrep Advance 3K Omega Filter Plate (Pall Corporation, Port Washington, NY) by centrifugation at 3500 x g for 60 min prior to LC MS/MS analysis.

Liquid Chromatography

[00131] Metabolites were separated on a 2.1 x 50 mm, 3 μιη Thermo Scientific Hypercarb column (T = 30°C) using a Dionex Ultimate 3000 UHPLC. The step gradient was run from 98% A (10 mM ammonium acetate, pH 9.5) and 2% B (acetonitrile) to 64 % A and 36% B over 6.3 min. The step gradient began at 2% B (0.6 mL min._! flow rate) from 0-0.45 min, was increased from 2% to 36%o B (0.6 mL min.i flow rate) from 0.45-6.3 min, was increased from 36% to 95% B (0.8 mL min.i flow rate) from 6.3-6.4 min, and was held until 8.4 min. Re-equilibration was performed at 2% B from 8.4-8.5 min (0.7 mL min.i flow rate) and was held until 1 1.5 min. The flow returned to 0.6 mL min.i at 11.6 min and was held until 1 1.7 min. The samples were inj ected (2 μΤ) on a LEAP CTC PAL autosampler maintained at 5 °C the entire run.

Mass Spectrometry

The instrument method was created with Xcalibur 3.0 and data acquisition was performed by TraceFinder 3.2. Quantitation of pyridine nucleotides was achieved using single reaction monitoring (SRM) on a Thermo Scientific Quantiva triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). The mass spectrometer was operated in positive ion mode using electrospray ionization with an ESI capillary voltage of 3500V. The ion transfer tube temperature was 300 °C and vaporizer temperature was 350 °C. The ESI source sheath gas was set to 10, the auxiliary gas was set to 10, and the sweep gas was set to 1. The mass spectrometer was operated with a mass resolution of 0.7 Da, a cycle time of 0.3 s, and nitrogen collision gas of 1.5 mTorr for generation and detection of product ions of each nucleotide. SRM transitions were 123. 1 80.1 for NAM, 127.1 84.1 for d₄-NAM, 137.2 94. 1 for 1-methyl NAM, 335.2 - 123.1 for NMN, 339.2 -» 123.1 for ¹⁸0₂-NMN, 348.1 - 136.1 for AMP, 405.0 - 97.1 for UDP, 404. 1 -» 1 12.2 for CDP, 484.0 - 12.1 for CTP, 496.0 -» 119.1 for ¹³C₉-¹⁵N₃-CTP, 428.0 -» 136.1 for ADP, 496.0 -» 102.1 for ¹³C₉-¹⁵N₂-UTP, 485.0 -> 97.0 for UTP, 444.1 -» 152.1 for GDP, 508. 1 - 136.1 for ATP, 523.1 - 146.1 for ¹³Ci₀-¹⁵N₅-ATP, 744.2 -» 136.1 for NADP, 524.0 - 152.1 for GTP, 539.0 - 162.1 for ¹³Ci₀-¹⁵N₅-GTP, 664.2 - 136.1 for NAD, and 668.2 136.1 for ¹⁸0₂-NAD. Collision energies to produce product ions ranged from 16 to 46 V with RF lens values ranging from 43 to 85 V.

Data Processing

[00132] The raw data was processed using Xcalibur 3.0. Calibration curves (R2 = 0.99 or greater) were either fitted with a linear or a quadratic curve with a 1/X or 1/X2 weighting. Results

[00133] Incubation of NAMPT with ATP (2 mM) produced a time-dependent increase of ADP formation (FIG. 1 A). The ability of NAMPT to hydrolyze other NTPs (2 mM each) including CTP, GTP and UTP, was also evaluated. Interestingly, NAMPT was promiscuous with respect to its NTP substrate. Under the experimental conditions, the preferred order for NTP hydrolysis was ATP > GTP ~ UTP > CTP (FIG. 1A; Table 1). Table 1 shows NTPase activity (μηιοΐ NDP per μιηοΐ NAMPT per hour) in the absence (None) and presence of PNP (50 μΜ), NMN (10 μΜ), FK-866 (1 μΜ), CHS-828 (1 μΜ) or GNI-50 (1 μΜ). All reactions contained an NTP concentration of 2 mM. Mean (sd) values are shown. The number of replicates were N=4 except the NTPase values for "None" conditions where ATPase, GTPase, UTPase and CTPase activities were derived from 31, 16, 17 and 16 replicates, respectively.

[00134] The kinetic behavior of the ATP and GTP substrates for the NAMPT-mediated NTPase reaction was also studied (FIG. IB). The V_max values for ATP and GTP were similar (32 ± 2 and 29 ± 3 μιηοΐ NDP / μηιοΐ NAMPT / h, respectively). The K_m values for ATP and GTP were 0.48 ± 0.09 and 1.4 ± 0.4 mM, respectively. Hence, NAMPT displays ~3-fold weaker apparent affinity for GTP as compared to ATP.

Table 1 : Impact of NAMPT ligands on the NTPase activities of NAMPT

[00135] NAMPT-mediated NMN synthesis from NAM and PRPP was increased in the presence of ATP (FIG. 2A). Remarkably, GTP, CTP and UTP did not promote NAMPT-mediated NMN production despite being hydrolyzed by NAMPT (FIG. 2A). Indeed, the presence of UTP exerted a slight but significant inhibitory effect on NAMPT-mediated NMN production.

