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WO2016085990A1 - Compositions et procédés relatifs à l'inhibition de l'activité sérine hydroxyméthyltransférase 2 - Google Patents

Compositions et procédés relatifs à l'inhibition de l'activité sérine hydroxyméthyltransférase 2 Download PDF

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Publication number
WO2016085990A1
WO2016085990A1 PCT/US2015/062442 US2015062442W WO2016085990A1 WO 2016085990 A1 WO2016085990 A1 WO 2016085990A1 US 2015062442 W US2015062442 W US 2015062442W WO 2016085990 A1 WO2016085990 A1 WO 2016085990A1
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Prior art keywords
cancer
shmt2
activity
agent
cells
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English (en)
Inventor
Nouri Neamati
Shili XU
Shuzo TAMURA
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University of Michigan System
University of Michigan Ann Arbor
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University of Michigan System
University of Michigan Ann Arbor
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91028—Hydroxymethyl-, formyl-transferases (2.1.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to small molecule compounds capable of inhibiting serine hydroxylmethyltransferase 2 (SHMT2) activity, methods for the discovery of such compounds, and related research and therapeutic uses for such compounds.
  • SHMT2 serine hydroxylmethyltransferase 2
  • the present invention provides compounds capable of inhibiting SHMT2 activity, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with diseases and other pathophysiological states caused by or associated with aberrant SHMT2 activity (e.g., cancer).
  • Serine hydroxymethyltransferase is an enzyme which plays an important role in cellular one-carbon pathways by catalyzing the reversible, simultaneous conversions of L- serine to glycine (retro-aldol cleavage) and tetrahydrofolate to 5, 10- methylenetetrahydrofolate (hydrolysis) (see, e.g., Appaji Rao N, et al. (2003) Biochim.
  • the present invention relates to small molecule compounds capable of inhibiting serine hydroxylmethyltransferase 2 (SHMT2) activity, methods for the discovery of such compounds, and related research and therapeutic uses for such compounds.
  • SHMT2 serine hydroxylmethyltransferase 2
  • the present invention provides compounds capable of inhibiting SHMT2 activity, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with diseases and other pathophysiological states caused by or associated with aberrant SHMT2 activity (e.g., cancer).
  • the present invention provides methods for inhibiting SHMT2 activity, comprising exposing one or more cells having SHMT2 activity to one or more agents presented in Table 1 and Table 5 (see, Examples). Such methods are not limited to a particular type of cell or cells.
  • the cells are either in vitro, ex vivo, or in vivo cells. In some embodiments, the cells are human cells.
  • the present invention provides methods for treating a subject suffering from condition characterized by aberrant SHMT2 activity, comprising
  • an agent e.g., compound presented in Table 1 and/or Table 5.
  • the subject is a human subject.
  • administration of the agent results in inhibition of SHMT2 activity.
  • Such methods are not limited to a particular type of condition characterized by aberrant SHMT2 activity.
  • SHMT2 expression and/or activity is associated with several types of cancer.
  • the condition is cancer (e.g., lung cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioma, liver cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, urothelial cancer) (e.g., any type of cancer associated with SHMT2 expression and/or activity).
  • the methods further comprise coadministration of the agent presented in Table 1 and/or Table 5 with an anti-cancer agent.
  • the present invention provides methods for prophylactically treating a subject at risk for developing a condition characterized by aberrant SHMT2 activity, comprising administering to the subject a therapeutically effective amount of an agent presented in Table 1 and/or Table 5.
  • the subject is a human subject.
  • administration of the agent results in inhibition of SHMT2 activity.
  • Such methods are not limited to a particular type of condition characterized by aberrant SHMT2 activity.
  • the condition is cancer (e.g., lung cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioma, liver cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, urothelial cancer) (e.g., any type of cancer associated with SHMT2 expression and/or activity).
  • the methods further comprise co-administration of the agent presented in Table 1 and/or Table 5 with an anti-cancer agent.
  • the present invention provides assays comprising cells expressing recombinant SHMT2 and MTHFD, wherein exposure of the assay to an agent capable of inhibiting SHMT2 activity results in a fluorescently detectable event.
  • the present invention provides methods for screening agents able capable of inhibiting SHMT2 activity, comprising administering a candidate agent to a sample comprising cells expressing SHMT2 and MTHFD, and detecting the presence or absence of fluorescence resulting from a SHMT2 and MTHFD coupling reaction, wherein detection of fluorescence indicates the candidate agent is able to inhibit SHMT2 activity.
  • the SHMT2 expressed in the sample is recombinant SHMT2.
  • the MTHFD expressed in the sample is recombinant MTHFD.
  • the candidate agent is a peptide.
  • the candidate agent is a small molecule.
  • FIG. 1 A-B shows the expression and purification of SHMT2 and MTHFD proteins.
  • SHMT2 protein was expressed in the E.coli BLR(DE3) strain, and purified using a Ni2+ NTA affinity column.
  • B. MTHFD protein was expressed in the E.coli BL21(DE3) strain, and purified using a anion exchanging (Q) column. The resulting SHMT2 and MTHFD proteins were over 90% pure as was determined by SDS-PAGE.
  • FIG. 2A-H shows the designand optimization of the SHMT2 activity assay for HTS.
  • FIG. 3A-B shows identification of the first-in-class SHMT2 inhibitors.
  • A. Approaches to identifying SHMT2 inhibitors.
  • B. Primary screens of a total of 6560 compounds for their abilities to inhibit SHMT2 activity. Two primary screens were performed at final compound concentrations at 26.5 ⁇ and 6.5 ⁇ , respectively, providing the top 200 compounds for dose response examination.
