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WO2025024030A2 - Colles moléculaires ciblant un régulateur transcriptionnel d'états métaboliques aberrants - Google Patents

Colles moléculaires ciblant un régulateur transcriptionnel d'états métaboliques aberrants Download PDF

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Publication number
WO2025024030A2
WO2025024030A2 PCT/US2024/029868 US2024029868W WO2025024030A2 WO 2025024030 A2 WO2025024030 A2 WO 2025024030A2 US 2024029868 W US2024029868 W US 2024029868W WO 2025024030 A2 WO2025024030 A2 WO 2025024030A2
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Prior art keywords
optionally substituted
compound
zbtb11
heterocycloalkyl
cells
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WO2025024030A3 (fr
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Fleur Marcia FERGUSON
Jiewei Jiang
Nathan Tran
Eric Wang
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Sanford Burnham Prebys Medical Discovery Institute
University of California Berkeley
University of California San Diego UCSD
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Sanford Burnham Prebys Medical Discovery Institute
University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the present invention relates to transcriptional regulation for therapeutic applications.
  • OxPhos oxidative phosphorylation
  • IACS- 010759 which inhibits an unidentified subunit of complex I, the first component of the mitochondrial electron transport chain — have achieved success in preclinical models of GBM. Although this validates the pathway, IACS-010759 has not advanced to GBM clinical trials but has been pursued as a treatment for other cancer types. Unfortunately, in those trials, doselimiting peripheral neuropathy was observed. To tackle cancer drug resistance in GBM, validation of mechanistically orthogonal novel therapeutic targets to exploit adaptive metabolic vulnerabilities in the OxPhos pathway is necessary.
  • pancreatic ductal adenocarcinomas are driven by mutant K-Ras.
  • K-Ras inhibitors Upon treatment with K-Ras inhibitors, PDAC cancer cells undergo metabolic reprogramming towards an oxidative phosphorylation-dependent, drug-resistant state.
  • direct inhibition of Complex I is poorly tolerated in patients due to on-target induction of peripheral neuropathy.
  • the inventors have developed molecular glue degraders against ZBTB11, a C2H2 zinc finger transcription factor that regulates the nuclear transcription of components of the mitoribosome and electron transport chain.
  • ZBTB11 degraders leverage the differences in mitochondrial biogenesis demand between human neurons and rapidly-dividing pancreatic cancer cells to selectively target the K-Ras inhibitor resistant state in PDAC.
  • Combination treatment of both K-Ras inhibitor-resistant cell lines and multidrug resistant patient-derived organoids resulted in superior anti-cancer activity compared to single agent treatment, while sparing hiPSC-derived neurons.
  • Proteomic and stable isotope tracing studies revealed mitoribosome depletion and impairment of the TCA-cycle as key events that mediate this response.
  • the instant disclosure validates ZBTB11 as a vulnerability in K-Ras inhibitorresistant PDAC and provides a suite of molecular glue degrader tool compounds with which to investigate its function.
  • pancreatic ductal adenocarcinomas make up >90% of all pancreatic cancers and are the third leading cause of cancer deaths in the US.
  • PDAC pancreatic ductal adenocarcinomas
  • K-Ras inhibitors include sotorasib and adagrasib, which were FDA approved for treatment of K-Ras G12C mutant NSCLC in 2021 and 2022 respectively. 10 11 Although encouraging responses to sotorasib in PDAC have been reported, resistance swiftly arises.
  • Adaptive K-Ras inhibitor resistance is accompanied by significant metabolic reprogramming of cancer cells, including hyperactivation of the PI3K/AKT pathway 17,18 , elevated TCA cycle flux 19 , Myc amplification 20 , and upregulation of mitochondrial biogenesis 21 , culminating in heightened reliance on oxidative phosphorylation (OXPHOS). 21,22
  • OXPHOS oxidative phosphorylation
  • K-Ras G12D is the most common K-Ras mutation in PDAC 3 and inhibitors that selectively target K-Ras G12D are currently the subject of Ph I clinical trials in PDAC patients (NCT05737706). It was recently demonstrated that acquired resistance to the K-Ras G12D inhibitor MRTX1133 develops in human PDAC cells and is associated with feedback activation of ERBB/AKT signaling and enhanced OXPHOS (Fig. 3). 14 Resistance to the mechanistically orthogonal pan K-Ras(ON) inhibitor RM-7977 has been recently reported in murine models of PDAC, and is associated with Myc amplification 20 .
  • OXPHOS inhibitors have properties limiting their clinical use, including insufficient potency 24 and poor selectivity 23,26 .
  • Others such as the mitochondrial complex I inhibitor IACS-010759, display dose-limiting on-target toxicity in the clinic due to their action on actively respiring mitochondria in peripheral neurons.
  • 27 To identify mechanistically orthogonal approaches to combating OXPHOS-mediated adaptive K-Ras inhibitor resistance, public datasets were evaluated to find targets that met the following four criteria: 1) Upregulated at the protein level during the development K-Ras inhibitor resistance in PDAC cells at at least one time point. 22 2) Essential for cell survival in glycolysis suppression media (low glucose, plus galactose), which forces dependency on OXPHOS.
  • ZBTB11 is a zinc finger transcription factor that co-operates with NRF2 to regulate nuclear expression of components of complex I and the mitoribosome, thereby maintaining functional homeostasis in mitochondria. 31 ' 33 ZBTB11 is transiently upregulated in PDAC cells during the development of K-Ras inhibitor resistance, potentially to help sustain their increased demands on mitochondrial biogenesis. 22 Although ZBTB11 is a pan-essential gene 34 ' 36 , it was hypothesized that partial loss of ZBTB11 would impair K-Ras inhibitor resistant PDAC fitness, but have minimal effects on post-mitotic neurons due to differences in the mitochondrial biogenesis demand between these cell types.
  • ZBTB1 1 is a C2H2 zinc finger protein that contains a CXXCG sequence motif in its beta-hairpin loop, making it potentially chemically targetable via molecular glue degrader-mediated recruitment to the E3 -ligase CRL4 CRBN .
  • ZBTB11 is validated as a mechanistically orthogonal target for addressing OXPHOS-dependent drug resistant states in PDAC, using both genetic and pharmacological approaches.
  • the additional therapeutic intervention comprises chemotherapy, targeted therapy, immunotherapy, a kinase inhibitor, Ras pathway inhibitors, DNA damaging agents, and/or a PARP inhibitor.
  • the cancer relies on elevated oxidative phosphorylation for survival.
  • ZBTB11 Zinc Finger and BTB Domain Containing 11
  • ZBTB11-related disease or disorder is characterized by elevated oxidative phosphorylation.
  • a method of treating a disease or disorder associated with elevated oxidative phosphorylation in a subject in need thereof comprising administering to the subject a compound which inhibits or degrades Zinc Finger and BTB Domain Containing 11 (ZBTB11). Also provided herein is a method of inhibiting oxidative phosphorylation in a subject, comprising administering to the subject a compound which inhibits or degrades Zinc Finger and BTB Domain Containing 11 (ZBTB11).
  • the compound used in a method provided herein is a degrader of ZBTB11.
  • the compound binds the E3-ligase CRL4-CRBN and promotes interaction with ZBTB11 by binding at the interface of ZBTB11 and CRBN, leading to ZBTB11 ubiquiti nation and degradation.
  • the compound is a compound of Formula (I)
  • a 1 is optionally substituted aryl or heteroaryl
  • a 2 is optionally substituted aryl or heteroaryl
  • R 1 is H, optionally substituted alkyl, halogen, a bond with A 1 , or optionally substituted alkylene forming a bond with A 1 ;
  • each R 2 and R 3 is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, optionally substituted alkylene forming a bond with an R B , or R 2 and R 3 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl;
  • each R 4 and R 5 when present, is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, or two R 4 or two R ? are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl;
  • each R A if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, or optionally substituted aryl or heteroaryl; and R B , if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl; or a bond with an optionally substituted alkylene of R 2 or R 3 ; or a pharmaceutically acceptable salt thereof.
  • each X 1 is O.
  • X 2 is -NH-.