Suppression of ATP-stimulated NMN formation by a 4-fold molar excess of GTP, CTP or UTP revealed mutually-exclusive binding of NTPs to the NAMPT active site (FIG. 2B).

[00136] The ATPase activity of NAMPT is believed to involve a putative phosphoenzyme intermediate at His247. To reinforce the link between NAMPT and the observed NTPase activity, a NAMPT mutant possessing a His-to-Ala substitution at position 247 (NAMPT -H247A) was produced. NAMPT-H247A had very low NMN-forming activity, as demonstrated by its markedly reduced NMN-producing activity (<5%) compared to WT NAMPT. Importantly, the ATPase and GTPase activities of NAMPT-H247A were both reduced to <5% of that displayed by WT NAMPT (FIG. 1 C). These data firmly established that GTP hydrolysis is a catalytic activity that is intrinsic to NAMPT.

[00137] The ATPase activity of NAMPT is stimulated by PPi and PNP. PNP (assayed at 50 μΜ) had a striking stimulatory effect (7.5-fold) on the ATPase of NAMPT (Table 1). There was little to no impact of PNP on the GTPase and UTPase activities catalyzed by NAMPT; however, the CTPase activity of NAMPT was increased 3.3 -fold in the presence of PNP (Table 1). The product of the NAMPT enzymatic reaction, NMN, also stimulated the NTPase activity of NAMPT as assessed by NDP production (Table 1). The stimulatory effect of NMN (assayed at only 10 μΜ) on the CTPase activity of NAMPT was very striking (15.4-fold). NMN also stimulated the UTPase (7.3-fold), ATPase (5.2-fold) and GTPase (2.6-fold) activities of NAMPT. The NMN analog, nicotinic acid mononucleotide (NaMN), failed to stimulate the ATPase activity of NAMPT. This finding was consistent with the strict specificity of NAMPT for NAM versus nicotinic acid.

[00138] FK-866, CHS-828, and GNI-50 are small molecule inhibitors of NAMPT-mediated NMN formation. FK-866, CHS-828, and GNI-50 (each assayed at 1 μΜ) were very potent inhibitors of NAMPT-mediated NMN production, and abolished NMN production by NAMPT.

[00139] Surprisingly, addition of FK-866 to NAMPT elicited pronounced stimulation of NAMPT NTPase activity (Table 1). The stimulatory effect of FK-866 on NAMPT-mediated NTP hydrolysis exhibited the following potency rank order: CTP (25.8-fold) > UTP (7.9-fold) > GTP (4.7-fold) > ATP (2.7-fold). Concentrations as low as 250 nM FK-866 exerted a pronounced stimulatory effect on the NAMPT-mediated ATPase and GTPase activities (FIG. 3A). There was little additional increased hydrolysis of ATP or GTP as the FK-866

concentration was raised to 2 μΜ. These data showed that FK-866 was very potent at stimulating the ATPase and GTPase activities of NAMPT, as was also the case for inhibition of NAMPT-mediated NMN formation.

[00140] The impact of FK-866 on the Vmax and Km values for ATP and GTP was examined next (FIG. IB). The V_max and K_m values for ATP in the presence of 1 μΜ FK-866 were 437 ± 78 μηιοΐ ADP / μηιοΐ NAMPT / h and 6.0 ± 1.0 mM, respectively. Hence, FK-866 increased significantly the V_max and K_m values for ATP by 14-fold (p = 0.002) and 12-fold (p=0.01), respectively. This greatly increased Vmax value for ATP hydrolysis in the presence of FK-866 was not expected. The V_max and K_m values for GTP in the presence of 1 μΜ FK-866 were 154 ± 9 μηιοΐ ADP / μιηοΐ NAMPT / h and 1.0 ± 0.2 mM, respectively. Hence, FK-866 significantly increased the Vmax for the GTPase activity by 5-fold (p<0.001). In contrast to ATP, the apparent affinity of GTP for NAMPT was not impacted by the presence of FK-866 (p = 0.17).

[00141] In contrast to FK-866, CHS-828 (1 μΜ) had a much different impact on the NAMPT- mediated NTPase activities (Table 1). CHS-828 inhibited NAMPT -mediated ATPase activity (43% reduction) and had no appreciable effect on the GTPase activity. CHS-828 stimulated NAMPT-mediated hydrolysis of CTP (10.8-fold) and UTP (1.8-fold) but to a lesser extent than produced by FK-866. Interestingly, the stimulatory effect of GNI-50 (1 μΜ) on the NTPase activity of NAMPT was more uniformly balanced (Table 1). The ATPase, GTPase, UTPase and CTPase activities of NAMPT were increased by 2.0-, 4.9-, 4.2-, and 7.8-fold, respectively, in the presence of 1 μΜ GNI-50.

[00142] The opposing effects of FK-866 and CHS-828 on NAMPT-mediated ATPase activity led to the question of whether CHS-828 could block the stimulatory effect of FK-866 (FIG. 3B). A 10-fold molar excess of CHS-828 (relative to FK-866) blocked the stimulatory effects of FK- 866 on NAMPT-mediated ATP hydrolysis. This result is consistent with findings from X-ray crystallography studies showing mutually-exclusive binding of FK-866 and CHS-828 to NAMPT. Discussion

[00143] The reciprocal impact of FK-866, CHS-828 and GNI-50 on NMN formation (inhibition) and NTPase activity (activation) was surprising, as was finding the disparate effects of the FK- 866, CHS-828 and GNI-50 on the NTPase activities of NAMPT. The results described above shed important new light on the enzymatic activity catalyzed by NAMPT. Firstly, they show that NAMPT-mediated NTP hydrolysis is promiscuous rather than selective for ATP. Secondly, despite this promiscuity, only ATP hydrolysis drives NMN production by NAMPT. Thirdly, inhibitors of NAMPT-mediated NMN formation such as FK-866, CHS-828 and GNI-50 are potent activators of NTP hydrolysis by NAMPT. However, each of these NAMPT inhibitors exhibited unique profiles with respect to their abilities to stimulate the ATPase, CTPase, GTPase and UTPase activities of NAMPT. Together, these findings have unveiled novel and surprising catalytic functions and regulatory mechanisms for NAMPT that can have important biological and pharmacological implications.