  • FIG. 4A-C shows generation of NSCLC cell lines with Dox-inducible overexpression of SHMT2.
  • A. Dox-induced SHMT2, LacZ, and GFP in indicated engineered NSCLC cell lines. Cells were incubated with Dox at 200 ng/mL for 72 h, prior to harvest for Western blotting analysis. Histogram shows the fold increase of SHMT2 expression after 72 h Dox treatment from three experiments. Bar, SD.
  • B Dox-induced GFP and LacZ expression in control cell lines. Dox-induced GFP expression was monitored using fluorescent microscopy. The activity of LacZ-encoded ⁇ -galactosidase was measured by the conversion of colorless X-Gal into an insoluble blue product that was visualized under bright-field microscopy.
  • C. Dox induced SHMT2 overexpression in a time-dependent manner. Cells were treated with 200 ng/mL. Dox for indicated days, followed by Western blotting analysis of SHMT2 abundance.
  • FIG. 5A-B shows generation of NSCLC cell lines with Dox-inducible knockdown of SHMT2.
  • A. Dox-induced SHMT2 knockdown in indicated engineered NSCLC cell lines. Cells were incubated with Dox at 1 ⁇ g/mL for 72 h, prior to harvest for Western blotting analysis.
  • B. Dox-induction of SHMT2 knockdown in a time-dependent manner. Cells were treated with 1 ⁇ g/mL Dox for indicated days, followed by Western blotting analysis of SHMT2 abundance.
  • FIG. 6A-B NSCLC cell lines with Dox-inducible expression of LacZ or GFP as control cell lines.
  • the term "therapeutically effective dose” is meant a dose that produces the desired effect for which it is administered.
  • the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
  • derivative of a small molecule refers to a chemically modified small molecule wherein the chemical modification takes place either at a functional group of the small molecule (e.g., compound) or on, for example, an aromatic ring.
  • the term "subject” refers to organisms to be treated by the methods and agents of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.
  • mammals e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like.
  • subject generally refers to an individual who will receive or who has received treatment (e.g., administration of an agent of the present invention and optionally one or more other agents) for a condition characterized by aberrant SHMT2 activity (e.g., cancer (e.g., lung cancer)).
  • treatment e.g., administration of an agent of the present invention and optionally one or more other agents
  • a condition characterized by aberrant SHMT2 activity e.g., cancer (e.g., lung cancer)
  • the term "host cell” refers to any eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • eukaryotic or prokaryotic cell e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
  • co-administration refers to the administration of at least two agent(s) (e.g., an agent of the present invention) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are co- administered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).
  • the term "toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975]).
  • salts of the compounds of the present invention may be derived from inorganic or organic acids and bases.
  • acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p- sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their
  • bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NW , wherein W is C 1-4 alkyl, and the like.
  • salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate,
  • salts include anions of the compounds of the present invention compounded with a suitable cation such as Na + , NH 4 + , and NW 4 + (wherein W is a C 1-4 alkyl group), and the like.
  • salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable.
  • salts of acids and bases that are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • non-specific binding and “background binding” when used in reference to the interaction of a molecule with a protein or enzyme refers to an interaction that is not dependent on the presence of a particular structure.
  • the term “modulate” refers to the activity of an agent (e.g., a small molecule of the present invention) to affect (e.g., to promote or retard) an aspect of cellular function, including, but not limited to, enzymatic activity, maturation, cell growth, replication, proliferation, and the like.
  • an agent e.g., a small molecule of the present invention
  • affect e.g., to promote or retard an aspect of cellular function, including, but not limited to, enzymatic activity, maturation, cell growth, replication, proliferation, and the like.
  • test compound refers to any chemical entity, pharmaceutical, drug, and the like, that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample.
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • the present invention relates to small molecule compounds capable of inhibiting serine hydroxymethyltransferase 2 (SHMT2) activity, methods for the discovery of such compounds, and related research and therapeutic uses for such compounds.
  • SHMT2 serine hydroxymethyltransferase 2
  • the present invention provides compounds capable of inhibiting SHMT2 activity, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with diseases and other pathophysiological states caused by or associated with aberrant SHMT2 activity (e.g., cancer).
  • compositions and methods of the present invention are described in more detail in the following sections: I. Targeting SHMT2 Activity; II. Exemplary Agents; III. Pharmaceutical Compositions, Formulations, and Exemplary Administration Routes and Dosing Considerations; IV. Drug Screens; and V. Therapeutic Applications.
  • SHMTs Serine hydroxymethyltransferases
  • Metabolic reprogramming alters nutrient uptake and use, and supports the high rate of cell proliferation in cancer. Although targeting cancer cell metabolism is an emerging and exciting area for targeted therapeutics, a broad-spectrum inhibitor of metabolic pathways will cause serious side effects. Therefore, the discovery of cancer cells' dependence on a single nutrient such as glycine and the role of SHMT2, a key mitochondrial enzyme in its biosynthesis, provide unique opportunities to design safe targeted therapeutics.
  • metabolic reprogramming provides cancer cells with survival advantages by altering nutrient uptake and use to require increased building blocks for new cellular components, including proteins, lipids, and nucleic acids.
  • the metabolites themselves can also be oncogenic by rewiring signaling pathways, dysregulating epigenetics, and suppressing cell differentiation, and therefore they are regarded to be oncometabolites.
  • SHMTs simultaneously catalyzed the conversion of L-serine to glycine, and tetrahydrofolate (THF) to 5, 10-methylenetetrahydro folate (5, 10-CH2-THF). Elevated expression of SHMT2 is associated with fast proliferation in cancer cells and with poor prognosis and survival probability in cancer patients, whereas silencing SHMT2 significantly impairs cancer cell growth and proliferation.