  • X 3 is -
  • a 1 is optionally substituted phenyl or pyridyl. In embodiments, A 1 has the structure In embodiments, A 2 is optionally substituted phenyl. In embodiments,
  • a 2 is phenyl substituted by one substituent R 6 and further optionally substituted by one or more R 7 , wherein:
  • R 6 is -CF3; and eac optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, or optionally substituted aryl or heteroaryl; and wherein each R c is independently optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, or optionally substituted aryl or heteroaryl;
  • R 1 is H, optionally substituted alkyl, halogen, or optionally substituted alkylene forming a bond with A 1 ;
  • each R 2 and R 3 is independently optionally substituted alkyl, optionally substituted alkylene forming a bond with an R B , or R 2 and R 3 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl;
  • each R 4 and R 5 when present, is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl,
  • R 8 is H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, -CN, or halogen; m is 1, 2, or 3; n is 0, 1, 2, or 3; and
  • each X 1 is O.
  • a 2 is substituted 0, 1 or 2 R 7 .
  • each R 7 is independently chloro or -CF3.
  • R 1 is H.
  • R 2 and R 3 are each independently methyl, ethyl, propyl, or taken together with the carbon to which they are attached to form a cyclopropyl, cyclobutyl, or cyclopentyl.
  • R B is H.
  • n is 0.
  • m is 2.
  • each R 5 is H.
  • R 8 is H.
  • the compound according to Formula (I) has a structure according to Formula (laa) or Formula (lab), or a pharmaceutically acceptable salt thereof: wherein,
  • R 8 is H or Ci-6 alkyl; p is 1, 2 or 3; and R 1A is H or C1-6 alkyl.
  • composition comprising the compound described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • a method of treating a disease or disorder in a subject in need thereof comprising administering to the subject a compound described herein, or a pharmaceutical composition described herein.
  • the compound degrades Zinc
  • FIG. 1 OXPHOS is upregulated in K-Ras inhibitor resistant PDAC cells, and countered by ZBTB11 knockdown.
  • FIG. 1 Metabolomic profiling of parental and K-Ras G12C inhibitor resistant PDAC cell lines.
  • Panel B MSEA identifies amino acid metabolic pathways as major metabolic shifts between parental and sotorasib-resistant MIA PaCa-2 cells.
  • FIG. 3 Metabolic profiling of parental and K-Ras G12D inhibitor resistant PDAC cell lines.
  • Panel B MRTX1133-resistant SUIT-2 cells perform higher basal and maximal levels of OXPHOS than parental SUIT-2 cells.
  • Panel C MRTX1133-resistant SUIT-2 cells perform higher basal and maximal levels of OXPHOS than parental SUIT-2 cells.
  • FIG. 4 CRISPRi ZBTB11 knockdown impacts OXPHOS-related genes.
  • FIG. 5 Development of a ZBTB11 molecular glue degrader.
  • Panel A Depiction of ZBTB11 domains and the ZBTBl l-HiBiT assay.
  • Panel C Chemical structure of screening hits and optimized ZBTB 11 degrader and negative controls.
  • Panel D Chemical structure of screening hits and optimized ZBTB 11 degrader and negative controls.
  • FIG. 6 Characterization of the ZBTB11 degrader pharmacology.
  • Panel A Cellular CRBN engagement. HEK293 cells were transiently transfected with NanoLuc-CRBN and treated with the NanoBRETTM Tracer Reagent and indicated compound for 2 hrs.
  • Panel C Construct design for ZBTB11 ZF fingerprinting experiments. Panels D-E.
  • ZBTB11 ZF fingerprinting reveals ZF10 as the primary CRBN target degron.
  • MOLT-4 cell lines with stable expression of the indicated constructs were generated. Cells were treated with 1 pM screening hit compound ALV-05-184 for 5 hrs.
  • Panel F IKZFl-HiBiT degradation in Jurkat knock-in cells. Cells were treated for 8 hrs with the indicated compound.
  • Panel G GSPTl-HiBiT degradation in Jurkat knock-in cells. Cells were treated for 8 hrs with the indicated compound.
  • Panels A, D-G. Data is depicted as the average +/- S.D. of n 3 biological replicates and is normalized to DMSO vehicle treatment control.
  • FIG. 7 Further characterization of ZBTB11 degrader compound and controls.
  • Panel B ZBTBl l-HiBiT degradation by JWJ-01-306 achieves optimal kinetics by 2 hrs. MIA PaCa-2 knock-in cells were treated for the indicated time.
  • Panel C ZBTBl l-HiBiT protein levels decrease following JWJ-01-306 washout. MIA PaCa-2 knock-in cells were treated for 2 hrs with 10 pM JWJ-01-306, followed by three media washes to remove drug.
  • FIG. 8 JWJ-01-306 combination treatment overcomes acquired resistance to K- Ras inhibitors in PDAC via metabolic pathway reprogramming.
  • Panel A. JWJ-01-306 synergizes with sotorasib to inhibit proliferation of sotorasib-resistant MIA PaCa-2 cells. Cells were treated with the indicated compounds and cell numbers were measured using trypan blue exclusion. Data is depicted as the average +/- S.D. of n 3 biological replicates.
  • Panel B JWJ- 01-306 effectively inhibits proliferation of SUIT-2 cells. Cells were treated with the indicated compounds and cell confluence was measured using a Cellcyte X live cell analyzer. Data is depicted as the average +/- S.D.
  • FIG. 10 JWJ-01-306 leads to reduction in ZBTB11 downstream mitochondrial proteins.
  • Panel A Comparison between MitoCarta 3.0 database and significantly regulated protein hits identified in proteomics reveals enrichment of mitochondria-related proteins.
  • Panel B Protein abundances of ZBTB11 -regulated targets decrease with treatment of 10 pM JWJ-01- 306. Each boxplot represents the median value, with the bounds indicating the 25 th and 75 th percentiles. Significance level is marked with asterisks (two-tailed moderated t-test, * p-value ⁇ 0.05, ** p-value ⁇ 0.01, *** p-value ⁇ 0.001).
  • FIG. 11 Metabolic reprogramming of K-Ras inhibitor resistant PDAC cells by ZBTB11 degradation.
  • Panel A. ZBTB11 degradation reduces rates of OXPHOS in sotorasib- resistant MIA PaCa-2 cells. Cells were treated with 10 pM of the indicated compound for 24 hrs.
  • Panel B. ZBTB11 degradation reduces ATP production by OXPHOS in sotorasib-resistant MIA PaCa-2 cells.
  • Panel C ZBTB1 1 degradation reduces rates of OXPHOS in MRTX1133-resistant SUIT-2 cells. Cells were treated with 10 pM of the indicated compound for 24 hrs.
  • ZBTB11 degradation reduces ATP production by OXPHOS in MRTX1133-resistant SUIT -2 cells.
  • Panel E Overexpression of ZBTB 1 1 WT or ZBTB11 K866T rescues JWJ-01-306-mediated reduction of OXPHOS. Sotorasib-resistant MIA PaCa-2 cell lines with stable expression of NanoLuc-ZBTB 11 WT or NanoLuc-ZBTB l 1 K866T were generated. Cells were treated with 10 pM JWJ-01-306 for 24 hrs.
  • Panel F Overexpression of ZBTB 1 1 WT or ZBTB11 K866T rescues JWJ- 01-306-mediated reduction of ATP production by OXPHOS.
  • Panel G Overexpression of ZBTB 1 1 WT or ZBTB11 K866T rescues JWJ- 01-306-mediated reduction of ATP production by OXPHOS.
  • ZBTB11 degradation induces downregulation of ZBTB11 -regulated genes.
  • Sotorasib-resistant MIA PaCa-2 cells were treated with 10 pM of the indicated compound for 24 hrs.
  • data is shown for DMSO, JWJ-01-306, JWJ-01-368, and JWJ-01-334 from left to right.
  • Panel H Overexpression of ZBTB1 1 WT or ZBTB11 K866T rescues JWJ-01-306-mediated downregulation of ZBTB11- regulated genes.
  • Cells were treated with 10 pM JWJ-01-306 for 24 hrs.
  • MSEA identifies the TCA cycle as the major metabolic pathway disrupted by ZBTB11 degradation.
  • Metabolites were then evaluated for pathway enrichment using KEGG pathway analysis and filtered for pathways containing a minimum of four hits. Full datasets in Dataset 5.