[00144] The results show for the first time the ability of NAMPT to hydrolyze other NTPs.

Nicotinate phosphoribosyltransferase (NaPRTase) is another member of the type II

phosphoribosyltransferase family that shares a conserved overall structure and common catalytic mechanism with NAMPT despite very limited sequence similarity. ATP also stimulates the catalytic activity of NaPRTase via the generation of a putative phosphohistidine intermediate. The promiscuous nucleotide specificity of NAMPT revealed by our investigation is concordant with analogous studies of NaPRTase, but there is an important fundamental difference. While NAMPT hydrolyzed all of the tested NTPs, only ATP stimulated NAMPT-mediated NMN formation.

[00145] The exclusive ability of ATP as compared to the other NTPs to promote NAMPT- mediated NMN formation is surprising. The current NAMPT reaction scheme depicts ATP hydrolysis as the first catalytic step which gives rise to a phosphoenzyme intermediate. The next step in the proposed catalytic mechanism is ADP release. This reaction scheme predicts that any NTP hydrolyzed by NAMPT should produce an identical phosphoenzyme intermediate with indistinguishable propensities to promote NMN formation. CTP, GTP and UTP are hydrolyzed by NAMPT but do not promote NMN production. Hence, the interaction of ATP with NAMPT and/or production of enzyme-bound ADP appear to stage NAMPT for accelerated NMN formation.

[00146] The promiscuous NTPase activity of NAMPT is especially noteworthy in light of the discovery provided herein that it was stimulated by NAMPT ligands including NMN, FK-866, CHS-828, and GNI-50.

Example 2: Identification of Ap4 formation modulatory activity

[00147] A calibration curve for adenosine 5'-tetraphosphate (Ap4) (Jena Bioscience) was prepared in 200 μΐ_^ of 0.5 M perchloric acid (PC A) and 100 mM ammonium formate at Ap4 concentrations of 0.04 μΜ, 0.08 μΜ, 0.16 μΜ, 0.3 μΜ, 0.625 μΜ, 1.25 μΜ, 2.5 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ, 62.5 μΜ, 125 μΜ, 250 μΜ, and 500 μΜ. Calibration curve and study samples were run on an Accela HPLC/Thermo Scientific triple quadrupole mass spectrometer by positive electrospray ionization. 5 μL samples were injected onto a 3 x 50 mm, 3 μιη Hypercarb column and separated using a linear gradient from 98% A (10 mM ammonium bicarbonate, pH 9.5) and 2% B (acetonitrile (ACN)) to 5% A and 95% B at 6.4 minutes at 0.6 mL/min. Multiple reaction monitoring was used to monitor transitions for Ap4 (m/z 588 136) and 13C1015N5-ATP (m/z 523 -> 146) as an internal standard. Linear calibration curves were obtained using Xcalibur software to quantitate Ap4 in biological samples. Inorganic phosphate (Pi) was assayed using the PiColorLock™ Gold phosphate detection reagent (Innova Biosciences).

[00148] To test the effects of NAMPT inhibitors on AP4 formation, human NAMPT (200 nM) was incubated with ATP (2 mM) and vehicle (1% DMSO) or one of the NAMPT inhibitors FK- 866, CHS-828, or GNI-50 (1 μΜ each) for increasing times at 37 °C in TMD buffer. Samples (50 μΐ) were removed at the indicated times and quenched with 50 μΐ of 1M perchloric acid (PCA) and submitted for LC MS/MS to detect ADP and Ap4. Another sample (80 μL) was removed, frozen and subsequently assayed for Pi using the Pi ColorLock™ Gold assay kit. [00149] As described above, ADP, Pi, and Ap4 concentrations were measured after incubating NAMPT with ATP and a NAMPT inhibitorsFK-866, CHS-828, or GNI-50. Surprisingly, these NAMPT inhibitors not only differentially modulated Ap4 formation, but this effect did not have a direct correlation with NTPase modulatory activity as observed through NAMPT' s ATPase activity. Incubation of human NAMPT (200 nM) with ATP (2 mM) produced a time-dependent increase of Ap4 formation (1.04 μΜ Ap4 per hr) (see FIG. 4C). The presence of NAMPT inhibitor FK-866 (at 1 μΜ) had essentially no impact on Ap4 production (0.88 μΜ Ap4 per hr). However, the NAMPT inhibitors CHS-828 and GNI-50 were markedly different from each other and from FK-866 with respect to their impact on NAMPT-mediated Ap4 production (see FIG. 4C). CHS-828 (1 μΜ) nearly abolished Ap4 generation (0.88 μΜ per hr), while GNI-50 stimulated Ap4 production approximately 1 9-fold (1.93 μΜ per hr). Table 2 shows the impact of the NAMPT inhibitors on ADP, P and Ap4 formation. Notably, although FK-866 had the largest impact on ADP production (a 5.39-fold increase in ADP production as compared to vehicle control), it resulted in less than half the Ap4 of GNI-50. Moreover, Ap4 decreased with FK-866 as compared to vehicle control but increased with GNI-50 as compared to vehicle control. Table 2: Impact of NAMPT inhibitors on ADP, P_;, and Ap4 formation by NAMPT

