Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors.

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

MC180295 is a highly potent and selective CDK9 inhibitor with preclinical in vitro and in vivo efficacy in cancer.

BACKGROUND: Inhibition of cyclin-dependent kinase 9 (CDK9), a novel epigenetic target in cancer, can reactivate epigenetically silenced genes in cancer by dephosphorylating the SWI/SNF chromatin remodeler BRG1. Here, we characterized the anti-tumor efficacy of MC180295, a newly developed CDK9 inhibitor. METHODS: In this study, we explored the pharmacokinetics of MC180295 in mice and rats, and tested the anti-tumor efficacy of MC180295, and its enantiomers, in multiple cancer cell lines and mouse models. We also combined CDK9 inhibition with a DNA methyltransferase (DNMT) inhibitor, decitabine, in multiple mouse models, and tested MC180295 dependence on T cells. Drug toxicity was measured by checking body weights and complete blood counts. RESULTS: MC180295 had high specificity for CDK9 and high potency against multiple neoplastic cell lines (median IC50 of 171 nM in 46 cell lines representing 6 different malignancies), with the highest potency seen in AML cell lines derived from patients with MLL translocations. MC180295 is a racemic mixture of two enantiomers, MC180379 and MC180380, with MC180380 showing higher potency in a live-cell epigenetic assay. Both MC180295 and MC180380 showed efficacy in in vivo AML and colon cancer xenograft models, and significant synergy with decitabine in both cancer models. Lastly, we found that CDK9 inhibition-mediated anti-tumoral effects were partially dependent on CD8 + T cells in vivo, indicating a significant immune component to the response. CONCLUSIONS: MC180380, an inhibitor of cyclin-dependent kinase 9 (CDK9), is an efficacious anti-cancer agent worth advancing further toward clinical use.

Haploinsufficiency of ZNF251 causes DNA-PKcs-dependent resistance to PARP inhibitors in BRCA1-mutated cancer cells.

Poly (ADP-ribose) polymerase (PARP) inhibitors represent a promising new class of agents that have demonstrated efficacy in treating various cancers, particularly those that carry BRCA1/2 mutations. The cancer associated BRCA1/2 mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPis) have been applied to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, resistance to PARPis is common and can occur through multiple mechanisms, including the restoration of HR and/or the stabilization of replication forks. To gain a better understanding of the mechanisms underlying PARPi resistance, we conducted an unbiased CRISPR-pooled genome-wide library screen to identify new genes whose deficiency confers resistance to the PARPi olaparib. Our study revealed that ZNF251, a transcription factor, is a novel gene whose haploinsufficiency confers PARPi resistance in multiple breast and ovarian cancer lines harboring BRCA1 mutations. Mechanistically, we discovered that ZNF251 haploinsufficiency leads to constitutive stimulation of DNA-PKcs-dependent non-homologous end joining (NHEJ) repair of DSBs and DNA-PKcs-mediated fork protection in BRCA1-mutated cancer cells (BRCA1mut + ZNF251KD). Moreover, we demonstrated that DNA-PKcs inhibitors can restore PARPi sensitivity in BRCA1mut + ZNF251KD cells ex vivo and in vivo. Our findings provide important insights into the mechanisms underlying PARPi resistance and highlight the unexpected role of DNA-PKcs in this phenomenon.

DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17).

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation.

Design, synthesis, and evaluation of (2S,4R)-Ketoconazole sulfonamide analogs as potential treatments for Metabolic Syndrome.

Metabolic Syndrome, also referred to as 'Syndrome X' or 'Insulin Resistance Syndrome,' remains a major, unmet medical need despite over 30years of intense effort. Recent research suggests that there may be a causal link between this condition and abnormal glucocorticoid processing. Specifically, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis leads to increased systemic cortisol concentrations. Cushing' syndrome, a disorder that is also typified by a marked elevation in levels of cortisol, produces clinical symptomology that is similar to those observed in MetS, and they can be alleviated by decreasing circulating cortisol concentrations. As a result, it has been suggested that decreasing systemic cortisol concentration might have a positive impact on the progression of MetS. This could be accomplished through inhibition of enzymes in the cortisol synthetic pathway, 11ß-hydroxylase (Cyp11B1), 17α-hydroxylase-C17,20-lyase (Cyp17), and 21-hydroxylase (Cyp21). We have identified a series of novel sulfonamide analogs of (2S,4R)-Ketoconazole that are potent inhibitors of these enzymes. In addition, selected members of this class of compounds have pharmacokinetic properties consistent with orally delivered drugs, making them well suited to further investigation as potential therapies for MetS.