  • Panels A-F Cellular respiration rates were evaluated in a mitochondrial stress test with the indicated drugs using a Seahorse analyzer. Oxygen Consumption Rates (OCR) and Extracellular Acidification Rates (ECAR) from the mitochondrial stress test were used to calculate ATP production rates.
  • FIG. 12 Metabolic reprogramming of parental and K-Ras inhibitor resistant PDAC cells by ZBTB11 degradation.
  • Panel A. ZBTB11 degradation reduces rates of OXPHOS in parental MIA PaCa-2 cells. Cells were treated with 10 pM of the indicated compound for 24 hrs.
  • Panel B. ZBTB11 degradation reduces ATP production by OXPHOS in parental MIA PaCa- 2 cells.
  • Panel C. ZBTB11 degradation induces compensatory genetic upregulation of ZBTB11 and downregulation of ZBTB11 -regulated genes. Parental MIA PaCa-2 cells were treated with 10 pM of the indicated compound for 24 hrs.
  • Panel D. ZBTB11 degradation reduces rates of OXPHOS in parental SUIT -2 cells.
  • Panel E. ZBTB11 degradation reduces ATP production by OXPHOS in parental SUIT-2 cells.
  • Panel F. ZBTB11 degradation induces compensatory genetic upregulation of ZBTB11 and downregulation of ZBTB11 -regulated genes.
  • Parental SUIT-2 cells were treated with 10 pM of the indicated compound for 24 hrs.
  • Panel G. ZBTB1 1 degradation induces compensatory genetic upregulation of ZBTB11 and downregulation of ZBTB11 -regulated genes.
  • MRTX1133-resistant SUIT-2 cells were treated with 10 pM of the indicated compound for 24 hrs.
  • Panel H. ZBTB11 degradation induces compensatory genetic upregulation of ZBTB11.
  • Sotorasib-resistant MIA PaCa-2 cells were treated with 10 pM of the indicated compound for 24 hrs.
  • Cellular respiration rates were evaluated in a mitochondrial stress test with the indicated drugs using a Seahorse analyzer.
  • Oxygen Consumption Rates (OCR) and Extracellular Acidification Rates (ECAR) from the mitochondrial stress test were used to calculate ATP production rates.
  • FIG. 13 ZBTB11 degradation spares neurons and deepens the response of PDAC patient-derived organoids to K-Ras inhibitors.
  • Panel A Mitochondrial activity and oxidative stress assays show differential pharmacology of Complex I and ZBTB11 perturbation.
  • hiPSC- derived neurons were treated for 24 h with DMSO, 1 pM IACS 010759, 1 pM JWJ-01-306 or 1 pM JWJ-01-368.
  • Panel B Neurotoxicity assays show differential pharmacology of Complex I and ZBTB11 perturbation.
  • hiPSC-derived neurons were treated for 72 h with DMSO, 1 pM IACS 010759, 1 pM JWJ-01-306 or 1 pM JWJ-01-368.
  • Panel C Proliferation assays show ZBTB11 + K-Ras inhibitor combination has superior antiproliferative effects in PDAC patient- derived organoids. Cells were treated with DMSO vehicle, 1 pM JWJ-01-306, 200 nM MRTX1133 or 1 pM JWJ-01-306 + 200 nM MRTX1133 (COMBO) and cellular confluence observed using brightfield imaging at the indicated timepoints.
  • the present invention provides in embodiments therapeutic compounds as described herein, pharmaceutical compositions comprising the compounds, and methods of use for treatment of various diseases and disorders in which inhibition and/or degradation of ZBTB11 can be an effective treatment, such as cancer.
  • the compounds can induce targeted protein degradation of a transcriptional regulator of mitochondrial biogenesis, such as ZBTB 11.
  • Mitochondrial biogenesis, and downstream OxPhos metabolism is adaptively upregulated by drug-resistant cancers, including KRAS G12C inhibitors in pancreatic cancer, and BRAF inhibitors in melanoma. Beyond this, numerous cancers have demonstrated this phenomena, including KRAS inhibitor resistant lung cancers, and TMZ resistant glioblastomas.
  • the instant ZBTB11 molecular glues combat this effect, sensitizing the cancers to chemotherapy or targeted therapy to overcome their drug resistance.
  • the invention provides that the compounds are small molecule molecular glue degraders that works via a molecular glue mechanism whereby the molecules bind the E3- ligase CRL4-CRBN and promotes interaction with ZBTB11 by binding at the interface of ZBTB 11 and CRBN, leading to ZBTB 11 ubiquitination and degradation.
  • the invention provides that the compounds, and pharmaceutical compositions containing the compounds, can be used to treat cancer for tumors that rely on elevated OxPhos for survival, and for drug-resistant tumors that evade chemotherapy via up regulation of mitochondrial biogenesis.
  • the invention provides methods of treating cancer in a subject, comprising administering to a subject in need thereof an effective amount of a composition as described herein. In embodiments the method further comprises co-administering a chemotherapeutic agent with the compounds described herein.
  • derivative refers to a chemical substance related structurally to another substance, or a chemical substance that can be made from another substance (i.e., the substance it is derived from), e.g., through chemical or enzymatic modification.
  • the term “attached” or “conjugated” refers to interactions and/or states in which material or compounds are connected or otherwise joined with one another. These interactions and/or states are typically produced by, e.g., covalent bonding, ionic bonding, chemisorption, physisorption, and combinations thereof.
  • Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways.
  • compositions comprising, “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components.
  • a composition, and/or a method that “comprises” a list of elements is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the composition and/or method.
  • the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified.
  • “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component).
  • the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.
  • transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a fusion protein, pharmaceutical composition, and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’. It is understood that aspects and embodiments of the invention described herein include “consisting” and/or “consisting essentially of’ aspects and embodiments.
  • a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.
  • Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and/or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and/or to the other particular value.
  • the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • any reference to "one embodiment” or “an embodiment” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment.
  • the appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and/or non-human mammals.
  • the term “pharmaceutically acceptable diluent or excipient” or “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and/or vehicle with which the compounds of the disclosure, is administered.
  • Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier.
  • Methods for producing compositions in combination with carriers are known to those of skill in the art.
  • the language “pharmaceutically acceptable diluent or excipient” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • Formulations may also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents or sterile, pyrogen-free, water.
  • exemplary administration forms may include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • compositions of the disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired.
  • the pharmaceutical compositions are typically suitable for parenteral administration, wherein administration includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intranasal, intratracheal, intraarterial, intrathecal, intradermal, intrasynovial injection or infusions.
  • the pharmaceutical compositions of the present disclosure comprise intravenous administration.
  • the term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals.
  • the combination partners show a cooperative, e.g., synergistic effect.
  • co-admini strati on” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
  • pharmaceutical combination as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • fixed combination means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • non-fixed combination means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.
  • cocktail therapy e g., the administration of three or more active ingredients.
  • the disclosure provides a method of treating a subject in need thereof, comprising administering to the subject an effective amount of the compounds of the disclosure or the pharmaceutical composition of the disclosure.
  • subject refers to an animal. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • administering comprises administering a therapeutically effective amount to a human subject.
  • the term “amount” refers to “an amount effective” or “an effective amount” of a cell to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
  • “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions.
  • an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease.
  • an effective amount may be given in single or divided doses.
  • the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated.
  • “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • the terms “prevent,” “preventing” and “prevention” refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof.
  • the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein.
  • the terms encompass the inhibition or reduction of a symptom of the particular disease.
  • subjects with familial history of a disease are potential candidates for preventive regimens.
  • subjects who have a history of recurring symptoms are also potential candidates for prevention.
  • a “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • alkyl refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • An alkyl comprising up to 10 carbon atoms is referred to as a Ci-Cio alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a Ci-Ce alkyl.
  • Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly.
  • Alkyl groups include, but are not limited to, Ci-Cio alkyl, C1-C9 alkyl, Ci-Cs alkyl, C1-C7 alkyl, Ci- C 6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C 2 -Cs alkyl, C 3 -C 8 alkyl and C 4 - C alkyl.
  • alkyl group may be optionally substituted.
  • Alkylene refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, and likewise can be saturated or unsatured.
