Structure-activity relationship study of central pyridine-derived TYK2 JH2 inhibitors: Optimization of the PK profile through C4' and C6 variations.

Efforts directed at improving potency and preparing structurally different TYK2 JH2 inhibitors from the first generation of compounds such as 1a led to the SAR study of new central pyridyl based analogs 2-4. The current SAR study resulted in the identification of 4h as a potent and selective TYK2 JH2 inhibitor with distinct structural differences from 1a. In this manuscript, the in vitro and in vivo profiles of 4h are described. The hWB IC50 of 4h was shown as 41 nM with 94% bioavailability in the mouse PK study.

Discovery of BMS-986339, a Pharmacologically Differentiated Farnesoid X Receptor Agonist for the Treatment of Nonalcoholic Steatohepatitis.

While several farnesoid X receptor (FXR) agonists under clinical investigation for the treatment of nonalcoholic steatohepatitis (NASH) have shown beneficial effects, adverse effects such as pruritus and elevation of plasma lipids have limited their clinical efficacy and approvability. Herein, we report the discovery and preclinical evaluation of compound 32 (BMS-986339), a nonbile acid FXR agonist with a pharmacologically distinct profile relative to our previously reported agonist BMS-986318. Compound 32 exhibited potent in vitro and in vivo activation of FXR, albeit with a context-dependent profile that resulted in tissue-selective effects in vivo. To our knowledge, this is the first report that demonstrates differential induction of Fgf15 in the liver and ileum by FXR agonists in vivo. Compound 32 demonstrated robust antifibrotic efficacy despite reduced activation of certain genes in the liver, suggesting that the additional pharmacology of BMS-986318 does not further benefit efficacy, possibly presenting an opportunity for reduced adverse effects. Further evaluation in humans is warranted to validate this hypothesis.

Discovery of Non-Nucleotide Small-Molecule STING Agonists via Chemotype Hybridization.

The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.

Signal transfer in human protein tyrosine phosphatase PTP1B from allosteric inhibitor P00058.

Protein tyrosine phosphatases constitute a family of cytosolic and receptor-like signal transducing enzymes that catalyze the hydrolysis of phospho-tyrosine residues of phosphorylated proteins. PTP1B, encoded by PTPN1, is a key negative regulator of insulin and leptin receptor signaling, linking it to two widespread diseases: type 2 diabetes mellitus and obesity. Here, we present crystal structures of the PTP1B apo-enzyme and a complex with a newly identified allosteric inhibitor, 2-(2,5-dimethyl-pyrrol-1-yl)-5-hydroxy-benzoic acid, designated as P00058. The inhibitor binding site is located about 18 Å away from the active center. However, the inhibitor causes significant re-arrangements in the active center of enzyme: residues 45-50 of catalytic Tyr-loop are shifted at their Cα-atom positions by 2.6 to 5.8 Å. We have identified an event of allosteric signal transfer from the inhibitor to the catalytic area using molecular dynamic simulation. Analyzing change of complex structure along the fluctuation trajectory we have found the large Cα-atom shifts in external strand, residues 25-40, which occur at the same time with the shifts in adjacent catalytic p-Tyr-loop. Coming of the signal to this loop arises due to dynamic fluctuation of protein structure at about 4.0 nanoseconds after the inhibitor takes up its space. Communicated by Ramaswamy H. Sarma.

Azatricyclic Inverse Agonists of RORγt That Demonstrate Efficacy in Models of Rheumatoid Arthritis and Psoriasis.

Structure-activity relationship studies directed toward the replacement of the fused phenyl ring of the lead hexahydrobenzoindole RORγt inverse agonist series represented by 1 with heterocyclic moieties led to the identification of three novel aza analogs 5-7. The hexahydropyrrolo[3,2-f]quinoline series 5 (X = N, Y = Z=CH) showed potency and metabolic stability comparable to series 1 but with improved in vitro membrane permeability and serum free fraction. This structural modification was applied to the hexahydrocyclopentanaphthalene series 3, culminating in the discovery of 8e as a potent and selective RORγt inverse agonist with an excellent in vitro profile, good pharmacokinetic properties, and biologic-like in vivo efficacy in preclinical models of rheumatoid arthritis and psoriasis.