[00150] The values in Table 4represent the mean production rates of ADP, Pi, or Ap4, or calculated values using those production rates, in μΜ/hr. Values in parentheses represent standard deviations. Pi + Ap4 is the sum of Pi and Ap4 production rates, which corresponds to the ADP value if the Pi liberated during Ap4 hydrolysis has only two possible fates: free Pi and Ap4. ADP/(Pi + Ap4) is the ratio between ADP formation and combined Pi and Ap4 formation.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for preparing a pharmaceutical composition comprising a nicotinamide phosphoribosyltransferase (NAMPT) modulatory compound having a nucleoside triphosphatase (NTPase) modulatory activity, said method comprising:

a) administering the NAMPT modulatory compound to a subject; and

b) determining a modulation of NTPase activity of NAMPT, wherein the ability of the NAMPT modulatory compound to modulate NTPase activity of NAMPT is indicative of the NAMPT modulatory compound having an NTPase modulatory activity; and

c) formulating the NAMPT modulatory compound having an NTPase modulatory activity with a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the modulation of NTPase activity is an increase in NTPase activity.

3. The method of claim 1, wherein the modulation of NTPase activity is a decrease in NTPase activity.

4. The method of any one of claims 1-3, wherein determining the modulation of NTPase activity comprises using mass spectrometry.

5. The method of claim 4, wherein the mass spectrometry is used to detect nucleoside diphosphate production.

6. The method of claim 5, wherein the nucleoside diphosphate is selected from the group consisting of ADP, GDP, CDP, or UDP.

7. The method of claim 6, wherein the NDP is ADP.

8. The method of claim 6, wherein the NDP is GDP.

9. The method of claim 6, wherein the NDP is CDP.

10. The method of claim 6, wherein the NDP is UDP.

11. The method of any one of claims 1-3, wherein determining the modulation of NTPase activity comprises using a colorimetric assay.

12. The method of claim 11, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

13. The method of claim 12, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

14. The method of claim 13, wherein the NDP is ADP.

15. The method of claim 13, wherein the NDP is GDP.

16. The method of claim 13, wherein the NDP is CDP.

17. The method of claim 13, wherein the NDP is UDP.

18 A method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound having an NTPase modulatory activity, the NAMPT modulatory compound having been administered to a subject and determined to modulate NTPase activity of NAMPT in a biological sample obtained from the subject, wherein the ability of the NAMPT modulatory compound to modulate NTPase activity of NAMPT in the biological sample is indicative of the NAMPT modulatory compound having an NTPase modulatory activity, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

19. The method of claim 18, wherein the NTPase modulatory activity is an increase in NTPase activity.

20. The method of claim 18, wherein the NTPase modulatory activity is a decrease in NTPase activity.

21. The method of any one of claims 18-20, wherein the NTPase modulatory activity was determined using mass spectrometry.

22. The method of claim 21, wherein the mass spectrometry was used to detect NDP production.

23. The method of claim 22, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

24. The method of claim 23, wherein the NDP is ADP.

25. The method of claim 23, wherein the NDP is GDP.

26. The method of claim 23, wherein the NDP is CDP.

27. The method of claim 23, wherein the NDP is UDP.

28. The method of any one of claims 18-20, wherein the NTPase modulatory activity was determined using a colorimetric assay.

29. The method of claim 28, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

30. The method of claim 29, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

31. The method of claim 30, wherein the NDP is ADP.

32. The method of claim 30, wherein the NDP is GDP.

33. The method of claim 30, wherein the NDP is CDP.

34. The method of claim 30, wherein the NDP is UDP.

35. A method for optimizing an NTPase modulatory activity of a NAMPT modulatory compound, wherein said method comprises determining the NTPase modulatory activity of the NAMPT modulatory compound.

36. The method of claim 35, further comprising modifying the NAMPT modulatory compound to optimize the NTPase modulatory activity.

37. The method of claim 35 or 36, wherein optimizing the NTPase modulatory activity comprises increasing the NTPase modulatory activity.

38 The method of claim 35 or 36, wherein optimizing the NTPase modulatory activity comprises decreasing the NTPase modulatory activity.

39. The method of any one of claims 35-38, wherein determining the NTPase modulatory activity comprises using mass spectrometry.

40. The method of claim 39, wherein the mass spectrometry is used to detect NDP production.

41. The method of claim 40, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

42. The method of claim 41, wherein the NDP is ADP.

43. The method of claim 41, wherein the NDP is GDP.

44. The method of claim 41, wherein the NDP is CDP.

45. The method of claim 41, wherein the NDP is UDP.

46. The method of any one of claims 35-38, wherein determining the modulation of NTPase activity comprises using a colorimetric assay.

47. The method of claim 46, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

48. The method of claim 47, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

49. The method of claim 48, wherein the NDP is ADP.

50. The method of claim 48, wherein the NDP is GDP.

51. The method of claim 48, wherein the NDP is CDP.

52. The method of claim 48, wherein the NDP is UDP.

53. A method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound, wherein an NTPase modulatory activity of the NAMPT modulatory compound has been identified, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

54. The method of claim 53, wherein the NTPase modulatory activity is an increase in NTPase activity.

55 The method of claim 53, wherein the NTPase modulatory activity is a decrease in NTPase activity.

56. The method of any one of claims 53-55, wherein the NTPase modulatory activity was determined using mass spectrometry.