  • the alkylene is -CH2-, -CH2CH2-, or -CH 2 CH 2 CH 2 -.
  • the alkylene is -CH2-.
  • the alkylene is -CH2CH2-.
  • the alkylene is -CIUCIUCIU-. Unless stated otherwise specifically in the specification, an alkylene group may be optionally substituted.
  • Heteroalkyl refers to an alkyl group wherein one or more of the carbons of the alkyl group is replaced with a heteroatom.
  • exemplary heteroatoms include N, O, Si, P, B, and S atoms, preferably N, O and S. Note that valency of heteroatoms may not be identical to that of a carbon atom, so, for example, a methylene (CH2) of an alkyl may be replaced with an NH group, S group, O group, or the like in a heteroalkyl.
  • Heteroalkylene refers to an alkylene group wherein one or more of the carbons of the alkylene group is replaced with a heteroatom.
  • Exemplary heteroatoms include N, O, Si, P, B, and S atoms, preferably N, O and S.
  • an aryl group comprises a partially reduced cycloalkyl group defined herein (e.g., 1,2-dihydronaphthalene).
  • An aryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which may include fused, spiro or bridged ring systems.
  • cycloalkyl refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • cycloalkyls are saturated or partially unsaturated.
  • cycloalkyls are spirocyclic or bridged compounds.
  • cycloalkyls are fused with an aromatic ring (in which case the cycloalkyl is bonded through a non-aromatic ring carbon atom).
  • Cycloalkyl groups include groups having from 3 to 10 ring atoms.
  • Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms.
  • Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • the monocyclic cycloalkyl is cyclopentyl.
  • the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl.
  • the monocyclic cycloalkyl is cyclopentenyl.
  • Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, decalinyl, 3,4- dihydronaphthalenyl-l(2H)-one, spiro[2.2]pentyl, norbornyl and bicycle[l.l.l]pentyl.
  • a cycloalkyl group may be optionally substituted.
  • heterocycloalkyl refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen, and sulfur.
  • the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems.
  • the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized.
  • the nitrogen atom may be optionally quatemized.
  • the heterocycloalkyl radical is partially or fully saturated.
  • heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,
  • heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 12 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 1 or 2 N atoms. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 3 or 4 N atoms.
  • heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted.
  • heteroaryl refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • heteroaryl is monocyclic or bicyclic.
  • monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline,
  • monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl.
  • bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine.
  • heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furyl.
  • a heteroaryl contains 0-6 N atoms in the ring.
  • a heteroaryl contains 1-4 N atoms in the ring. In some embodiments, a heteroaryl contains 4-6 N atoms in the ring. In some embodiments, a heteroaryl contains 0-4 N atoms, 0-1 0 atoms, 0-1 P atoms, and 0- 1 S atoms in the ring. In some embodiments, a heteroaryl contains 1-4 N atoms, 0-1 0 atoms, and 0-1 S atoms in the ring. In some embodiments, heteroaryl is a C1-C9 heteroaryl. In some embodiments, monocyclic heteroaryl is a C1-C5 heteroaryl.
  • monocyclic heteroaryl is a 5-membered or 6-membered heteroaryl.
  • a bicyclic heteroaryl is a C6-C9 heteroaryl.
  • a heteroaryl group comprises a partially reduced cycloalkyl or heterocycloalkyl group defined herein (e.g., 7,8-dihydroquinoline).
  • a heteroaryl radical can be a monocyclic or polycyclic (e g., bicyclic, tricyclic, or tetracyclic) ring system, which may include fused, spiro or bridged ring systems.
  • optional substituents are independently selected from D, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, - NH(cyclopropyl), -CH3, -CH2CH3, -CF3, -OCH3, and - OCF3.
  • substituted groups are substituted with one or two of the preceding groups.
  • the compounds which are capable of inhibiting or degrading ZBTB11.
  • the compounds provided herein are capable of degrading ZBTB11.
  • the compounds act as a molecular glue with an E3-ligase.
  • the E3 ligase comprises cereblon (CRBN).
  • the compounds herein bind the E3 -ligase CRL4-CRBN.
  • the compounds herein promote interaction between ZBTB11 and the E3-ligase CRL4-CRBN.
  • the compounds bind at the interface of ZBTB11 and CRBN.
  • the compounds are effective to induce ubiquitination and degradation of ZBTB11.
  • the compounds described herein are effective in the methods of treatment described herein.
  • a compound according to the instant disclosure is a compound of Formula (I):
  • a 1 is optionally substituted aryl or heteroaryl
  • a 2 is optionally substituted aryl or heteroaryl
  • R 1 is H, optionally substituted alkyl, halogen, a bond with A 1 , or optionally substituted alkylene forming a bond with A 1 ;
  • each R 2 and R 3 is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, optionally substituted alkylene forming a bond with an R B , or R 2 and R 3 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl;
  • each R 4 and R 5 when present, is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, or two R 4 or two R 5 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl;
  • R A if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, or optionally substituted aryl or heteroaryl;
  • R B if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl; or a bond with an optionally substituted alkylene of R 2 or R 3 ; or a pharmaceutically acceptable salt thereof.
  • each X 1 is independently O or S. In embodiments, each X 1 is O.
  • X 2 is — O — , — N(R A ) — , — S — , —
  • X 2 is — N(R A ) — .
  • X 2 is -NH- or -N(CH3)-.
  • X 2 is -NH-.
  • each R A if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, or optionally substituted aryl or heteroaryl. In embodiments, each R A , if present, is H, optionally substituted alkyl, or optionally substituted cycloalkyl. In embodiments, each R A , if present, is H or optionally substituted alkyl. In embodiments, each R A , if present, is H or C1-C3 alkyl. In embodiments, each R A , if present, is H or -CH3. In embodiments, each R A , if present, is H.
  • X 3 is optionally substituted heterocycloalkylene.
  • X 3 is — N(R B ) — .
  • X 3 is - NH- or -N(CH 3 )-.
  • X 3 is -N(R B ), wherein R B is a bond with an optionally substituted alkylene of R 2 or R 3 .
  • R B if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl; or a bond with an optionally substituted alkylene of R 2 or R 3 .
  • R B if present, is H, optionally substituted alkyl, optionally substituted cycloalkyl, or a bond with an optionally substituted alkylene of R 2 or R 3 .
  • R B if present, is H or optionally substituted alkyl.
  • R B if present, is H or C1-C3 alkyl.
  • R B if present, is H or -CH3. In embodiments, R B , if present, is H. In embodiments, R B , if present, is a bond with an optionally substituted alkylene of R 2 or R 3 .
  • a 1 is optionally substituted aryl or heteroaryl. In embodiments, A 1 is optionally substituted 6-membered aryl or heteroaryl. In embodiments, A 1 is optionally substituted phenyl or pyridyl. In embodiments, A 1 is optionally substituted phenyl.
  • a 1 has the structure of optionally substituted
  • a 1 has the structure of [0084]
  • the aryl or heteroaryl of A 1 is optionally substituted with R 8 , wherein R 8 is H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, -CN, or halogen.
  • R 8 is H, optionally substituted alkyl, -CN, or halogen.
  • R 8 is H or C1-C3 alkyl.
  • a 2 is optionally substituted aryl or heteroaryl. In embodiments, A 2 is optionally substituted 6-membered aryl or heteroaryl. In embodiments, A 2 is optionally substituted phenyl or pyridyl. In embodiments, A 2 is optionally substituted phenyl.
  • the R 6 is -CF3 or chloro. In embodiments, the R 6 is -CF3. In embodiments, each R c , if present, is H or optionally substituted alkyl. In embodiments, each R c , if present, is H.
  • each R 7 is independently -CN, halogen, or optionally substituted alkyl. In embodiments, each R 7 is independently -CN, halogen, or fluoroalkyl. In embodiments, each R 7 is independently -CN, halogen, or -CF3. In embodiments, each R 7 is independently -CF3 or halogen. In embodiments, each R 7 is independently -CF3 or chloro. In embodiments, one R 7 is present. In embodiments, no R 7 are present.
  • R 1 is H, optionally substituted alkyl, halogen, a bond with A 1 , or optionally substituted alkylene forming a bond with A 1 .
  • R 1 is H or optionally substituted alkyl.