Synthesis and biological evaluation of novel pyrrolidine acid analogs as potent dual PPARα/γ agonists.

The design, synthesis and structure-activity relationships of a novel series of 3,4-disubstituted pyrrolidine acid analogs as PPAR ligands is outlined. In both the 1,3- and 1,4-oxybenzyl pyrrolidine acid series, the preferred stereochemistry was shown to be the cis-3R,4S isomer, as exemplified by the potent dual PPARα/γ agonists 3k and 4i. The N-4-trifluoromethyl-pyrimidinyl pyrrolidine acid analog 4i was efficacious in lowering fasting glucose and triglyceride levels in diabetic db/db mice.

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: identification of a potent and selective 5-HT6 receptor antagonist showing activity in rat social recognition test.

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ∼30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.

Novel brain penetrant benzofuropiperidine 5-HT6 receptor antagonists.

7-Arylsulfonyl substituted benzofuropiperidine was discovered as a novel scaffold for 5HT(6) receptor antagonists. Optimization by substitution at C-1 position led to identification of selective, orally bioavailable, brain penetrant antagonists with reduced hERG liability. An advanced analog tested in rat social recognition model showed significant activity suggesting potential utility in the enhancement of short-term memory.

2-Arylbenzoxazoles as novel cholesteryl ester transfer protein inhibitors: optimization via array synthesis.

2-Arylbenzoxazole 5 was identified as a hit from a fluorescence-based high-throughput screen for CETP inhibitors. The synthesis and SAR investigation employing array synthesis of the A- and B-rings are described.

A selective small molecule agonist of the melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice.

It is well established that melanocortins are peptides that have potent anti-inflammatory activity. Recent research has focused on understanding which of the known melanocortin receptors mediates the anti-inflammatory actions of the melanocortins. The aim of this study was to assess the anti-inflammatory activity of a synthetic MC-1R agonist. BMS-470539 is a potent, selective, full agonist of human and murine MC-1R with EC(50) values in a cAMP accumulation assay of 16.8 and 11.6 nM, respectively. BMS-470539 dose-dependently inhibited TNF-alpha-induced activation of a NF-kappaB transcriptional reporter in human melanoma cells, which endogenously express MC-1R. In vivo studies with BMS-470539 demonstrated that subcutaneous administration of BMS-470539 resulted in a dose-dependent inhibition of LPS-induced TNF-alpha production in BALB/c mice. In this model, the compound had an ED(50) of approximately 10 micromol/kg and a pharmacodynamic half-life of approximately 8 h. Pharmacokinetic analysis of the compound indicated that the compound had a t(1/2) of 1.7 h. In a model of lung inflammation, administration of 15 micromol/kg BMS-470539 resulted in a 45% reduction in LPS-induced leukocyte infiltration (an infiltrate comprised primarily of neutrophils). The compound was also effective in a model of delayed-type hypersensitivity, reducing paw swelling by 59%, comparable with that seen with 5 mg/kg dexamethasone. These studies demonstrate that a selective small molecule agonist of the melanocortin-1 receptor is a potent anti-inflammatory agent in vivo and provides compelling evidence for the involvement of this receptor in the modulation of inflammation.

Discovery of tyrosine-based potent and selective melanocortin-1 receptor small-molecule agonists with anti-inflammatory properties.

The melanocortin-1 receptor (MC-1R) is a G-protein-coupled receptor involved in inflammation and skin pigmentation. Compound 2 is the first highly potent and selective MC-1R small-molecule agonist reported. Compound 2 showed efficacy in an acute model of inflammation, which has demonstrated the role of MC-1R in modulation of inflammation.