  • the SHMT2-catalyzed reaction provides important building blocks for cellular renewal and proliferation. While glycine can be used for protein synthesis or de novo purine biosynthesis, 5,10-CH 2 -THF brings the one- carbon unit into the folate cycle for purine synthesis. Accumulating evidence from metabolomics and oncogenomics indicates that SHMT2 is a promising drug target for the development of novel and effective cancer therapy.
  • HTS high throughput screening
  • HTS for SHMT2 inhibitors was performed.
  • Two primary screens of 6560 compounds capable of inhibiting SHMT2 activity were performed at final compound concentrations of 26.5 ⁇ (Fig. 3B) and 6.5 ⁇ (Fig. 3C), with Z-factors of 0.84 and 0.83, respectively (see, also, Tables 3 and 4 described in Example 2) (see, also, Table 6 described in Example 2 and accompanying description).
  • the present invention provides SHMT2 inhibiting agents (e.g., small molecules identified as capable of inhibiting SHMT2 activity).
  • the SHMT2 inhibiting agent is one of the small molecules presented in Table 1 and Table 5, including salts, esters, and prodrugs thereof, and including both R and S enantiomeric forms and racemic mixtures thereof.
  • the agents of the present invention are useful in the preparation of medicaments to treat a variety of conditions associated with SHMT2 activity (e.g., cancer (e.g., lung cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioma, liver cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, urothelial cancer) (e.g., any type of cancer associated with SHMT2 expression and/or activity)).
  • cancer e.g., lung cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioma, liver cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, urothelial cancer
  • any type of cancer associated with SHMT2 expression and/or activity e.g., any type of cancer associated with SHMT
  • agents are also useful for preparing
  • medicaments for treating other disorders wherein the effectiveness of the compounds are known or predicted.
  • the methods and techniques for preparing medicaments of an agent of the present invention are well-known in the art. Exemplary pharmaceutical formulations and routes of delivery are described below.
  • any one or more of the agents described herein, including the many specific embodiments, are prepared by applying standard pharmaceutical manufacturing procedures. Such medicaments can be delivered to the subject by using delivery methods that are well-known in the pharmaceutical arts.
  • compositions are administered alone, while in some other embodiments, the compositions are preferably present in a pharmaceutical formulation comprising at least one active ingredient/agent, as defined above, together with a solid support or alternatively, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents.
  • Each carrier must be "acceptable” in the sense that it is compatible with the other ingredients of the formulation and not injurious to the subject.
  • Contemplated formulations include those suitable oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous,
  • formulations are conveniently presented in unit dosage form and are prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association (e.g., mixing) the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, wherein each preferably contains a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient is presented as a bolus, electuary, or paste, etc.
  • tablets comprise at least one active ingredient and optionally one or more accessory agents/carriers are made by compressing or molding the respective agents.
  • compressed tablets are prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose)surface-active or dispersing agent.
  • a binder e.g., povidone, gelatin, hydroxypropylmethyl cellulose
  • lubricant e.g., inert diluent
  • preservative e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose
  • Molded tablets are made by molding in a suitable machine a mixture of the powdered compound (e.g., active ingredient) moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions for topical administration are optionally formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • topical formulations comprise patches or dressings such as a bandage or adhesive plasters impregnated with active ingredient(s), and optionally one or more excipients or diluents.
  • the topical formulations include a compound(s) that enhances absorption or penetration of the active agent(s) through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide (DMSO) and related analogues.
  • DMSO dimethylsulfoxide
  • the aqueous phase of a cream base includes, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • a polyhydric alcohol i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • oily phase emulsions of this invention are constituted from known ingredients in a known manner.
  • This phase typically comprises a lone emulsifier (otherwise known as an emulgent), it is also desirable in some embodiments for this phase to further comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier so as to act as a stabilizer.
  • a lipophilic emulsifier so as to act as a stabilizer.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
  • oils or fats for the formulation is based on achieving the desired properties (e.g., cosmetic properties), since the solubility of the active
  • creams should preferably be a non-greasy, non-staining and washable products with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent.
  • Formulations for rectal administration may be presented as a suppository with suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate.
  • Formulations suitable for nasal administration include coarse powders having a particle size, for example, in the range of about 20 to about 500 microns which are administered in the manner in which snuff is taken, i.e., by rapid inhalation (e.g., forced) through the nasal passage from a container of the powder held close up to the nose.
  • suitable formulations wherein the carrier is a liquid for administration include, but are not limited to, nasal sprays, drops, or aerosols by nebulizer, an include aqueous or oily solutions of the agents.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations are presented/formulated in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above-recited, or an appropriate fraction thereof, of an agent.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. It also is intended that the agents, compositions and methods of this invention be combined with other suitable compositions and therapies. Still other formulations optionally include food additives (suitable sweeteners, flavorings, colorings, etc.), phytonutrients (e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, and other acceptable compositions (e.g., conjugated linoelic acid), extenders, and stabilizers, etc.
  • food additives suitable sweeteners, flavorings, colorings, etc.
  • phytonutrients e.g., flax seed oil
  • minerals e.g., Ca, Fe, K, etc.
  • vitamins e.g., conjugated linoelic acid
  • extenders e.g., conjugated linoelic
  • the agents of the present invention are provided in unsolvated form or are in non-aqueous solutions (e.g., ethanol).
  • the agents may be generated to allow such formulations through the production of specific crystalline polymorphs compatible with the formulations.