  • R 1 is H.
  • 1 is 0 or 1. In embodiments, 1 is 1.
  • each R 2 and R 3 is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, optionally substituted alkylene forming a bond with an R B , or R 2 and R 3 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl.
  • R 2 and R 3 are each independently H, optionally substituted alkyl, or optionally substituted cycloalkyl.
  • R 2 and R 3 are each independently H, C1-C3 alkyl, or C3-C6 cycloalkyl.
  • R 2 is alkylene forming a bond with an R B and R 3 is H. In embodiments, R 2 is C1-C4 alkylene forming a bond with an R B and R 3 is H. In embodiments, R 2 is C2 alkylene forming a bond with an R B and R 3 is H.
  • m is 0, 1, 2, or 3. In embodiments, m is 1 or 2. In embodiments, m is 2. In embodiments, each R 5 , when present, is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, or two R 5 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl. In embodiments, each R 5 , when present, is independently H or optionally substituted alkyl. In embodiments, each R 5 , when present, is independently H or C1-C3 alkyl. In embodiments, each R ⁇ when present, is H.
  • n is 0, 1, 2, or 3. In embodiments, n is 0, 1 or 2. In embodiments, n is 0. In embodiments, n is 1 or 2. In embodiments, n is 2. In embodiments, each R 4 , when present, is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, or two R 4 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl. In embodiments, each R 4 , when present, is independently H or optionally substituted alkyl. In embodiments, each R 4 , when present, is independently H or C1-C3 alkyl. In embodiments, each R 4 , when present, is H.
  • the compound having a structure according to Formula (I) conforms to the substructure of Formula (la):
  • a 2 is phenyl substituted by one substituent R 6 and further optionally substituted by one or more R 7 , wherein:
  • R 1 is H, optionally substituted alkyl, halogen, or optionally substituted alkylene forming a bond with A 1 ; each R 2 and R 3 is independently optionally substituted alkyl, optionally substituted alkylene forming a bond with an R B , or R 2 and R 3 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl; each R 4 and R 5 , when present, is independently H, optionally substituted alkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, or two R 4 or two R 5 are taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl or heterocycloalkyl
  • R 8 is H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, optionally substituted aryl or heteroaryl, -CN, or halogen; m is 1, 2, or 3; n is 0, 1, 2, or 3; and
  • R B is H or a bond with an optionally substituted alkylene of R 2 or R 3 ; or a pharmaceutically acceptable salt thereof, and .
  • the compound of Formula (la) is not a compound provided in US Patent Application Publication No. US2023/0192644. [0097] In embodiments, any one of the variables set forth in Formula (la) may be the same as those defined in relation to Formula (I).
  • a compound according to the instant disclosure useful in the inhibition and/or degradation of ZBTB11 is one selected from Table 1 below.
  • the instant disclosure provides additional compound analogs to those specifically exemplified herein, such as those in Table 2 below. It is predicted that the compounds in Table 2 below may also exhibit ZBTB11 degradation activity. Table 2 - Additional Potential ZBTB11 Degraders
  • n is preferably an integer from 1 to 3.
  • a pharmaceutical composition comprising a compound as described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of the relevant compound.
  • the therapeutically effective amount of a disclosed compound will depend on the route of administration, the species of subject and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed compounds is understood by those of skill in the art.
  • the pharmaceutical composition can also further comprise an additional therapeutic agent (e.g., any of those described herein in reference to combination therapies).
  • harmaceutical compositions for administration to a subject can include at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • the pharmaceutically acceptable carriers useful for these formulations are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.
  • parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • injectable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional nontoxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • compositions disclosed herein include those formed from pharmaceutically acceptable salts and/or solvates of the disclosed compounds.
  • the compound can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound.
  • Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.
  • local anesthetics for example, benzyl alcohol
  • isotonizing agents for example, sodium chloride, mannitol, sorbitol
  • adsorption inhibitors for example, solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione)
  • solubility enhancing agents for example, cyclodextrins and derivatives thereof
  • stabilizers for example, serum albumin
  • reducing agents for example, glutathione
  • compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • pharmaceutically acceptable vehicles for solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use.
  • Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by fdtered sterilization.
  • dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile-fdtered solution thereof.
  • the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the compound can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought.
  • a prophylactically or therapeutically effective amount of the compound and/or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof.
  • the compound can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol).
  • the therapeutically effective dosage of the compound can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein.
  • Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject.
  • Suitable models in this regard include, for example, murine, rat, avian, dog, sheep, porcine, feline, non-human primate, and other accepted animal model subjects known in the art.
  • effective dosages can be determined using in vitro models.
  • an effective amount or effective dose of the compound may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.
  • kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects.
  • Kits for diagnostic use are also provided.
  • these kits include a container or formulation that contains one or more of the compounds described herein.
  • this component is formulated in a pharmaceutical preparation for delivery to a subject.
  • the compound is optionally contained in a bulk dispensing container or unit or multi-unit dosage form.
  • Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator.
  • Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used.
  • a method of treating a ZBTB11 -related disease or disorder in a subject in need thereof comprising administering to the subject a compound which inhibits or degrades ZBTB11.
  • a method of treating a disease or disorder characterized by elevated oxidative phosphorylation in the subject the method comprising administering to the subject a compound which inhibits or degrades ZBTB11.
  • the compound is delivered in a therapeutically effective amount.
  • a disease or disorder associated with ZBTB11 includes those in which ZBTB11 acts to modulate oxidative phosphorylation in the subject.
  • ZBTB11 acts in diseased tissue to regulate oxidative phosphorylation.
  • the disease or disorder is characterized by elevated oxidative phosphorylation.
  • the disease or disorder is one in which diseased tissue (e.g., cancer cells) rely on elevated oxidative phosphorylation for survival.
  • the disease or disorder to be treated by a ZBTB11 inhibitor or degrader e.g., a compound as described herein
  • a ZBTB11 inhibitor or degrader e.g., a compound as described herein
  • the disease or disorder develops the elevated oxidative phosphorylation state in response to a mutation.
  • the disease or disorder develops the elevated oxidative phosphorylation state due to treatment with a therapeutic intervention (e.g., treatment of cancer with a K-Ras inhibitor).
  • the disease or disorder to be treated by targeting ZBTB11 is one which is resistant or refractory to treatment with another therapeutic intervention.
  • the disease or disorder is resistant or refractory to treatment with the other therapeutic intervention in the absence of a ZBTB11 inhibitor or degrader.
  • the disease or disorder is refractory due at least partially to enhanced reliance on oxidative phosphorylation upon treatment with the other therapeutic intervention.
  • the disease or disorder treated with a ZBTB11 inhibitor or degrader is further treated with an additional therapeutic intervention.
  • the method of treatment comprises administering an additional therapeutic intervention.
  • the additional therapeutic intervention comprises chemotherapy, targeted therapy, immunotherapy, a kinase inhibitor, a Ras pathway inhibitor, DNA damaging agents, and/or a PARP inhibitor.
  • the additional therapeutic intervention is a K-Ras inhibitor (e.g., sotorasib and/or adagrasib).
  • the additional therapeutic intervention is an anti-angiogenic therapy.
  • the additional therapeutic intervention is a BCL2 inhibitor, tyrosine kinase inhibitor, or BRAF inhibitor.
  • the additional therapeutic intervention is one which results in elevated oxidative phosphorylation in the subject.
  • the additional therapeutic intervention is one to which resistance can be developed due to enhanced oxidative phosphorylation in the relevant tissue.
  • the disease or disorder to be treated with a ZBTB11 inhibitor or degrader is cancer.
  • the cancer is a drugresistant cancer.
  • the cancer is one which exhibits elevated oxidative phosphorylation.
  • the cancer relies on elevated oxidative phosphorylation for survival.
  • the cancer is pancreatic cancer.
  • the cancer is a K-Ras mutant cancer.
  • the cancer is pancreatic dual adenocarcinoma.
  • Example 1 Identification of ZBTB11 as an Orthogonal Therapeutic Target [00120]
  • K-Ras inhibitor resistance reliably generates a high-OXPHOS state, and to develop in-house PDAC models, 3 independent K-RasG12C MIA PaCa-2 lines were cultured with escalating sublethal concentrations of sotorasib (AMG-510) to generate sotorasib-resistant cell lines Rl, R2 and R3 (Fig. 1 Panel A, Fig. 2 Panel A).