Tricyclic-Carbocyclic RORγt Inverse Agonists-Discovery of BMS-986313.

SAR efforts directed at identifying RORγt inverse agonists structurally different from our clinical compound 1 (BMS-986251) led to tricyclic-carbocyclic analogues represented by 3-7 and culminated in the identification of 3d (BMS-986313), with structural differences distinct from 1. The X-ray co-crystal structure of 3d with the ligand binding domain of RORγt revealed several key interactions, which are different from 1. The in vitro and in vivo PK profiles of 3d are described. In addition, we demonstrate robust efficacy of 3d in two preclinical models of psoriasis-the IMQ-induced skin lesion model and the IL-23-induced acanthosis model. The efficacy seen with 3d in these models is comparable to the results observed with 1.

Substituted diaryl ether compounds as retinoic acid-related orphan Receptor-γt (RORγt) agonists.

The discovery of a series of substituted diarylether compounds as retinoic acid related orphan receptor γt (RORγt) agonists is described. Compound 1 was identified from deck mining as a RORγt agonist. Hit-to-lead optimization led to the identification of lead compound 5, which possesses improved potency (10x). Extensive SAR exploration led to the identification of a potent and selective compound 22, that demonstrated an improved pharmacokinetic profile and a dose-dependent pharmacodynamic response. However, when dosed in a MC38 syngeneic tumor model, no evidence of efficacy was observed. ©2020 Elsevier Science Ltd. All rights reserved.

Using yeast surface display to engineer a soluble and crystallizable construct of hematopoietic progenitor kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular kinase that plays an important role in modulating tumor immune response and thus is an attractive target for drug discovery. Crystallization of the wild-type HPK1 kinase domain has been hampered by poor expression in recombinant systems and poor solubility. In this study, yeast surface display was applied to a library of HPK1 kinase-domain variants in order to select variants with an improved expression level and solubility. The HPK1 variant with the most improved properties contained two mutations, crystallized readily in complex with several small-molecule inhibitors and provided valuable insight to guide structure-based drug design. This work exemplifies the benefit of yeast surface display towards engineering crystallizable proteins and thus enabling structure-based drug discovery.

Discovery of BMS-986202: A Clinical Tyk2 Inhibitor that Binds to Tyk2 JH2.

A search for structurally diversified Tyk2 JH2 ligands from 6 (BMS-986165), a pyridazine carboxamide-derived Tyk2 JH2 ligand as a clinical Tyk2 inhibitor currently in late development for the treatment of psoriasis, began with a survey of six-membered heteroaryl groups in place of the N-methyl triazolyl moiety in 6. The X-ray co-crystal structure of an early lead (12) revealed a potential new binding pocket. Exploration of the new pocket resulted in two frontrunners for a clinical candidate. The potential hydrogen bonding interaction with Thr599 in the pocket was achieved with a tertiary amide moiety, confirmed by the X-ray co-crystal structure of 29. When the diversity search was extended to nicotinamides, a single fluorine atom addition was found to significantly enhance the permeability, which directly led to the discovery of 7 (BMS-986202) as a clinical Tyk2 inhibitor that binds to Tyk2 JH2. The preclinical studies of 7, including efficacy studies in mouse models of IL-23-driven acanthosis, anti-CD40-induced colitis, and spontaneous lupus, will also be presented.

Novel Tricyclic Pyroglutamide Derivatives as Potent RORγt Inverse Agonists Identified using a Virtual Screening Approach.

