Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer.

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both  ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). SUMMARY: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Benzimidazolone as potent chymase inhibitor: modulation of reactive metabolite formation in the hydrophobic (P1) region.

A new class of chymase inhibitor featuring a benzimidazolone core with an acid side chain and a P(1) hydrophobic moiety is described. Incubation of the lead compound with GSH resulted in the formation of a GSH conjugate on the benzothiophene P(1) moiety. Replacement of the benzothiophene with different heterocyclic systems such as indoles and benzoisothiazole is feasible. Among the P(1) replacements, benzoisothiazole prevents the formation of GSH conjugate and an in silico analysis of oxidative potentials agreed with the experimental outcome.

E7766, a Macrocycle-Bridged Stimulator of Interferon Genes (STING) Agonist with Potent Pan-Genotypic Activity.

A strategy for creating potent and pan-genotypic stimulator of interferon genes (STING) agonists is described. Locking a bioactive U-shaped conformation of cyclic dinucleotides by introducing a transannular macrocyclic bridge between the nucleic acid bases leads to a topologically novel macrocycle-bridged STING agonist (MBSA). In addition to substantially enhanced potency, the newly designed MBSAs, exemplified by clinical candidate E7766, exhibit broad pan-genotypic activity in all major human STING variants. E7766 is shown to have potent antitumor activity with long lasting immune memory response in a mouse liver metastatic tumor model. Two complementary stereoselective synthetic routes to E7766 are also described.

The discovery of thienopyridine analogues as potent IkappaB kinase beta inhibitors. Part II.

An SAR study that identified a series of thienopyridine-based potent IkappaB Kinase beta (IKKbeta) inhibitors is described. With focuses on the structural optimization at C4 and C6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C4, whereas polar groups with proper orientation at C6 efficiently enhance compound potency. The most potent analogues inhibit IKKbeta with IC50s as low as 40 nM, suppress LPS-induced TNF-alpha production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-kappaB reporter gene assay, demonstrating that they directly interfere with the NF-kappaB signaling pathway.

Estrogen Receptor Covalent Antagonists: The Best Is Yet to Come.

The development of tamoxifen and subsequent estrogen receptor alpha (ERα) antagonists represents a tremendous therapeutic breakthrough in the treatment of breast cancer. Despite the ability of ERα antagonists to increase survival rates, resistance to these therapies is an all-too-common occurrence. The majority of resistant tumors, including those with hotspot mutations in the ligand-binding domain of ERα, remain dependent on ERα signaling, indicating that either a more potent or novel class of antagonist could have clinical benefit. With this thought in mind, we developed a novel ERα antagonist that exhibits enhanced potency due to its ability to covalently target a unique cysteine in ER. This review describes the design of this antagonist, H3B-5942, and discusses opportunities for future improvements, which could reduce the risk of escape mutations to this therapeutic modality.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RCM macrocyclization made practical: an efficient synthesis of HCV protease inhibitor BILN 2061.

We report here that dramatic improvement of the key RCM reaction in the synthesis of HCV protease inhibitor BILN2061 can be achieved by N-substitution of the diene substrate with an electron-withdrawing group. Mechanistic studies using 1H NMR spectroscopy showed an unprecedented switch of the initiation sites and the correlation between such switch and the results of RCM, from the unmodified to the modified substrates. We also provided theoretical evidence that such modification may also increase the thermodynamic preference of the macrocyclic product over the diene substrate.

Synthesis and SAR studies of indole-based MK2 inhibitors.

Chemistry has been developed to specifically functionalize two structurally similar classes of indole-based MK2 inhibitors at positions prompted by a combination of X-ray crystallographic and computer assisted drug design. A gain in molecular potency was obtained by introducing aminomethyl groups to the lactam rings of 6-arylcarbamoyl-tetrahydro-beta-carbolinone and 6-arylcarbamoyl-dihydropyrazino[1,2-a]indolone MK2 inhibitors. In addition, improvements in molecular potency were achieved by expansion of the lactam from a 6- to 7-membered ring leading to 7-arylcarbamoyl-tetrahydro-[1,4]diazepino[1,2-a]indolones.

Celecoxib is a substrate of CYP2D6: Impact on celecoxib metabolism in individuals with CYP2C9*3 variants.

Celecoxib was characterized as a substrate of human cytochrome P450 (CYP) 2D6 in vitro. In recombinant CYP2D6, celecoxib hydroxylation showed atypical substrate inhibition kinetics with apparent Km, Ki, and Vmax of 67.2 µM, 12.6 µM, and 1.33 µM/min, respectively. In human liver microsomes (HLMs), a concentration-dependent inhibition of celecoxib hydroxylation by quinidine was observed after CYP2C9 and CYP3A4 were inhibited. In individual HLMs with variable CYP2D6 activities, a significant correlation was observed between celecoxib hydroxylation and CYP2D6-selective dextromethorphan O-demethylation when CYP2C9 and CYP3A4 activities were suppressed (r = 0.97, P < 0.0001). Molecular modeling showed two predominant docking modes of celecoxib with CYP2D6, resulting in either a substrate or an inhibitor. A second allosteric binding antechamber, which stabilized the inhibition mode, was revealed. Modeling results were consistent with the observed substrate inhibition kinetics. Using HLMs from individual donors, the relative contribution of CYP2D6 to celecoxib metabolism was found to be highly variable and dependent on CYP2C9 genotypes, ranging from no contribution in extensive metabolizers with CYP2C9*1*1 genotype to approximately 30% in slow metabolizers with allelic variants CYP2C9*1*3 and CYP2C9*3*3. These results demonstrate that celecoxib may become a potential victim of CYP2D6-associated drug-drug interactions, particularly in individuals with reduced CYP2C9 activity.