  • the present invention provides instructions for administering an agent to a subject.
  • the present invention provides instructions for using the compositions contained in a kit for the treatment of conditions characterized by the dysregulation of apoptotic processes in a cell or tissue (e.g., providing dosing, route of administration, decision trees for treating physicians for correlating patient-specific characteristics with therapeutic courses of action).
  • the present invention provides instructions for using the compositions contained in the kit to treat a variety of disorders associated with SHMT2 activity (e.g., cancer (e.g., lung cancer)).
  • Various delivery systems are known and can be used to administer therapeutic agents (e.g., exemplary agents as described in Section II above) of the present invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and the like.
  • Methods of delivery include, but are not limited to, intra-arterial, intramuscular, intravenous, intranasal, and oral routes.
  • the agents identified can be administered to subjects or individuals susceptible to or at risk of developing a variety of conditions associated with SHMT2 activity (e.g., cancer (e.g., lung cancer)).
  • a subject such as a mouse, a rat or a human patient
  • the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tissue sample is removed from the patient and the cells are assayed for sensitivity to the agent.
  • Therapeutic amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • in vivo administration is effected in one dose, continuously or intermittently throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and vary with the composition used for therapy, the purpose of therapy, the target cell being treated, and the subject being treated. Single or multiple administrations are carried out with the dose level and pattern being selected by the treating physician.
  • Suitable dosage formulations and methods of administering the agents are readily determined by those of skill in the art.
  • the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
  • the effective amount may be more or less than when the agent is used alone.
  • the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
  • an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including, but not limited to, oral, rectal, nasal, topical (including, but not limited to, transdermal, aerosol, buccal and sublingual), vaginal, parental (including, but not limited to, subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It is also appreciated that the preferred route varies with the condition and age of the recipient, and the disease being treated.
  • the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient.
  • Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
  • the use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.
  • the present invention also includes methods involving co-administration of the agents described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering an agent of this invention. In co-administration procedures, the agents may be administered concurrently or sequentially. In one
  • the agents described herein are administered prior to the other active agent(s).
  • the pharmaceutical formulations and modes of administration may be any of those described above.
  • the two or more co-administered chemical agents, biological agents or radiation may each be administered using different modes or different formulations.
  • the agent or agents to be co-administered depends on the type of condition being treated. For example, when the condition being treated is associated with SHMT2 activity (e.g., cancer), the agent is known to treat cancer (e.g., lung cancer). A number of suitable therapeutic or anticancer agents are contemplated for use in the methods provided herein.
  • the methods provided herein can include but are not limited to, administration of numerous therapeutic agents such as: agents that induce apoptosis; polynucleotides (e.g., anti- sense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-a) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans- retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucleotides
  • anticancer agents comprise agents that induce or stimulate apoptosis.
  • Agents that induce or stimulate apoptosis include, for example, agents that interact with or modify DNA, such as by intercalating, cross-linking, alkylating, or otherwise damaging or chemically modifying DNA.
  • Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to
  • TRAIL-Rl or TRAIL-R2 kinase inhibitors
  • kinase inhibitors e.g., epidermal growth factor receptor (EGFR) kinase inhibitor.
  • Additional anticancer agents include: vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet- derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (CO
  • HEXADROL hydroxychloroquine
  • METICORTEN ORADEXON
  • ORASONE oxyphenbutazone
  • PEDIAPRED phenylbutazone
  • PLAQUENIL prednisolone
  • prednisone prednisone
  • PRELONE PRELONE
  • TANDEARIL cancer chemotherapeutic drugs
  • CAMPTOSAR CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP- 16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.
  • compositions and methods provided herein include one or more agents provided herein and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).
  • at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).
  • Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and
  • methylmelamines e.g., hexamethylmelamine and thiotepa
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)
  • triazenes e.g., dacarbazine (DTIC;
  • antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g., methotrexate
  • amethopterin a pesticide that influences the expression of a pesticide.
  • pyrimidine analogs e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)
  • purine analogs e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2 ' -deoxycoformycin)).
  • chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin
  • vinca alkaloids e.g., vinblastine (VLB), vincristine
  • epipodophyllotoxins e.g., etoposide and teniposide
  • antibiotics e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin
  • mitomycin C (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g.,
  • methylhydrazine derivatives e.g., procarbazine (N-methylhydrazine;
  • adrenocortical suppressants e.g., mitotane ( ⁇ , ⁇ '-DDD) and aminoglutethimide
  • 1 1) adrenocorticosteroids e.g., prednisone
  • progestins e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate
  • 13) estrogens e.g., diethylstilbestrol and ethinyl estradiol
  • antiestrogens e.g., tamoxifen
  • 15) androgens e.g., testosterone propionate and fluoxymesterone
  • 16) antiandrogens e.g., flutamide
  • gonadotropin-releasing hormone analogs e.g., leuprolide
  • any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention.
  • the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies.
  • Table 2 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.
  • Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-0- tetradecanoylphorbol- 13 -acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI- PEG 20, AE-941, AG-013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB- 10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin- 1 , buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, comb
  • VNP40101M volociximab
  • vorinostat VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.
  • anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al, 2002.
  • methods provided herein comprise administering one or more agents provided herein with radiation therapy.
  • the methods provided herein are not limited by the types, amounts, or delivery and administration systems used to deliver therapeutic dose of radiation to an animal.
  • the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof.
  • the radiation is delivered to the animal using a linear accelerator.
  • the radiation is delivered using a gamma knife.
  • the source of radiation can be external or internal to the animal.
  • External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals.
  • Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive.
  • Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.