Employing a virtual screening approach, we identified the pyroglutamide moiety as a nonacid replacement for the cyclohexanecarboxylic acid group which, when coupled to our previously reported conformationally locked tricyclic core, provided potent and selective RORγt inverse agonists. Structure-activity relationship optimization of the pyroglutamide moiety led to the identification of compound 18 as a potent and selective RORγt inverse agonist, albeit with poor aqueous solubility. We took advantage of the tertiary carbinol group in 18 to synthesize a phosphate prodrug, which provided good solubility, excellent exposures in mouse PK studies, and significant efficacy in a mouse model of psoriasis.

Tricyclic sulfones as potent, selective and efficacious RORγt inverse agonists - Exploring C6 and C8 SAR using late-stage functionalization.

In order to rapidly develop C6 and C8 SAR of our reported tricyclic sulfone series of RORγt inverse agonists, a late-stage bromination was employed. Although not regioselective, the bromination protocol allowed us to explore new substitution patterns/vectors that otherwise would have to be incorporated at the very beginning of the synthesis. Based on the SAR obtained from this exercise, compound 15 bearing a C8 fluorine was developed as a very potent and selective RORγt inverse agonist. This analog's in vitro profile, pharmacokinetic (PK) data and efficacy in an IL-23 induced mouse acanthosis model will be discussed.

Discovery of 2,6-difluorobenzyl ether series of phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfones as surprisingly potent, selective and orally bioavailable RORγt inverse agonists.

In an effort to discover oral inverse agonists of RORγt to treat inflammatory diseases, a new 2,6-difluorobenzyl ether series of cyclopentyl sulfones were found to be surprisingly more potent than the corresponding alcohol derivatives. When combined with a more optimized phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfone template, the 2,6-difluorobenzyl ethers yielded a set of very potent RORγt inverse agonists (e.g., compound 26, RORγt Gal4 EC50 11 nM) that are highly selective against PXR, LXRα and LXRß. After optimizing for stability in human and mouse liver microsomes, compounds 29 and 38 were evaluated in vivo and found to have good oral bioavailability (56% and 101%, respectively) in mice. X-ray co-crystal structure of compound 27 in RORγt revealed that the bulky benzyl ether group causes helix 11 of the protein to partially uncoil to create a new, enlarged binding site, which nicely accommodates the benzyl ether moiety, leading to net potency gain.

Discovery of BMS-986251: A Clinically Viable, Potent, and Selective RORγt Inverse Agonist.

Novel tricyclic analogues were designed, synthesized, and evaluated as RORγt inverse agonists. Several of these compounds were potent in an IL-17 human whole blood assay and exhibited excellent oral bioavailability in mouse pharmacokinetic studies. This led to the identification of compound 5, which displayed dose-dependent inhibition of IL-17F production in a mouse IL-2/IL-23 stimulated pharmacodynamic model. In addition, compound 5 was studied in mouse acanthosis and imiquimod-induced models of skin inflammation, where it demonstrated robust efficacy comparable to a positive control. As a result of this excellent overall profile, compound 5 (BMS-986251) was selected as a clinically viable developmental candidate.

Substituted benzyloxytricyclic compounds as retinoic acid-related orphan receptor gamma t (RORγt) agonists.

Substituted benzyloxy aryl compound 2 was identified as an RORγt agonist. Structure based drug design efforts resulted in a potent and selective tricyclic compound 19 which, when administered orally in an MC38 mouse tumor model, demonstrated a desired pharmacokinetic profile as well as a dose-dependent pharmacodynamic response. However, no perceptible efficacy was observed in this tumor model at the doses investigated.

Discovery of BMS-986260, a Potent, Selective, and Orally Bioavailable TGFßR1 Inhibitor as an Immuno-oncology Agent.

Novel imidazole-based TGFßR1 inhibitors were identified and optimized for potency, selectivity, and pharmacokinetic and physicochemical characteristics. Herein, we report the discovery, optimization, and evaluation of a potent, selective, and orally bioavailable TGFßR1 inhibitor, 10 (BMS-986260). This compound demonstrated functional activity in multiple TGFß-dependent cellular assays, excellent kinome selectivity, favorable pharmacokinetic properties, and curative in vivo efficacy in combination with anti-PD-1 antibody in murine colorectal cancer (CRC) models. Since daily dosing of TGFßR1 inhibitors is known to cause class-based cardiovascular (CV) toxicities in preclinical species, a dosing holiday schedule in the anti-PD-1 combination efficacy studies was explored. An intermittent dosing regimen of 3 days on and 4 days off allowed mitigation of CV toxicities in one month dog and rat toxicology studies and also provided similar efficacy as once daily dosing.