  • the cellular metabolic state was characterized using Seahorse bioenergetic analysis, where a mitochondrial stress test revealed increased basal and maximal oxidative phosphorylation in the resistant cells compared to parental cell controls, as well as increased extracellular acidification rate (ECAR) resulting in overall higher concentrations of ATP produced per minute per cell (Fig. 1 Panels B- C).
  • Seahorse bioenergetic analysis was performed on previously reported K-RasG12D MTXR SUIT2 lines which are resistant to MRTX1133, and compared them to parental SUIT2 cells (Fig. 3 Panels A-C). Increased basal and maximal oxidative phosphorylation was also observed, and overall higher concentrations of ATP produced per minute per cell in MTXR cells.
  • ZBTB 11 regulates nuclear transcription of components of the mitoribosome and complex I in murine ES cells and HEK293 cells 33 to modulate OXPHOS, but its functions in PDAC have not been characterized.
  • inducible ZBTB 11 CRISPRi cell lines were developed to evaluate effects of ZBTB 11 depletion genetically, confirming knockdown by immunoblot and qPCR (Fig. 4 Panels A-B, D).
  • mitoribosome genes MRPL48, MRPL44, MRPL1, MRPL30
  • 4 complex I genes NDUF37, NDUFA12, NDUFC2, NDUFAF1
  • qPCR was performed to quantify their mRNA levels 1 and 3 days after hZBTBl 1 CRISPRi knockdown. Reduction of the mitoribosome mRNA set was observed 24 hrs after dCas9-KRAB induction, and reduction of the complex I mRNA set after 3 days (Fig. 1 Panel D, Fig. 4 Panels C, E).
  • ZBTB11 is thus concluded to be a transcriptional regulator of mitochondrial genes and OXPHOS in K-Ras mutant PDAC, consistent with its reported role in other organisms and cell types.
  • ZBTB 11 harbors the CXXCG beta-hairpin motif found in many CRBN molecular glue targets, no CRBN-recruiting degraders of ZBTB 11 have been reported. 38
  • CRISPR/cas9 was used to knock in a HiBiT tag at the c-termini of the endogenous ZBTB 11 locus and used this assay to profile a diverse in-house library of CRBN-binding molecular glue candidates (Fig. 5 Panel A).
  • Several multi -targeted hits able to deplete ZBTB 11 by greater than 30% were identified, from the same chemical series (Fig. 5 Panels B-C).
  • Molecular glue degraders depend on formation of ternary complexes for their cellular activity.
  • ZBTB 11 and CRBN To measure intracellular ternary complex between molecules, ZBTB 11 and CRBN, a nanoBRET assay was developed and used it to confirm that JWJ-01-306 promotes a ZBTB 11 :molecular glue:CRBN ternary complex (Fig. 5 Panel F).
  • ternary complex molecular modeling protocols was established (Fig. 5 Panel G).
  • ZBTB 11 contains 12 C2H2 zinc finger domains, 7 of which harbor a CXXCG motif (ZF1, ZF3, ZF4, ZF6, ZF9, ZF10, and ZF11).
  • NLuc tagged ZF constructs were generated, and quantified the change in individual ZF-nLuc fusion protein levels upon treatment with ZBTB 11 degrader molecules was quantified, to elucidate that zinc finger 10 (ZF10) contains the primary JWJ/CRBN degron (Fig. 6 Panels C-E).
  • ZF10 zinc finger 10
  • Fig. 6 Panels C-E zinc finger 10
  • an AlphaFold2 40 model of ZBTB 11 ZF10 was generated and this structure was used to perform Rosetta proteincomplex docking to JWJ-01-306 bound CRBN. 41 Manual inspection of the clusters revealed a binding mode consistent with previously reported cryo-EM structures of CRBN-binding molecular glue degraders.
  • the glutarimide binds in the tri -tryptophan pocket where it makes two H-bonds to His378 and one H-bond to the backbone amide of Ser379 in CRBN (Fig. 5 Panel G).
  • the aniline ring makes an edge-to-face TL interaction with His353 in CRBN, and 3- substitution orients the benzylic cyclobutane ring towards the interface of ZBTB11 ZF10 and CRBN, making hydrophobic contact with CRBN Pro352.
  • ZBTB11 residue Lys866 and CRBN residue Glu377 form a cation-7r-anion stacking interaction with the 4-C1, 3-CFa substituted phenyl ring in JWJ degraders, that bridges the ternary complex. 43 These interactions agree with trends in the ZBTB11 degrader structure activity relationships.
  • JWJ-01-334 To aid interpretation of phenotypic data and confirm observed effects are on- target, two negative control compounds were developed: JWJ-01-334 and JWJ-01-368. To rescue all CRBN-dependent pharmacology, JWJ-01-334, which contains an N-Methyl glutarimide that prevents binding to CRBN in cellular CRBN target engagement assays 44 , and rescues ZBTB11 ternary complex formation and degradation (Fig. 5 Panels D, F, Fig. 6 Panel A) was synthesized.
  • JWJ-01-368 which lacks the aromatic CF3 substituent crucial for achieving the cation-7t-anion interaction that promotes ZBTB1 1 :JWJ:CRBN ternary complex formation, was synthesized.
  • JWJ-01 -368 efficiently binds CRBN (Fig. 6 Panel A) but fails to promote both ZBTB11 ternary complex formation (Fig. 5 Panel F) and ZBTB11 degradation (Fig. 5 Panel D).
  • JWJ-01-306 and JWJ-01-368 were evaluated in Mia PaCa-2 cells by global proteomics analysis, following a 5 hr treatment with 10 pM compound (Fig. 5 Panel H and Fig. 7 Panel A, Dataset 2).
  • JWJ-01-306 treated cells potent ZBTB11 downregulation was observed, and off-target degradation of three related C2H2 ZF transcription factors; ZFP91, ZBTB21 and WIZ.
  • JWJ-01-368 treated cells ZBTB11 degradation was not observed, indicating JWJ-01-368 is a suitable control compound for rescue of ZBTB11 -specific pharmacology (Fig. 7 Panel A, Dataset 2).
  • JWJ-01-306 Plasma protein binding analysis revealed that JWJ-01-306 is over 99% protein bound (Dataset 3). Taken together, these data indicate that the free drug levels of JWJ-01-306 in vivo are unlikely to be sufficient to support ZBTB11 degradation at reasonable dosing regimes, and that further chemistry optimization is needed to improve the stability and physicochemical properties of JWJ-01-306. Therefore, JWJ-01-306 is recommended as a cell-based tool compound for target validation and mechanistic studies.
  • K-Ras inhibitor-resistant MIA PaCa-2 cells showed significantly impaired proliferation only in the presence of sotorasib+JWJ-01-306 (Fig. 8 Panel A), but not sotorasib+JWJ-01-368.
  • proliferation of parental cells was completely impaired by high-concentration (500 nM) of MRTX1133 and by JWJ-01-306 as a single agent within 5 days (10 pM, Fig. 8 Panel B).
  • JWJ-01-306 Gratifyingly, cell growth in MRTX1133- resistant SUIT2 lines was blocked by JWJ-01-306 single agent treatment and MRTX1133 + JWJ-01-306, but not MRTX1133 alone or MRTX1133 + JWJ-01-368 (Fig. 8 Panel B).
  • Protein sets primarily responsive to Sotorasib include upregulation of mitochondrial ATP synthesis, and changes to glycine, serine and threonine metabolism pathway proteins, consistent with the observed metabolic and metabolomic differences observed between WT and Sotorasib resistant cell lines (Fig. 8 Panel C cluster 8, Fig. 9 cluster 5). Finally, clusters representing proteins that change only in response to the combination treatment are enriched for nucleoside metabolism, ER membrane, and gene expression (upre, DNA replication (downregulated, Fig. 8 Panel C cluster 4). To further elucidate the effects of ZBTB11 on mitochondrial proteins, proteins downregulated in JWJ-01- 306 treatment conditions were evaluated and compared to the mitocarta 3.0 database of mitochondrial proteins (Fig. 10 Panel A).