57. The method of claim 56, wherein the mass spectrometry was used to detect NDP production.

58. The method of claim 57, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

59. The method of claim 58, wherein the NDP is ADP.

60. The method of claim 58, wherein the NDP is GDP.

61. The method of claim 58, wherein the NDP is CDP.

62. The method of claim 58, wherein the NDP is UDP.

63. The method of any one of claims 53-55, wherein the NTPase modulatory activity was determined using a colorimetric assay.

64. The method of claim 63, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

65 The method of claim 64, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

66. The method of claim 65, wherein the NDP is ADP.

67. The method of claim 65, wherein the NDP is GDP.

68. The method of claim 65, wherein the NDP is CDP.

69 The method of claim 65, wherein the NDP is UDP.

70. A method for identifying a NAMPT modulatory compound, said method comprising: a) treating NAMPT with a test compound;

b) determining a modulation of NMN formation by the NAMPT; and

c) determining a modulation of NTPase activity of NAMPT;

wherein the modulation of NMN formation of b) and the modulation of NTPase activity of c) identifies the test compound as an NAMPT modulatory compound.

71. The method of claim 70, wherein the determining a modulation of NMN formation by the NAMPT comprises comparing:

i) NMN formation by the NAMPT that has been treated with the test compound; and ii) NMN formation by NAMPT that has not been treated with the test compound.

72. The method of claim 70, wherein the determining a modulation of NMN formation by the NAMPT comprises comparing:

i) NMN formation by the NAMPT before it has been treated with the test compound; and ii) NMN formation by the NAMPT after it has been treated with the test compound.

73. The method of claim 70, wherein the determining a modulation of NTPase activity of the NAMPT comprises comparing:

i) NTPase activity of the NAMPT that has been treated with the test compound; and ii) NTPase activity of NAMPT that has not been treated with the test compound.

74 The method of claim 70, wherein the determining a modulation of NTPase activity of the NAMPT comprises comparing:

i) NTPase activity of the NAMPT before it has been treated with the test compound; and ii) NTPase activity of the NAMPT after it has been treated with the test compound.

75. The method of any one of claims 70-72, wherein the modulation of NMN formation is a decrease in NMN formation.

76. The method of any one of claims 70-72, wherein the modulation of NMN formation is no change in NMN formation.

77. The method of claim 75 or 76, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

78. The method of claim 77, wherein the patient is a cancer patient.

79. The method of any one of claims 70-72, wherein the modulation of NMN formation is an increase in NMN formation.

80. The method of any one of claims 70-79, wherein the modulation of NTPase activity is an increase in NTPase activity.

81. The method of any one of claims 70-79, wherein the modulation of NTPase activity is no change in NTPase activity.

82. The method of claim 80 or 81, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

83. The method of claim 82, wherein the patient is a cancer patient.

84. The method of any one of claims 70-79, wherein the modulation of NTPase activity is a decrease in NTPase activity.

85. The method of any one of claims 70-84, wherein determining the modulation of NTPase activity comprises using mass spectrometry.

86. The method of claim 85, wherein the mass spectrometry is used to detect NDP production.

87. The method of claim 86, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

88. The method of claim 87, wherein the NDP is ADP.

89. The method of claim 87, wherein the NDP is GDP.

90. The method of claim 87, wherein the NDP is CDP.

91. The method of claim 87, wherein the NDP is UDP.

92. The method of any one of claims 70-84, wherein screening the test compound for modulation of NTPase activity comprises using a colorimetric assay.

93. The method of claim 92, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

94. The method of claim 93, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

95. The method of claim 94, wherein the NDP is ADP.

96. The method of claim 94, wherein the NDP is GDP.

97. The method of claim 94, wherein the NDP is CDP.

98. The method of claim 94, wherein the NDP is UDP.

99. A method for determining an NTPase modulatory activity of an NAMPT modulatory compound, said method comprising:

a) treating NAMPT with the NAMPT modulatory compound; and

b) determining a modulation of NTPase activity of NAMPT.

100. The method of claim 99, wherein the determining an NTPase modulatory activity of an NAMPT modulatory compound comprises comparing:

101. The method of claim 99, wherein the determining a modulation of NTPase activity of the NAMPT comprises comparing:

102. The method of any one of claims 99-101, wherein the modulation of NTPase activity is an increase in NTPase activity.

103. The method of any one of claims 99-101, wherein the modulation of NTPase activity is no change in NTPase activity.

104. The method of claim 102 or 103, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

105. The method of claim 104, wherein the patient is a cancer patient.

106. The method of any one of claims 99-101, wherein the modulation of NTPase activity is a decrease in NTPase activity.

107. The method of any one of claims 99-106, wherein determining the modulation of NTPase activity comprises using mass spectrometry

108. The method of claim 107, wherein the mass spectrometry is used to detect NDP production.

109. The method of claim 108, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

110. The method of claim 109, wherein the NDP is ADP.

111. The method of claim 109, wherein the NDP is GDP.

112. The method of claim 109, wherein the NDP is CDP.

113. The method of claim 109, wherein the NDP is UDP.

114. The method of any one of claims 99-106, wherein determining the modulation of NTPase activity comprises using a colorimetric assay.

