The SWI/SNF multisubunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase ...domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutant tumors, including SMARCA4-deficient lung cancer; however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic, and pharmacologic tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g., lung, synovial sarcoma, leukemia, and rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacologic efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation, and target gene expression studies. Furthermore, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacologic studies exemplify a general strategy for multidomain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy.
The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene ...expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC K d = 31 nM and ITC K d = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.
Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the ...treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection.
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Structure based design of a novel class of aminopyrimidine MTH1 (MutT homolog 1) inhibitors is described. Optimization led to identification of IACS-4759 (compound 5), a sub-nanomolar ...inhibitor of MTH1 with excellent cell permeability and good metabolic stability in microsomes. This compound robustly inhibited MTH1 activity in cells and proved to be an excellent tool for interrogation of the utility of MTH1 inhibition in the context of oncology.
Blocking Plasmodium falciparum human-to-mosquito transmission is essential for malaria elimination, nonetheless drugs killing the pathogenic asexual stages are generally inactive on the parasite ...transmissible stages, the gametocytes. Due to technical and biological limitations in high throughput screening of non-proliferative stages, the search for gametocyte-killing molecules so far tested one tenth the number of compounds screened on asexual stages. Here we overcome these limitations and rapidly screened around 120,000 compounds, using not purified, bioluminescent mature gametocytes. Orthogonal gametocyte assays, selectivity assays on human cells and asexual parasites, followed by compound clustering, brought to the identification of 84 hits, half of which are gametocyte selective and half with comparable activity against sexual and asexual parasites. We validated seven chemotypes, three of which are, to the best of our knowledge, novel. These molecules are able to inhibit male gametocyte exflagellation and block parasite transmission through the Anopheles mosquito vector in a standard membrane feeding assay. This work shows that interrogating a wide and diverse chemical space, with a streamlined gametocyte HTS and hit validation funnel, holds promise for the identification of dual stage and gametocyte-selective compounds to be developed into new generation of transmission blocking drugs for malaria elimination.
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•Development and optimization of a “Direct-to-Biology” workflow, with synthesis and biological evaluation in plate.•Introduction of structural diversity through SNAr to get analogues ...with good chemical purity and reliable biological data.•Fast evaluation of a variety of structurally diverse fragments leading to nanomolar SHP2 allosteric inhibitors.
Tyrosine phosphatase SHP2 is a proto-oncogenic protein involved in cell growth and differentiation via diverse intracellular signaling pathways. With the scope of identifying new SHP2 allosteric inhibitors, we report here the development and optimization of a high-throughput “Direct-to-Biology” (D2B) workflow including the synthesis and the biological evaluation of the reaction crude, thus eliminating the need for purification. During this labor-saving procedure, the structural diversity was introduced through a SNAr reaction. A wide array of analogues with good chemical purity was generated, allowing the obtention of reliable biological data which validated this efficient technique. This approach enabled the fast evaluation of a variety of structurally diverse fragments leading to nanomolar SHP2 allosteric inhibitors and a new series bearing a novel bicyclo3.1.0hexane moiety.
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Boc-protected (3R,4S)-3,4-dihydroxypiperidine diol 1 is an enantiomerically pure, highly valuable synthetic intermediate. Herein we report the development of two scalable synthetic ...strategies avoiding the use of hazardous sodium azide, as well as the formation of azido intermediates. Starting from the readily available 2-deoxy-d-ribose, it was possible to produce a substantial amount of diol 1 following two distinct routes, without a significant decrease in performance when scaling up from 1- to 230-mmol.
Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is the first reported nonreceptor oncogenic tyrosine phosphatase connecting multiple signal transduction cascades and exerting ...immunoinhibitory function through the PD-1 checkpoint receptor. As part of a drug discovery program aimed at obtaining novel allosteric SHP2 inhibitors, a series of pyrazopyrazine derivatives bearing an original bicyclo3.1.0hexane basic moiety on the left-hand side region of the molecule were identified. We report herein the discovery process, the in vitro pharmacological profile, and the early developability features of compound 25, one of the most potent members of the series.
Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential ...immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo3.1.0hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.
Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction ...with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS.
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