Discovery of Selective Estrogen Receptor Covalent Antagonists for the Treatment of ERαWT and ERαMUT Breast Cancer.

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Discovery and optimization of p38 inhibitors via computer-assisted drug design.

Integration of computational methods, X-ray crystallography, and structure-activity relationships will be disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.

H3B-6527 Is a Potent and Selective Inhibitor of FGFR4 in FGF19-Driven Hepatocellular Carcinoma.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999-7013. ©2017 AACR.

Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic "immuno-oncogenes" that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.

Synthesis, SAR, and series evolution of novel oxadiazole-containing 5-lipoxygenase activating protein inhibitors: discovery of 2-[4-(3-{(r)-1-[4-(2-amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915).

The synthesis, structure-activity relationship (SAR), and evolution of a novel series of oxadiazole-containing 5-lipoxygenase-activating protein (FLAP) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (IC50 < 100 nM). Optimization of binding and functional potencies, as well as physicochemical properties resulted in the identification of compound 69 (BI 665915) that demonstrated an excellent cross-species drug metabolism and pharmacokinetics (DMPK) profile and was predicted to have low human clearance. In addition, 69 was predicted to have a low risk for potential drug-drug interactions due to its cytochrome P450 3A4 profile. In a murine ex vivo whole blood study, 69 demonstrated a linear dose-exposure relationship and a dose-dependent inhibition of LTB4 production.

Design and synthesis of dipeptide nitriles as reversible and potent Cathepsin S inhibitors.

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.

Total synthesis of 6-deoxypladienolide D and Assessment of Splicing Inhibitory Activity in a Mutant SF3B1 cancer cell line.

A total synthesis of the natural product 6-deoxypladienolide D (1) has been achieved. Two noteworthy attributes of the synthesis are (1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and (2) the development of a scalable and cost-effective synthetic route to support drug discovery efforts. 6-Deoxypladienolide D (1) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing.

Discovery of potent, selective chymase inhibitors via fragment linking strategies.

Chymase plays an important and diverse role in the homeostasis of a number of cardiovascular processes. Herein, we describe the identification of potent, selective chymase inhibitors, developed using fragment-based, structure-guided linking and optimization techniques. High-concentration biophysical screening methods followed by high-throughput crystallography identified an oxindole fragment bound to the S1 pocket of the protein exhibiting a novel interaction pattern hitherto not observed in chymase inhibitors. X-ray crystallographic structures were used to guide the elaboration/linking of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over cathepsin G and that mitigated a number of liabilities associated with poor physicochemical properties of the series it was derived from.

Efficient chiral monophosphorus ligands for asymmetric Suzuki-Miyaura coupling reactions.

A series of novel P-chiral monophosphorus ligands exhibit efficiency in asymmetric Suzuki-Miyaura coupling reactions, enabling the construction of an array of chiral biaryl products in high yields and excellent enantioselectivities (up to 96% ee) under mild conditions. The carbonyl-benzooxazolidinone moiety in these chiral biaryl products allows facile derivatization for further synthetic applications. A computational study has revealed that a π-π interaction between the two coupling partners can enhance the enantioselectivity of the coupling reaction.

Torsion angle preference and energetics of small-molecule ligands bound to proteins.

Small organic molecules can assume conformations in the protein-bound state that are significantly different from those in solution. We have analyzed the conformations of 21 common torsion motifs of small molecules extracted from crystal structures of protein-ligand complexes and compared them with their torsion potentials calculated by an ab initio DFT method. We find a good correlation between the potential energy of the torsion motifs and their conformational distribution in the protein-bound state: The most probable conformations of the torsion motifs agree well with the calculated global energy minima, and the lowest torsion-energy state becomes increasingly dominant as the torsion barrier height increases. The torsion motifs can be divided into 3 groups based on torsion barrier heights: high (>4 kcal/mol), medium (2-4 kcal/mol), and low (<2 kcal/mol). The calculated torsion energy profiles are predictive for the most preferred bound conformation for the high and medium barrier groups, the latter group common in druglike molecules. In the high-barrier group of druglike ligands, >95% of conformational torsions occur in the energy region <4 kcal/mol. The conformations of the torsion motifs in the protein-bound state can be modeled by a Boltzmann distribution with a temperature factor much higher than room temperature. This high-temperature factor, derived by fitting the theoretical model to the experimentally observed conformation occurrence of torsions, can be interpreted as the perturbation that proteins inflict on the conformation of the bound ligand. Using this model, it is calculated that the average strain energy of a torsion motif in ligands bound to proteins is approximately 0.6 kcal/mol, a result which can be related to the lower binding efficiency of larger ligands with more rotatable bonds. The above results indicate that torsion potentials play an important role in dictating ligand conformations in both the free and the bound states.

Identification of a novel class of succinyl-nitrile-based Cathepsin S inhibitors.

The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the alpha-carbon of the P1 residue.