115. The method of claim 114, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

116. The method of claim 115, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

117. The method of claim 116, wherein the NDP is ADP.

118. The method of claim 116, wherein the NDP is GDP.

119. The method of claim 116, wherein the NDP is CDP.

120. The method of claim 116, wherein the NDP is UDP.

121. A method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising:

a) determining the mobility of one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound;

b) contacting the cells with the NAMPT modulatory compound;

c) determining the mobility of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and

d) comparing the mobility of the one or more cells before and after contacting the cells with the NAMPT modulatory compound;

wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound

122. The method of claim 121 , wherein determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay.

123. The method of claim 121 , wherein determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay.

124. The method of claim 121 , wherein determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

125. A method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising:

a) contacting one or more cells expressing NAMPT with the NAMPT modulatory compound; b) determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound;

c) determining the mobility of one or more cells that have not been contacted with the NAMPT modulatory compound; and

d) comparing the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound;

wherein said comparison identifies the cell mobility modulatory activity of the NAMPT modulatory compound.

126. The method of claim 125, wherein determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay.

127. The method of claim 125, wherein determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay.

128. The method of claim 125, wherein determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

129. The method of any one of claims 121-128, wherein the cell mobility modulatory activity is a decrease in cell mobility.

130. The method of any one of claims 121-128, wherein the cell mobility modulatory activity is no change in cell mobility.

131. The method of any one of claims 121-128, wherein the cell mobility modulatory activity is an increase in cell mobility.

132. The method of claim 129 or 130, further comprising administering the NAMPT modulatory compound to a patient in need thereof

133. The method of claim 132, wherein the patient is a cancer patient.

134. A method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising:

a) determining the metastatic state of one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound;

b) contacting the cells with the NAMPT modulatory compound;

c) determining the metastatic state of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and

d) comparing the metastatic state of the one or more cells before and after contacting the cells with the NAMPT modulatory compound;

wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound.

135. The method of claim 134, wherein determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay.

136. The method of claim 134, wherein determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay.

137. The method of claim 134, wherein determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

138. A method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound that modulates NAMPT NTPase activity, said method comprising:

a) contacting one or more cells expressing NAMPT with the NAMPT modulatory compound;

b) determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound;

c) determining the metastatic state of one or more cells that have not been contacted with the NAMPT modulatory compound; and

d) comparing the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound;

139. The method of claim 138, wherein determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay.

140. The method of claim 138, wherein determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay.

141. The method of claim 138, wherein determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

142. The method of any one of claims 134-1 1, wherein the cell metastasis modulatory activity is a decrease in metastatic activity.

143. The method of any one of claims 134-141, wherein the cell metastasis modulatory activity is no change in metastatic activity.

144. The method of any one of claims 134-1 1, wherein the cell metastasis modulatory activity is an increase in metastatic activity.

145. The method of claim 142 or 143, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

146. The method of claim 145, wherein the patient is a cancer patient.

147. The method of any one of the above claims, wherein the NTPase activity is selected from the group consisting of ATPase activity, GTPase activity, CTPase activity, UTPase activity, or a combination thereof.

148. The method of claim 147, wherein the NTPase activity is ATPase activity.

149. The method of claim 147, wherein the NTPase activity is GTPase activity.

150. The method of claim 147, wherein the NTPase activity is CTPase activity.

151. The method of claim 147, wherein the NTPase activity is UTPase activity.

152. The method of any one of the above claims, wherein the NAMPT modulatory compound is an NAMPT inhibitor.

153. The method of claim 152, wherein the NAMPT inhibitor is selected from the group consisting of FK-866, GNI-50, and CHS-828.

154. The method of claim 153, wherein the NAMPT inhibitor is FK-866.

155. The method of claim 153, wherein the NAMPT inhibitor is GNI-50.

156. The method of claim 153, wherein the NAMPT inhibitor is CHS-828.

157. The method of any one of the above claims, said method further comprising modifying the NAMPT modulatory compound to change its NTPase activity.

158. The method of claim 157, wherein said change to the NTPase activity is an increase in NTPase activity.

159. The method of claim 157, wherein said change to the NTPase activity is a decrease in NTPase activity.

160. A method for treating cancer, said method comprising administering an NAMPT modulatory compound of any one of the above claims.

161. A composition comprising an NAMPT modulatory compound of any one of claims 70-98.

162. A composition comprising an NAMPT modulatory compound of any one of claims 99- 152.

163. A method for preparing the composition of claim 161 or 162, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

164. A method for identifying a NAMPT modulatory compound, said method comprising: a) treating NAMPT with a test compound;

b) determining a modulation of NMN formation by the NAMPT; and

c) determining a modulation of guanosine triphosphatase (GTPase) activity of NAMPT; wherein the modulation of NMN formation of b) and the modulation of GTPase activity of c) identifies the test compound as an NAMPT modulatory compound.

165. The method of claim 164, wherein the determining a modulation of NMN formation by the NAMPT comprises comparing:

166. The method of claim 164, wherein the determining a modulation of NMN formation by the NAMPT comprises comparing:

167. The method of claim 164, wherein the determining a modulation of GTPase activity of the NAMPT comprises comparing:

i) GTPase activity of the NAMPT that has been treated with the test compound; and ii) GTPase activity of NAMPT that has not been treated with the test compound.

168. The method of claim 164, wherein the determining a modulation of GTPase activity of the NAMPT comprises comparing:

i) GTPase activity of the NAMPT before it has been treated with the test compound; and ii) GTPase activity of the NAMPT after it has been treated with the test compound.