Rationally Designed, Conformationally Constrained Inverse Agonists of RORγt-Identification of a Potent, Selective Series with Biologic-Like in Vivo Efficacy.

RORγt is an important nuclear receptor that regulates the production of several pro-inflammatory cytokines such as IL-17 and IL-22. As a result, RORγt has been identified as a potential target for the treatment of various immunological disorders such as psoriasis, psoriatic arthritis, and inflammatory bowel diseases. Structure and computer-assisted drug design led to the identification of a novel series of tricyclic RORγt inverse agonists with significantly improved in vitro activity in the reporter (Gal4) and human whole blood assays compared to our previous chemotype. Through careful structure activity relationship, several potent and selective RORγt inverse agonists have been identified. Pharmacokinetic studies allowed the identification of the lead molecule 32 with a low peak-to-trough ratio. This molecule showed excellent activity in an IL-2/IL-23-induced mouse pharmacodynamic study and demonstrated biologic-like efficacy in an IL-23-induced preclinical model of psoriasis.

Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Identification of potent, selective and orally bioavailable phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfone analogues as RORγt inverse agonists.

An X-ray crystal structure of one of our previously discovered RORγt inverse agonists bound to the RORγt ligand binding domain revealed that the cyclohexane carboxylic acid group of compound 2 plays a significant role in RORγt binding, forming four hydrogen bonding and ionic interactions with RORγt. SAR studies centered around the cyclohexane carboxylic acid group led to identification of several structurally diverse and more potent compounds, including new carboxylic acid analogues 7 and 20, and cyclic sulfone analogues 34 and 37. Notably, compounds 7 and 20 were found to maintain the desirable pharmacokinetic profile of 2.

Structure-based Discovery of Phenyl (3-Phenylpyrrolidin-3-yl)sulfones as Selective, Orally Active RORγt Inverse Agonists.

A new phenyl (3-phenylpyrrolidin-3-yl)sulfone series of RORγt inverse agonists was discovered utilizing the binding conformation of previously reported bicyclic sulfonamide 1. Through a combination of structure-based design and structure-activity relationship studies, a polar set of amides at N1-position of the pyrrolidine ring and perfluoroisopropyl group at para-position of the 3-phenyl group were identified as critical structural elements to achieve high selectivity against PXR, LXRα, and LXRß. Further optimization led to the discovery of (1R,4r)-4-((R)-3-((4-fluorophenyl)sulfonyl)-3-(4-(perfluoropropan-2-yl)phenyl)pyrrolidine-1-carbonyl)cyclohexane-1-carboxylic acid (26), which displayed excellent selectivity, desirable liability and pharmacokinetic properties in vitro, and a good pharmacokinetic profile in mouse. Oral administration of 26 demonstrated dose-dependent inhibition of IL-17 production in a mouse IL-2/IL-23-induced pharmacodynamic model and biologic-like efficacy in an IL-23-induced mouse acanthosis model.

Discovery of 4-Azaindole Inhibitors of TGFßRI as Immuno-oncology Agents.

The multifunctional cytokine TGFß plays a central role in regulating antitumor immunity. It has been postulated that inhibition of TGFß signaling in concert with checkpoint blockade will provide improved and durable immune response against tumors. Herein, we describe a novel series of 4-azaindole TGFß receptor kinase inhibitors with excellent selectivity for TGFß receptor 1 kinase. The combination of compound 3f and an antimouse-PD-1 antibody demonstrated significantly improved antitumor efficacy compared to either treatment alone in a murine tumor model.