  • the animal may optionally receive radiosensitizers (e.g., metronidazole,
  • misonidazole intra-arterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5- substituted-4-nitroimidazoles, 2H-isoindolediones, [[(2-bromoethyl)-amino]methyl]-nitro- lH-imidazole-l-ethanol, nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine- containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5- thiotretrazole derivative, 3-nitro-l,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopur
  • Radiotherapy any type of radiation can be administered to an animal, so long as the dose of radiation is tolerated by the animal without unacceptable negative side-effects.
  • Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g. , X-rays or gamma rays) or particle beam radiation therapy (e.g., high linear energy radiation).
  • Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e., gain or loss of electrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference in its entirety).
  • the effects of radiation can be at least partially controlled by the clinician.
  • the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.
  • the total dose of radiation administered to an animal is about .01 Gray (Gy) to about 100 Gy.
  • about 10 Gy to about 65 Gy e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy
  • a complete dose of radiation can be administered over the course of one day
  • the total dose is ideally fractionated and administered over several days.
  • radiotherapy is administered over the course of at least about 3 days, e.g., at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks).
  • a daily dose of radiation will comprise approximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2 Gy (e.g., 1.5-2 Gy).
  • the daily dose of radiation should be sufficient to induce destruction of the targeted cells.
  • radiation is not administered every day, thereby allowing the animal to rest and the effects of therapy to be realized.
  • radiation desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week.
  • radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the animal's responsiveness and any potential side effects.
  • Radiation therapy can be initiated at any time in therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of therapeutic period. For example, radiation is administered in weeks 1-6 or in weeks 2-6 of a therapeutic period comprising 6 weeks for treating, for instance, a solid tumor. Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks.
  • one or more agents provided herein and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc.
  • the agent is administered prior to therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of therapeutic or anticancer agent.
  • the agent is administered after therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of the anticancer agent.
  • the agent and therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., the agent is administered daily while therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks.
  • the agent is administered once a week while therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.
  • potential agents e.g., peptides, small molecules, peptidomimetics, and/or cyclic peptides
  • agents are selected for use in the methods of the present invention by measuring their SHMT2 inhibitory activity.
  • binding affinity screens are conducted in in vitro systems. In other embodiments, these screens are conducted in in vivo or ex vivo systems.
  • an optimized assay is used to screen for agents capable of inhibiting SHMT2 activity (see, e.g., Examples 2, 5, 6, 7).
  • the present invention provides assays capable of indicating inhibition of SHMT2 activity.
  • Such assays comprise cells expressing recombinant SHMT2 and MTHFD, wherein exposure of the assay to an agent capable of inhibiting SHMT2 activity results in a fluorescently detectable event.
  • the present invention provides methods for screening agents able capable of inhibiting SHMT2 activity, comprising administering a candidate agent to a sample expressing SHMT2 and MTHFD, and detecting the presence or absence of fluorescence resulting from a SHMT2 and MTHFD coupling reaction, wherein detection of fluorescence indicates the candidate agent is able to inhibit SHMT2 activity.
  • the SHMT2 expressed in the sample is recombinant SHMT2.
  • the MTHFD expressed in the sample is recombinant MTHFD.
  • the candidate agent is a peptide.
  • the candidate agent is a small molecule.
  • such assays and related methods are used to screen potential agents (e.g., peptides, small molecules, peptidomimetics, and/or cyclic peptides) for an ability to treat disorders associated with SHMT2 activity (e.g., cancer (e.g., lung cancer)).
  • agents e.g., peptides, small molecules, peptidomimetics, and/or cyclic peptides
  • SHMT2 activity e.g., cancer (e.g., lung cancer)
  • structure-based virtual screening methodologies are contemplated for predicting the inhibitory activity / binding affinity of potential agents (e.g., peptides, small molecules, peptidomimetics, and/or cyclic peptides) with SHMT2 for purposes of inhibiting its activity.
  • potential agents e.g., peptides, small molecules, peptidomimetics, and/or cyclic peptides
  • small molecule structures are predicted from a molecular modeling software (e.g., MacroModel, MOE, Glide, Gold, Autodock, DOCK, Unity, Cerius2, Daylight, PipelinePilot, ChemAxon, Sprout, Hook, MCSS, AMBER, BOSS).
  • the present invention also provides methods of modifying and derivatizing the agents of the present invention to increase desirable properties (e.g., ability to bind SHMT2) (e.g., ability to inhibit SHMT2 activity) (e.g., ability to bind / inhibit SHMT2 activity) or to minimize undesirable properties.
  • desirable properties e.g., ability to bind SHMT2
  • ability to inhibit SHMT2 activity e.g., ability to bind / inhibit SHMT2 activity
  • iterative design and chemical synthesis approaches are used to produce a library of derivatized child compounds from a parent compound.
  • the present invention provides methods (e.g., therapeutic applications) for treating and/or preventing disorders related to SHMT2 activity (e.g., cancer).
  • the methods involve administering one or more agents (see, e.g., Section II- Exemplary Agents) of the present invention to a subject having a disorder related to SHMT2 activity.
  • agents see, e.g., Section II- Exemplary Agents
  • inhibition of SHMT2 activity with one or more of the exemplary agents is associated with a general reduction of symptoms associated with types of cancer having aberrant SHMT2 activity (e.g., lung cancer).
  • compositions and methods of the present invention are used to treat diseased cells, tissues, organs, or pathological conditions and/or disease states in an animal (e.g., a mammalian patient including, but not limited to, humans and veterinary animals).
  • an animal e.g., a mammalian patient including, but not limited to, humans and veterinary animals.