169. The method of any one of claims 164-166, wherein the modulation of NMN formation is a decrease in NMN formation.

170. The method of any one of claims 164-166, wherein the modulation of NMN formation is no change in NMN formation.

171. The method of claim 169 or 170, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

172. The method of claim 171 , wherein the patient is a cancer patient.

173. The method of any one of claims 164-166, wherein the modulation of NMN formation is an increase in NMN formation.

174. The method of any one of claims 164-173, wherein the modulation of GTPase activity is an increase in GTPase activity.

175. The method of any one of claims 164-173, wherein the modulation of GTPase activity is no change in GTPase activity.

176. The method of claim 174 or 175, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

177. The method of claim 176, wherein the patient is a cancer patient.

178. The method of any one of claims 95-104, wherein the modulation of GTPase activity is a decrease in GTPase activity

179. The method of any one of the above claims, wherein determining the modulation of GTPase activity comprises using mass spectrometry.

180. The method of claim 179, wherein the mass spectrometry is used to detect GDP production.

181. The method of any one of claims 95-178, wherein screening the test compound for modulation of GTPase activity comprises using a colorimetric assay.

182. The method of claim 181 , wherein the colorimetric assay detects inorganic phosphate produced during GDP production.

183. A method for determining a GTPase modulatory activity of an NAMPT modulatory compound, said method comprising:

a) treating NAMPT with the NAMPT modulatory compound; and

b) determining a modulation of GTPase activity of NAMPT.

184. The method of claim 183, wherein the determining a GTPase modulatory activity of an NAMPT modulatory compound comprises comparing:

185. The method of claim 183, wherein the determining a modulation of GTPase activity of the NAMPT comprises comparing:

186. The method of any one of claims 183-185, wherein the modulation of GTPase activity is an increase in GTPase activity.

187. The method of any one of claims 183-185, wherein the modulation of GTPase activity is no change in GTPase activity.

188. The method of claim 186 or 187, further comprising administering the NAMPT modulatory compound to a patient in need thereof

189. The method of claim 188, wherein the patient is a cancer patient.

190. The method of any one of claims 183-185, wherein the modulation of GTPase activity is a decrease in GTPase activity.

191. The method of any one of claims 183-190, wherein determining the modulation of GTPase activity comprises using mass spectrometry.

192. The method of claim 191 , wherein the mass spectrometry is used to detect GDP production.

193. The method of any one of claims 183-190, wherein determining the modulation of GTPase activity comprises using a colorimetric assay.

194. The method of claim 193, wherein the colorimetric assay detects inorganic phosphate produced during GDP production.

195. A method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound that modulates NAMPT GTPase activity, said method comprising:

b) contacting the cells with the NAMPT modulatory compound;

196. The method of claim 195, wherein determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay.

197. The method of claim 196, wherein determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay.

198. The method of claim 196, wherein determining the mobility of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the mobility of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

199. The method of claim 195, wherein said one or more cells are from a non-primary cell line.

200. A method for identifying a cell mobility modulatory activity of an NAMPT modulatory compound with GTPase activity, said method comprising:

b) determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound;

201. The method of claim 200, wherein determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay.

202. The method of claim 200, wherein determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay.

203. The method of claim 200, wherein determining the mobility of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the mobility of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

204. The method of any one of claims 196-203, wherein the cell mobility modulatory activity is a decrease in cell mobility.

205. The method of any one of claims 196-203, wherein the cell mobility modulatory activity is no change in cell mobility.

206. The method of any one of claims 196-203, wherein the cell mobility modulatory activity is an increase in cell mobility.

207. The method of claim 204 or 205, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

208. The method of claim 207, wherein the patient is a cancer patient.

209. A method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound with GTPase activity, said method comprising:

b) contacting the cells with the NAMPT modulatory compound;

c) determining the metastatic state of the one or more cells after contacting the one or more cells with the NAMPT modulatory compound; and d) comparing the metastatic state of the one or more cells before and after contacting the cells with the NAMPT modulatory compound;

210. The method of claim 209, wherein determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in situ assay.

211. The method of claim 209, wherein determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vitro assay.

212. The method of claim 209, wherein determining the metastatic state of the one or more cells expressing NAMPT before contacting the one or more cells with the NAMPT modulatory compound and determining the metastatic state of the one or more cells expressing NAMPT after contacting the one or more cells with the NAMPT modulatory compound comprises performing an in vivo assay.

213. A method for identifying a cell metastasis modulatory activity of an NAMPT modulatory compound with GTPase activity, said method comprising:

d) comparing the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound; wherein said comparison identifies the cell metastasis modulatory activity of the NAMPT modulatory compound

214. The method of claim 213, wherein determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in situ assay.

215. The method of claim 213, wherein determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vitro assay.

216. The method of claim 213, wherein determining the metastatic state of the one or more cells that have been contacted with the NAMPT modulatory compound and determining the metastatic state of the one or more cells that have not been contacted with the NAMPT modulatory compound comprises performing an in vivo assay.

217. The method of any one of claims 209-216, wherein the cell metastasis modulatory activity is a decrease in metastatic activity.

218. The method of any one of claims 209-216, wherein the cell metastasis modulatory activity is no change in metastatic activity.

219. The method of any one of claims 209-216, wherein the cell metastasis modulatory activity is an increase in metastatic activity.