  • ZBTB11 was confirmed to be downregulated in JWJ-01-306 treated samples (Fig 10 Panel B) alongside a subset of ZBTB11 downstream proteins (NDUFS7, NDUFA12, NDUFC2, NDUFAF1, MRPL48, MRPL44, MRPL1, MRPL30, Fig 10 Panel B). K-Ras levels were also reduced in the 5 d samples in response to JWJ-01-306 but not Sotorasib (Fig. 10 Panel B).
  • Sotorasib-resistant MiaPaCa-2 cells that stably expressed ZBTB 1 1 WT (control) or ZBTB11 K866T were treated with 10 pM JWJ-01-306 for 24 hrs and assessed levels of relevant downstream transcripts by qPCR.
  • complete rescue of transcript loss by the degradationresistant ZBTB11 K866T mutant for 7/8 downstream genes, and partial rescue of MRPL1 (Fig. 11 Panel H) was observed.
  • ZBTB11 degraders propensity to cause drug-induced neuropathy
  • the neurotoxicity of ZBTB11 degraders was profiled using a suite of assays developed in human induced pluripotent stem cell (hiPSC)-derived neurons? 0 ' 53 Neuronal viability, morphology, and mitochondrial function was monitored, demonstrating that iPSC-derived neurons exposed to 1 pM IACS-010759 for 24 hrs exhibit significantly decreased mitochondrial membrane potential (MMP) and increased reactive oxygen species (ROS), consistent with inhibition of mitochondrial complex I and reported adverse events in clinical trials (Fig. 13 Panel A).
  • MMP mitochondrial membrane potential
  • ROS reactive oxygen species
  • molecular glue degraders of the transcription factor ZBTB11 are disclosed.
  • ZBTB11 -governed metabolic networks were identified in human PDAC.
  • JWJ- 01-306 directly targets the transcriptional upregulation of OXPHOS genes associated with cancer drug resistance. It was demonstrated that pharmacological degradation of ZBTB11 counters this metabolic reprogramming associated with acquired resistance to K-Ras inhibitor in multiple ex vivo models of PDAC, identifying ZBTB11 as a druggable therapeutic vulnerability in PDAC.
  • a major limitation for targeting aberrant OXPHOS are the neurotoxic side effects of Complex I inhibition, observed as on-target adverse events in clinical trials and later in focused murine studies.
  • fetal bovine serum FBS; Gibco #A5256701
  • penicillin/streptomycin P/S; Corning &30-002-CI
  • Wildtype and Crbn-/- MOLT-4, Jurkat, and SUIT-2 cells were cultured in RPMI1640 (Corning #10-040- CM) supplemented with 10% FBS and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO2. All cell lines were tested for mycoplasma contamination using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza #LT07-710).
  • IKZFl-HiBiT and GSPTl-HiBiT Jurkat knock- in cell lines were a generous gift from Dr. Nathanael Gray (Stanford University).
  • Organoid culture Patient-derived organoids were maintained in complete human feeding medium in a 37 °C incubator with 5% CO2. The protocol to propagate and sustain organoid cultures was described previously. 1 ’ 2
  • the complex was cooled to room temperature and then incubated for 20 min with Alt-RTM S.p. HiFi Cas9 Nuclease V3 (IDT # 1081060) to form the ribonucleoprotein (RNP) complex.
  • Alt-RTM Cas9 Electroporation Enhancer IDT #1075916 was electroporated into cells using a NeonTM Transfection System (Thermo Scientific #MPK5000). Cells were seeded into media containing Alt-RTM HDR Enhancer V2 (IDT #10007910).
  • HiBiT expression from individual clones was measured using the Nano-Gio® HiBiT Lytic Detection System (Promega #N3040) on a ClarioSTAR Plus microplate reader (BMG LabTech) with NanoLuc® module (470 nm with 80 nm bandpass). Correct insertion of the HiBiT tag into the genome of the knocked-in cells was confirmed by sequencing. DNA HDR template and guide RNA sequences are detailed in Supplementary Table 1.
  • MIA PaCa-2 ZBTB11 -HiBiT cells were cultured in stepwise increasing concentrations (5 nM to 10 pM) of sotorasib (MedChemExpress #HY-114277) to afford the MIA PaCa-2 AMG510R cell lines.
  • SUIT-2 MRTXR cell lines resistant to MRTX1133 were generated previously 4 .
  • MIA PaCa-2 AMG510R and SUIT-2 MRTXR cell lines were maintained in appropriate media supplemented with 10 pM of sotorasib or MRTX1133.
  • MIA PaCa-2 and SUIT-2 cells were resuspended to 1 x 10 3 cells in 50 pl of complete growth medium and plated in solid white 384-well tissue culture plates (Corning #3570). Adherent cells were incubated for 24 h to allow for cell adhesion and recovery. Compounds were dispensed in dose response as indicated using a D300e Digital Dispenser (Tecan #30100152). Cells were incubated for 3 days, and endpoint viability was measured with CellTiterGlo® Luminescent Cell Viability Assay (Promega #G7572) on a ClarioSTAR Plus microplate reader with NanoLuc® module.
  • CRBN engagement assay The NanoBRETTM TE Intracellular E3 Ligase Assay Kit for CRBN (Promega #N2910) was used to measure CRBN engagement by compounds. Briefly, HEK293 cells were transfected with NanoLuc-CRBN and DDB1 expression vectors using OptiMEMTM I Reduced Serum Medium with no phenol red (Gibco #11058021) and TransIT-2020 Transfection Reagent (Minis Bio #5400). After incubating for 24 h, transfected cells were resuspended to 6.8 x 10 3 cells in 34 pl of OptiMEMTM I Reduced Serum Medium with no phenol red and plated in solid white 384-well tissue culture plates.
  • NanoBRETTM Tracer Reagent and compounds were dispensed in dose response as indicated using a D300e Digital Dispenser. Cells were incubated for 2 h prior to addition of 17 pl of Complete Substrate Plus Inhibitor Solution and subsequent readout on a ClarioSTAR Plus microplate reader with NanoBRETTM module (donor: 460 nm with 80 nm bandpass, acceptor: >610 nm longpass).
  • NanoBRETTM MOLT-4 cells were resuspended to 1 x 10 4 cells in 50 pl of OptiMEMTM I Reduced Serum Medium with no phenol red and plated in solid white 384-well tissue culture plates.
  • Carfdzomib (MedChemExpress #HY-10455) and the HaloTag® NanoBRETTM 618 Ligand were dispensed to a final concentration of 100 nM using a D300e Digital Dispenser.
  • ZBTB11 ZF fingerprinting and mutagenesis Full length human ZBTB11, or select portions of its tandem zinc finger domains, were cloned into the pNLFl-N NanoLuc® Protein Fusion Vector. QuikChange Site-Directed Mutagenesis Kit (Agilent #200519) was used to introduce ZBTB11 K866T and T659K mutations. MOLT-4 and MIA PaCa-2 cells were transfected with NLuc-ZBTBl l fusion protein vectors using a NeonTM Transfection System. Following a two-day recovery period, transfected cells were subjected to hygromycin B selection for seven days to yield stable NLuc-ZBTBl l expression cell lines.
  • MIA PaCa-2 cells were resuspended to 5 x 10 4 cells in 1 ml of complete growth medium and plated in clear 12-well tissue culture plates (Corning #3506). Cells were then treated with DMSO, sotorasib (1 pM), JWJ-01-306 (10 pM), or sotorasib + JWJ- 01-306. Viable cells were passaged and quantified by trypan blue exclusion (Invitrogen T10282) twice weekly using a Countess 3 Automated Cell Counter (Invitrogen #AMQAX2000).
  • MIA PaCa-2 ZBTB11-HiBiT cells were transduced with lentiviruses to introduce Tet-On 3G-Neo, TRE-dCas9-KRAB-mCherry, and sgRNA-BFP-Puro (nontargeting or against ZBTB11; see Supplemental Table 1). Following transduction, cells were sorted for mCherry hlgh /BFP hl8h expression and selected with 500 pg/ml G418 and 5 pg/ml puromycin.
  • CRISPRi cells were then maintained in complete growth media supplemented with 200 pg/ml G418 and 2 pg/ml puromycin. CRISPRi-mediated knockdown was induced by treatment with 500 ng/ml doxycycline as indicated.