  • various diseases and pathologies are amenable to treatment or prophylaxis using the present methods and compositions.
  • the present invention provides methods (e.g., therapeutic applications) for treating and/or preventing disorders related to SHMT2 activity.
  • the methods involve administering one or more agents (see, e.g., Section II- Exemplary Agents) of the present invention to a subject having a disorder related to SHMT2 activity.
  • agents see, e.g., Section II- Exemplary Agents
  • inhibition of SHMT2 activity with one or more of the exemplary agents is associated with a general reduction of symptoms associated with types of cancer having aberrant SHMT2 activity.
  • compositions and methods are not limited to treating and/or preventing a particular disorder associated with SHMT2 activity.
  • the disorder associated with SHMT2 activity is cancer.
  • a non-limiting exemplary list of these diseases and conditions includes, but is not limited to, lung cancer, pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic
  • the cancer cells being treated are metastatic. In other embodiments, the cancer cells being treated are resistant to anticancer agents. In other embodiments, the disorder is any disorder having cells having aberrant SHMT2 activity.
  • the present invention also includes methods involving co-administration of the agents described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering an agent of this invention.
  • the agents may be administered concurrently or sequentially.
  • the agents described herein are administered prior to the other active agent(s).
  • the pharmaceutical formulations and modes of administration may be any of those described above.
  • the two or more co-administered chemical agents, biological agents or radiation may each be administered using different modes or different formulations.
  • the agent or agents to be co-administered depends on the type of condition being treated.
  • the agent is known to treat cancer (e.g., lung cancer).
  • This example demonstrates expression and purification of human recombinant SHMT2 protein and E.coli MTHFD protein.
  • the expression of human recombinant SHMT2 protein in E.coli BLR (DE3) was induced with 1 mM IPTG (Fig. 1A).
  • the His-tag recombinant SHMT2 protein was purified using a Ni 2+ -NTA affinity column, yielding ⁇ 50 mg recombinant SHMT2 protein from 1 L E.coli culture in LB medium.
  • the enzyme was greater than 90% pure as judged by SDS- PAGE.
  • E.coli MTHFD protein in E.coli BL21 was induced with 1 mM IPTG (Fig. IB).
  • the enzyme was purified using an anion exchanging (Q) column, yielding ⁇ 30 mg MTHFD protein from 1 L E.coli culture in LB medium.
  • the enzyme was greater than 90% pure as judged by SDS-PAGE.
  • This example describes the design and optimization of a SHMT2 activity assay for high-throughput screening (HTS).
  • HTS high-throughput screening
  • the SHMT2 activity assay is able to tolerate the DMSO concentrations used in the HTS (Fig. 2G). Under these optimized conditions, the assay in a full 384-well plate showed a Z-factor of 0.896 (Fig. 2H).
  • HTS for SHMT2 inhibitors was performed as illustrated in Figure 3A.
  • Two primary screens of 6560 compounds were performed at final compound concentrations of 26.5 ⁇ (Fig. 3B) and 6.5 ⁇ (Fig. 3C), with Z-factors of 0.84 and 0.83, respectively (Table 3).
  • the top 10 overlapping compounds (Table 4) in the primary screens were selected for dose- response tests.
  • This example describes human SHMT2 protein expression and purification.
  • E.coli BLR (DE3) cells harboring the His-SHMT2 expression plasmid (#25479, Addgene, Cambridge MA) were cultured at 37°C in 1 L LB medium containing 50 ⁇ g/mL kanamycin. When OD 6 oo reached -0.8, expression of His-SHMT2 (53 kDa) was induced by the addition of IPTG at the final concentration of 1 mM and maintained at 16°C overnight. Cells were then harvested by centrifugation at 8,326 g (7,000 rpm, F 12-6x5000LEX rotor, Thermo Scientific, Rockford, IL) for 15 minutes and stored at -80°C. Purification of His- SHMT2 was carried out at 4°C.
  • Frozen cell paste was thawed and resuspended in 30 mL buffer A (50 mM HEPES, pH 7.5, 200 mM NaCl, 10% (v/v) glycerol, 10 ⁇ PLP (Pyridoxal 5'-phosphate hydrate), 0.5 mM EDTA, 1 mM DTT, and 100 ⁇ PMSF. Cells were disrupted by ultrasonication. Cell debris was removed by centrifugation at 38,758 g (18,000 rpm, F21- 8> ⁇ 50y rotor, Thermo Scientific, Rockford, IL) for 60 min.
  • buffer A 50 mM HEPES, pH 7.5, 200 mM NaCl, 10% (v/v) glycerol, 10 ⁇ PLP (Pyridoxal 5'-phosphate hydrate), 0.5 mM EDTA, 1 mM DTT, and 100 ⁇ PMSF. Cells were disrupted by ultrasonication.
  • the clear supernatant was loaded onto a i-Sepharose column pre-equilibrated with buffer B (50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 10 mM imidazole).
  • buffer B 50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 10 mM imidazole.
  • the column was washed with buffer C (50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 20 mM imidazole) for three column volumes, and the buffer D (50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 40 mM imidazole) for three column volumes.
  • His-SHMT2 protein was eluted with buffer E (50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 300 mM imidazole). Fractions containing His-SHMT2 judged by SDS-PAGE were pooled. PLP was added to the eluted protein solution to a final concentration of 10 ⁇ , and incubate on ice for 15 min. The protein solution was dialyzed in buffer F (50 mM HEPES, pH 7, 200 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 mM EDTA) at 4°C overnight. SHMT2 protein aliquots were frozen at -80°C. Example 4.
  • buffer E 50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 300 mM imidazole.