220. The method of claim 217 or 218, further comprising administering the NAMPT modulatory compound to a patient in need thereof.

221. The method of claim 220, wherein the patient is a cancer patient.

222. The method of any one of claims 96-221, wherein the NAMPT modulatory compound is an NAMPT inhibitor.

223. The method of claim 222, wherein the NAMPT inhibitor is selected from the group consisting of FK-866, GNI-50, CHS-828, and GNI-50.

224. The method of claim 223, wherein the NAMPT inhibitor is FK-866.

225. The method of claim 223, wherein the NAMPT inhibitor is GNI-50.

226. The method of claim 223, wherein the NAMPT inhibitor is CHS-828.

227. The method of claim 223 , wherein the NAMPT inhibitor is GNI-50.

228. The method of any one of claims 96-227, said method further comprising modifying the NAMPT modulatory compound to change its GTPase activity.

229. The method of claim 228, wherein said change to the GTPase activity is an increase in GTPase activity.

230. The method of claim 228, wherein said change to the GTPase activity is a decrease in GTPase activity.

231. A method for treating cancer, said method comprising administering an NAMPT modulatory compound of any one of claims 96-230.

232. A composition comprising an NAMPT modulatory compound of any one of claims 96- 183.

233. A composition comprising an NAMPT modulatory compound of any one of claims 184- 230.

234. A method for preparing the composition of claim 232 or 233, said method comprising combining the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

235. A method for preparing a pharmaceutical composition comprising a nicotinamide phosphoribosyltransferase (NAMPT) modulatory compound having a modulatory activity on adenosine-5'-tetraphosphate (Ap4) formation, said method comprising:

a) administering the NAMPT modulatory compound to a subject; and

b) determining a modulation of Ap4 formation by NAMPT, wherein the ability of the NAMPT modulatory compound to modulate Ap4 formation by NAMPT is indicative of the NAMPT modulatory compound having a modulatory activity on Ap4 formation; and

236. A method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound having a modulatory activity on adenosine-5'-tetraphosphate (Ap4) formation, the NAMPT modulatory compound having been administered to a subject and determined to modulate Ap4 formation by NAMPT in a biological sample obtained from the subject, wherein the ability of the NAMPT modulatory compound to modulate Ap4 formation by NAMPT in the biological sample is indicative of the NAMPT modulatory compound having a modulatory activity on Ap4, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

237. A method for preparing a pharmaceutical composition comprising a NAMPT modulatory compound, wherein a modulatory activity on adenosine-5'-tetraphosphate (Ap4) formation by the NAMPT modulatory compound has been identified, said method comprising formulating the NAMPT modulatory compound with a pharmaceutically acceptable carrier.

238. A method for identifying a NAMPT modulatory compound, said method comprising: a) treating NAMPT with a test compound;

b) determining a modulation of adenosine-5'-tetraphosphate (Ap4) formation by NAMPT; and

c) determining a modulation of NTPase activity of NAMPT;

wherein the modulation of Ap4 formation of b) and the modulation of NTPase activity of c) identifies the test compound as an NAMPT modulatory compound.

239. A method for determining an NTPase modulatory activity of an NAMPT modulatory compound, said method comprising:

a) treating NAMPT with the NAMPT modulatory compound; and

b) determining a modulation of adenosine-5'-tetraphosphate (Ap4) formation by NAMPT.

240. The method of any one of claims 235-239, wherein the modulation of NTPase activity is an increase in NTPase activity.

241. The method of any one of claims 235-239, wherein the modulation of NTPase activity is a decrease in NTPase activity.

242. The method of any one of claims 235-241, wherein the modulation of Ap4 formation is an increase in Ap4 formation.

243. The method of any one of claims 235-241, wherein the modulation of Ap4 formation is a decrease in Ap4 formation.

244. The method of any one of claims 235-243, wherein determining the modulation of NTPase activity comprises using mass spectrometry.

245. The method of claim 244, wherein the mass spectrometry is used to detect nucleoside diphosphate production.

246. The method of claim 245, wherein the nucleoside diphosphate is selected from the group consisting of ADP, GDP, CDP, or UDP.

247. The method of claim 246, wherein the NDP is ADP.

248. The method of claim 246, wherein the NDP is GDP.

249. The method of claim 246, wherein the NDP is CDP.

250. The method of claim 246, wherein the NDP is UDP.

251. The method of any one of claims 235-243, wherein determining the modulation of Ap4 formation comprises using mass spectrometry.

252. The method of claim 251 , wherein the mass spectrometry is used to detect Ap4 production.

253. The method of any one of claims 235-243, wherein determining the modulation of NTPase activity comprises using a colorimetric assay.

254. The method of claim 253, wherein the colorimetric assay detects inorganic phosphate produced during NDP production.

255. The method of claim 254, wherein the NDP is selected from the group consisting of ADP, GDP, CDP, or UDP.

256. The method of claim 255, wherein the NDP is ADP.

257. The method of claim 255, wherein the NDP is GDP.

258. The method of claim 255, wherein the NDP is CDP.

259. The method of claim 255, wherein the NDP is UDP.

260. The method of any one of claims 235-243, wherein determining the modulation of Ap4 formation comprises using a colorimetric assay.

261. The method of claim 260, wherein the colorimetric assay detects inorganic phosphate consumed during Ap4 production.

262. A composition comprising an NAMPT modulatory compound of any one of claims 235- 261.

263. A method for preparing the composition of claim 262, said method comprising combining the NAMPT modulatory compound with a pharmaceutically acceptable carrier.