  • ZBTB11-HiBiT signal was detected using the Nano-Gio® HiBiT Blotting System (Promega #N2410). Membranes were detected on an Odyssey CL X detection system (LI-COR Biosciences) after incubation with IRDye®800-labeled goat anti-rabbit IgG (LI-COR # 926-32211) and IRDye®680-labeled goat anti-mouse IgG (LI-COR #926-68070) secondary antibodies. [00158] Quantitative PCR.
  • MIA PaCa-2 and SUIT-2 cells were resuspended to 5 x io 4 cells in 100 pl of complete growth medium supplemented with 10 pM of sotorasib or MRTX1133 for K-Rasz-resistant cells and plated in Seahorse XF24 V7 PS Cell Culture Microplates (Agilent #100777-004). Adherent cells were incubated for 24 h to allow for cell adhesion and recovery.
  • MEM containing 1 g/1 glucose and no glutamine was supplemented with 1 g/1 unlabeled glucose (Gibco #A24940-01), 1 mM unlabeled glutamine (Gibco #25030-149), 1 mM 13 C5-glutamine (Sigma-Aldrich #605166), 1% MEM non-essential amino acids (NEAA), 10% FBS, and 1% P/S.
  • the upper polar phase was transferred to a separate tube and dried in a Speedvac, derivatized with isobutylhydroxylamine (20 mg/ml solution in pyridine), and mixed with N-tert- Butyldimethylsilyl-N-methyltri fluoroacetamide.
  • the GC-MS interface temperature was 300°C and (electron impact) ion source temperature was 200°C, with 70 eV ionization voltage.
  • Standards were run in parallel with samples. Metabolites in samples and standards were detected by MS/MS using precursor and product ion masses, and collision energies. Sample metabolites were quantified using calibration curves made from the standards in Chromeleon software (Thermo Scientific), and further data processing to adjust for the relative quantities of metabolites in the standards, and for recovery of the internal standard, were done in MS Excel. Normalized metabolite abundances were then used as inputs into Metab o Analyst 5 with default settings and KEGG pathways to perform Metabolite Set Enrichment Analysis (MSEA).
  • MSEA Metabolite Set Enrichment Analysis
  • Equal volumes of cell lysate and SDS protein solubilization buffer (10% SDS, 100 mM triethylammonium bicarbonate (TEAB), pH 7.55) were mixed and reduced by incubation with 5 mM tris-(2-carboxyethyl)-phosphine (TCEP) for 30 min at room temperature (RT). Subsequent alkylation was performed by adding 15 mM chloroacetamide (CAA) and incubating for 30 min at RT. Phosphoric acid was added to a final concentration of 1.2%, followed by six volumes of binding buffer (90% methanol; 100 mM TEAB, pH 7.1).
  • the colloidal protein solution was loaded onto a S-Trap micro column (Protifi #C02-micro-10) and centrifuged 1500 g for 2 min.
  • the columns were washed with binding/wash buffer (100 mM TEAB in 90% MeOH) four times and sequencinggrade trypsin (Protnega #V5l 1 1) in digestion buffer (100 mM TEAB) was added in 1 :10 (w/w) ratio. Proteins were digested on-column for 3 h at 47 °C.
  • Peptides were eluted by a step-wise elution method using 50 mM TEAB, pH 8.5, followed by 0.2% formic acid (FA) in HrO, and finally 50% acetonitrile (ACN)/0 2% FA in H 2 O. Eluted peptides were then vacuum dried before further processing
  • the mass spectrometer was operated in positive data-independent acquisition mode with the following settings: 120,000 resolution, 350-2000 m/z scan range, injection time set at auto, FAIMS CV at -40 and AGC target of 4 * 10 5 for MSI scan.
  • MS2 scans were acquired at a resolution of 30,000 using the following settings: precursor range of 400-100 m/z, isolation window at 8 m/z with Im/z overlap, giving 75 windows. Fragmentation was done using HCD collision energy of 30% and AGC target at 800% and maximum injection time at 54 ms.
  • Lysates were diluted four-fold, and proteins were digested with trypsin at 37°C for 16 hours using a 50:1 (protein: enzyme) ratio, followed by quenching by lowering the pH to ⁇ 3 with 10% TFA.
  • Peptides were purified using SepPak Cl 8 solid-phase extraction (SPE) cartridges (Waters, Milford, MA) and dry-down, then stored at -80° before run on LC-MS/MS. All the samples were analyzed on a timsTOF Pro 2 (Bruker) coupled to a EvosepOne LC system (Evosep) using the dia-PASEF method as previously described 7 .
  • JWJ-01-306 formulation 5% DMSO/20% Solutol® HS 15/0.9% NaCl
  • Plasma protein binding A Rapid Equilibrium Dialysis (RED) device (Thermo Scientific #90006) was used to perform the plasma protein binding assay. Briefly, phosphate- buffered saline (PBS) and plasma were loaded into buffer and plasma wells; respectively. JWJ- 01-306 was added to the plasma to final concentrations of 0.1 pM and 1 pM, and the RED device was incubated on an orbital shaker for 4.5 h at 37 °C. Standards for reference compound warfarin and for JWJ-01-306 were prepared in a separate clear 96-well plate by mixing equal volumes of PBS and plasma, followed by serial 10-fold dilution of 10 pM compound.
  • PBS phosphate- buffered saline
  • JWJ- 01-306 was added to the plasma to final concentrations of 0.1 pM and 1 pM
  • Standards for reference compound warfarin and for JWJ-01-306 were prepared in a separate clear 96-well plate by mixing equal volumes of PBS and
  • a homology modeling approach was taken to generate a model of the CRBN:JWJ-O1-3O6:ZBTB11 ZF 10 ternary complex.
  • the CRBN:pomalidomide:IKZFl ZF2 complex was extracted from RCSB PDB entry 6H0F 9 .
  • ZBTB11 ZF10 was extracted from the ZBTB11 structure predicted by AlphaFold2 10 (AFDB accession AF-O95625-F1).
  • PyMOL command line ZBTB11 ZF10 and JWJ-01-306 were structurally aligned with IKZF1 ZF2 and pomalidomide; respectively.
  • Preparative HPLC was performed on a Waters Sunfire C18 column (19 mm x 50 mm, 5 pM) using a gradient of 15-95% methanol in water containing 0.05% trifluoroacetic acid (TFA) over 22 min (28 min run time) at a flow rate of 20 mL/min. Assayed compounds were isolated and tested as TFA salts. Purities of assayed compounds were in all cases greater than 95%, as determined by reverse-phase HPLC analysis.
  • TFA trifluoroacetic acid
  • the filtrate was saved and washed with brine twice.
  • the organic layer was dried over anhydrous MgSCL, concentrated under reduced pressure, and purified via flash column chromatography, which provided the imine intermediate as lightyellow oil.
  • the imine intermediate was re-dissolved in THF (4 mL) and 1 M HC1 solution (2 mL) was added. After 0.5 h, the mixture was neutralized with saturated NaHCCh solution and extracted with EtOAc twice. The combined organic layer was combined, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified via flash column chromatography, which yielded the title compound as pale-yellow solid (136 mg, 62%). !
  • LAH lithium aluminum hydride
  • the aniline intermediate was dissolved in 2 mL DCM and 0.5 mL TFA, and stirred at rt for 2 h. Upon complete consumption of starting material monitored by LCMS, 20 mL DCM and 20 mL IM NaOH solution were added. The or ganic solution was dried over anhydrous MgSO4 and concentrated under reduced pressure, which provided the title amine compound as light-yellow oil.
  • NCHS National Cancer Institute
  • CELMoD compounds are regulators of cereblon conformation. Science 378, 549-553, doi: 10.1126/science.add7574 (2022).

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Abstract

Composition et procédés d'utilisation pour traiter des troubles associés à ZBTB11 tels que le cancer comprenant des composés tels que décrits dans la description qui réalisent une dégradation de ZBTB11.
PCT/US2024/029868 2023-07-25 2024-05-17 Colles moléculaires ciblant un régulateur transcriptionnel d'états métaboliques aberrants Pending WO2025024030A2 (fr)

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