  • PLP was added to the eluted protein solution to a final concentration of 10 ⁇ , and incubat
  • This example describes E.coli MTHFD protein expression and purification.
  • E.coli MTHFD protein 35 kDa
  • the expression and purification of E.coli MTHFD protein 35 kDa were performed as described (see, e.g., Fu TF, di Salvo M, & Schirch V (2001) Analytical biochemistry 290(2):359-365), with some modifications.
  • the E.coli BL21 (DE3) cells harboring the E.coli MTHFD expression plasmid were cultured at 37°C in 1 L LB medium containing 100 ⁇ g/mL ampicillin. When OD 6 oo reached -0.8, expression of MTHFD was induced by the addition of IPTG at the final concentration of 1 mM and growth continues for an additional 4 h.
  • Ammonium sulfate was added to the cell lysate to 30% saturation (176 g/L) and cell debris was removed by centrifugation at 38,758 g (18,000 rpm, F21-8x50y rotor, Thermo Scientific, Rockford, IL) for 30 min. Ammonium sulfate was added to the supernatant to 55% saturation (additional 162 g/L). After stirring, the slurry was centrifuged at 38,758 g (18,000 rpm, F21-8x50y rotor, Thermo Scientific, Rockford, IL) for 30 min, and the pellet was dissolved in 10 mL buffer G.
  • the solution was desalted through dialysis in a YM-10 dialysis tube at 4°C for overnight vs. 1 L buffer G, with replacement for at least once.
  • the protein solution was loaded onto an anion exchange (Q) column equilibrated with buffer G.
  • the column was wash with 12 column volumes of buffer G, and the enzyme was eluted with a linear gradient of equal volumes of buffer G and buffer H (100 mM potassium phosphate, 400 mM NaCl, pH 6.5, 5 mM 2-mercaptoethanol, 0.1 mM EDTA).
  • Fractions containing MTHFD protein judged by SDS-PAGE were pooled and precipitated by 60% ammonium sulfate (390 g/L) at 4°C.
  • This example describes a SHMT2 activity assay.
  • the SHMT2 activity assay was performed using Corning 384-well low volume/non- binding/round bottom black plates (#3676, Corning Inc., Corning, NY) under regular aerobic condition in buffer J (50 mM HEPES, pH 7.5, 0.5 mM EDTA) containing 0.67 ⁇ SHMT2, 0.33 ⁇ MTHFD, 2.67 mM L-serine, 0.53 mM THF, and 0.33 mM NADP + in a final volume of 15 ⁇ ⁇ . Due to the instability, the NADP+ solution was prepared fresh for each experiment. A negative control was included in each assay by replacing SHMT2 with equal amount of buffer J, providing the background of the assay.
  • This example describes a MTHFD activity assay.
  • the MTHFD activity assay was performed using Corning 384-well low volume/non- binding/round bottom black plates under regular aerobic condition in buffer J containing 0.33 ⁇ MTHFD, 2.67 mM L-serine, 0.53 mM 5,10-CH 2 -THF, and 0.33 mM NADP + in a final volume of 15 ⁇ L. Due to the instability, the NADP+ solution was prepared fresh for each experiment. A negative control was included in each assay by replacing MTHFD with equal amount of buffer J, providing the background of the assay. After 5 min incubation at 25 "C, 5 ⁇ L of the AMP LITE Fluorimetric NADPH detection reagent was added to measure the amount of NADPH. After 5 min of incubation in dark at 25 "C, the increase in fluorescence intensity (Ex: 540 nm; Em: 590 nm) was measured using the Synergy HI microplate reader or the PHERAstar microplate reader.
  • This example describes HTS for SHMT2 inhibitors.
  • HTS HTS-derived neuropeptide kinase
  • 6560 compounds were primarily screened twice at final compound concentrations of 26.5 ⁇ and 6.5 ⁇ , respectively.
  • the top 200 overlapping compounds in the two primary screens were further tested in duplicate at eight concentrations each, 60, 36, 21.6, 13.0, 7.8, 4.7, 2.8, and 1.7 ⁇ in 384-well plates.
  • Example 8
  • Fig. 4 shows generation of NSCLC cell lines with Dox- inducible overexpression of SHMT2.
  • H226-SHMT2Dox, H226-LacZDox, H226-GFPDox, H460-SHMT2Dox, H460- LacZDox, H460-GFPDox, H1299-SHMT2Dox, H1299-LacZDox, H1299-GFPDox, A549- SHMT2Dox, A549-LacZDox, A549-GFPDox cells were treated with doxycycline (Dox) at 200 ng/mL for 72 h.
  • Whole cell lysates were prepare and examined by Western blotting using the indicated antibodies.
  • GPF and LacZ expression were also examined using fluorescence microscopy and a beta-Gal staining kit (Life technologies, K 1465 -01), respectively.
  • the kinetics of Dox-induced SHMT2 expression in H226-SHMT2Dox, H460-SHMT2Dox, H1299-SHMT2Dox, and A549-SHMT2Dox cells were measured by Western blotting after indicated Dox treatment periods.
  • Fig. 5 shows generation of NSCLC cell lines with Dox- inducible knockdown of SHMT2.
  • H226-shCTRLDox, H226-shSHMT2Dox, H460-shCTRLDox, H460- shSHMT2Dox, H1299-shCTRLDox, H1299-shSHMT2Dox, A549-shCTRLDox, A549- shSHMT2Dox cells were treated Dox (200 ng/mL) for 72 h, followed by Western blotting analysis.
  • Fig. 6 NSCLC cell lines with Dox-inducible expression of LacZ or GFP as control cell lines.
  • H226-LacZDox, H460-LacZDox, H1299-LacZDox, A549-LacZDox cells were treated with Dox (200 ng/mL) for the indicated periods of time.
  • the expression of LacZ was monitored using a beta-Gal staining kit (Life technologies, K1465-01).
  • H226-GFPDox, H460-GFPDox, H1299-GFPDox, A549-GFPDox cells were treated with Dox (200 ng/mL) for the indicated periods of time.
  • the expression of GFP was monitored using fluorescence microscopy.
  • Dox (200 ng/mL) induced LacZ expression in H226-LacZDox, H460-LacZDox, H1299-LacZDox, A549-LacZDox cells within 48 h; and induced GFP expression in H226- GFPDox, H460-GFPDox, H1299-GFPDox, A549-GFPDox cells within 48 h.

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Abstract

La présente invention concerne des composés à petite molécule capables d'inhiber l'activité sérine hydroxyméthyltransférase 2 (SHMT2), des procédés pour la découverte de ces composés et des utilisations en recherche et thérapeutiques de ces composés. En particulier, la présente invention concerne des composés capables d'inhiber l'activité SHMT2, et des procédés d'utilisation de ces composés en tant qu'agents thérapeutiques pour traiter un certain nombre d'affections associées à des maladies et d'autres états physiopathologiques causés par ou associés à une activité SHMT2 aberrante (par exemple, un cancer).
PCT/US2015/062442 2014-11-24 2015-11-24 Compositions et procédés relatifs à l'inhibition de l'activité sérine hydroxyméthyltransférase 2 Ceased WO2016085990A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112755188A (zh) * 2019-11-01 2021-05-07 中国科学院微生物研究所 Shmt2抑制剂在制备抑制膀胱癌细胞生长和转移的药物中的应用
WO2021122415A1 (fr) 2019-12-19 2021-06-24 Bayer Aktiengesellschaft Dérivés furoindazole
CN114432442A (zh) * 2022-01-24 2022-05-06 山东大学第二医院 表柔比星作为125i的增敏剂
JP2022532379A (ja) * 2019-05-14 2022-07-14 ザ スクリプス リサーチ インスティテュート 神経変性および代謝障害の処置のための化合物
WO2022179940A1 (fr) 2021-02-23 2022-09-01 Bayer Aktiengesellschaft Dérivés de furoindazole utilisés en tant qu'antagonistes de gpr84
WO2022229061A1 (fr) 2021-04-29 2022-11-03 Bayer Aktiengesellschaft Dérivés de furoindazole utilisés comme antagonistes ou inhibiteurs de gpr84
CN116751178A (zh) * 2023-06-20 2023-09-15 中国人民解放军军事科学院军事医学研究院 一种取代2h-苯并吡喃-3-甲酰苯胺类化合物及其制备方法与应用
WO2024083705A1 (fr) 2022-10-18 2024-04-25 Bayer Aktiengesellschaft Dérivés de furoindazole pour le traitement de la douleur
CN120247898A (zh) * 2025-03-31 2025-07-04 大连医科大学附属第二医院 一种噻吩甲酰胺类化合物及其用途

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WO2014150688A1 (fr) * 2013-03-15 2014-09-25 The General Hospital Corporation Glycine, métabolisme mitochondriale monocarboné et cancer

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WO2013120086A1 (fr) * 2012-02-10 2013-08-15 Whitehead Institute For Biomedical Research Inhibition du système de clivage de la glycine pour le traitement du cancer
WO2014150688A1 (fr) * 2013-03-15 2014-09-25 The General Hospital Corporation Glycine, métabolisme mitochondriale monocarboné et cancer

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022532379A (ja) * 2019-05-14 2022-07-14 ザ スクリプス リサーチ インスティテュート 神経変性および代謝障害の処置のための化合物
CN112755188A (zh) * 2019-11-01 2021-05-07 中国科学院微生物研究所 Shmt2抑制剂在制备抑制膀胱癌细胞生长和转移的药物中的应用
CN112755188B (zh) * 2019-11-01 2022-03-08 中国科学院微生物研究所 Shmt2抑制剂在制备抑制膀胱癌细胞生长和转移的药物中的应用
WO2021122415A1 (fr) 2019-12-19 2021-06-24 Bayer Aktiengesellschaft Dérivés furoindazole
CN115135656A (zh) * 2019-12-19 2022-09-30 拜耳公司 呋喃并吲唑衍生物
JP2023508908A (ja) * 2019-12-19 2023-03-06 バイエル、アクチエンゲゼルシャフト フロインダゾール誘導体
WO2022179940A1 (fr) 2021-02-23 2022-09-01 Bayer Aktiengesellschaft Dérivés de furoindazole utilisés en tant qu'antagonistes de gpr84
WO2022229061A1 (fr) 2021-04-29 2022-11-03 Bayer Aktiengesellschaft Dérivés de furoindazole utilisés comme antagonistes ou inhibiteurs de gpr84
CN114432442A (zh) * 2022-01-24 2022-05-06 山东大学第二医院 表柔比星作为125i的增敏剂
WO2024083705A1 (fr) 2022-10-18 2024-04-25 Bayer Aktiengesellschaft Dérivés de furoindazole pour le traitement de la douleur
CN116751178A (zh) * 2023-06-20 2023-09-15 中国人民解放军军事科学院军事医学研究院 一种取代2h-苯并吡喃-3-甲酰苯胺类化合物及其制备方法与应用
CN120247898A (zh) * 2025-03-31 2025-07-04 大连医科大学附属第二医院 一种噻吩甲酰胺类化合物及其用途

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