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.

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.

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

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.

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.

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.

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.

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.

RORγt is the master regulator of the IL-23/IL-17 axis, a pathway that is clinically validated for the treatment of various immunological disorders. Over the last few years, our group has reported different chemotypes that potently act as inverse agonists of RORγt. One of them, the tricyclic pyrrolidine chemotype, has demonstrated biologic-like preclinical efficacy and has led to our clinical candidate BMS-986251. In this letter, we discuss the invention of an annulation reaction which enabled the synthesis of a tricyclic exocyclic amide chemotype and the identification of compounds with RORγt inverse agonist activity. Preliminary structure activity relationships are disclosed.

TYK2 is a nonreceptor tyrosine kinase involved in adaptive and innate immune responses. A deactivating coding variant has previously been shown to prevent receptor-stimulated activation of this kinase and provides high protection from several common autoimmune diseases but without immunodeficiency. An agent that recapitulates the phenotype of this deactivating coding variant may therefore represent an important advancement in the treatment of autoimmunity. BMS-986165 is a potent oral agent that similarly blocks receptor-stimulated activation of TYK2 allosterically and with high selectivity and potency afforded through optimized binding to a regulatory domain of the protein. Signaling and functional responses in human TH17, TH1, B cells, and myeloid cells integral to autoimmunity were blocked by BMS-986165, both in vitro and in vivo in a phase 1 clinical trial. BMS-986165 demonstrated robust efficacy, consistent with blockade of multiple autoimmune pathways, in murine models of lupus nephritis and inflammatory bowel disease, supporting its therapeutic potential for multiple immune-mediated diseases.

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.

Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors.

Abstract Oral agents targeting Janus-associated kinases (JAKs) are among the most promising new agents in clinical development. To better understand the relationship between JAK inhibition and clinical outcome, compounds targeting JAKs are required to be analyzed in peripheral whole blood. To date, this type of analysis has been low throughput and is associated with high cost. Here, we developed a robust 384-well flow based assay approach to screen small molecules for inhibition of JAK/STAT signaling in whole blood using high-throughput flow cytometry. The platform provides a highly sensitive analysis of signaling events in low volumes of blood facilitating a robust target engagement assay to measure inhibition of proximal signaling by selective JAK inhibitors. Further, automation technologies and processes optimization were established to overcome bottlenecks in sample integrity, handling and multiparametric data analysis without affecting assay performance. The result was dramatically increased sample throughput when compared to conventional manual flow cytometric approaches enabling the identification of novel selective JAK/STAT inhibitors enabling the investigation of IO and Immunology targets earlier in drug discovery. Citation Format: mark fereshteh, Litao Zhang, Xin Li, Sha Li, Yi Fan, Gary Schieven. HT flow cytometry platform enabling measurement of immune signaling in whole blood and T-cells for drug discovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 355.

Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification, and optimization, as well as determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency. Here we describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines were selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance were developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) was applied to analyze the data and generate data reports. Using tool compounds, we have shown that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, we have demonstrated that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, to drive cellular structure-activity relationships, and to profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization.

ObjectivesDaphne odora var. marginata (D. marginata), an aiophyllus arbuscular plant, is one of the traditional Chinese medicines used to treat rheumatoid arthritis. This study investigated the therapeutic effects and mechanisms of daphnetin, an active monomer ingredient derived from D. marginata, on collagen-induced arthritis (CIA) in rats.MethodsThe effects of daphnetin on joint diseases were assessed by hematoxylin and eosin staining and radiographic and transmission electron microscopy. The protein and mRNA expression levels of T helper (Th)1/Th2/Th17-type cytokines in the spleen were determined by flow cytometry and quantitative real-time PCR.ResultsOur results showed that daphnetin significantly reduced paw swelling and was nontoxic in vivo at the tested doses. Synovial hyperplasia, joint destruction and chondrocyte degeneration in CIA rats were suppressed by daphnetin. Daphnetin treatment also reduced the levels of Th1/Th2/Th17 type cytokines in spleen lymphocytes in CIA rats. Moreover, the expression of Foxp3, which can down-regulate the activity of Th17 cells, was significantly increased in the daphnetin-treated groups.ConclusionsThese results suggest that daphnetin may have therapeutic effects in down-regulating Th17-type responses in CIA rats. The beneficial effects of daphnetin on CIA may be related to its inhibition of Th17 cell priming and activation.

Daphnetin extracted from Daphne odora Var. marginata contains coumarin compounds, which possess properties of analgesic and anti-inflammatory effects. In this study, we investigated the therapeutic effect of daphnetin on anti-arthritis and its role on the balance of Tregs and Th17, using a collagen-induced arthritis rat model. Collagen-induced arthritis rats were treated with daphnetin for 21 days. The therapeutic effects of daphnetin were evaluated by clinical symptoms and histopathology. The levels of Th17-, Treg-, Th2-, Th1-type cytokines in serum were determined by ELISA. The expression levels of related receptors RORγt, NF-κB, Foxp3 and CD77 in joint tissues were detected by immunohistochemistry. Our results showed that administration of daphnetin significantly alleviated the severity of the arthritis, as evidenced by the reduction of arthritis scores, suppression of the infiltration of inflammatory cells and prevention of synovial hyperplasia, thereby resulting in the joint destruction in the arthritis rats. Additionally, daphnetin treatment reduced the serum level of Th17-, Th2- and Th1-type in collagen-induced arthritis rats. Correspondingly, the expression of RORγt, NF-κB and CD77 in joint tissue of collagen-induced arthritis rats was remarkably decreased, while the expression of Foxp3 and IL-10 was remarkably increased after being administered with daphnetin. Collectively, this study demonstrated that administration of daphnetin attenuated the clinical symptoms and pathological destruction of arthritis joints. The therapeutic effects were associated with the up-regulation of Tregs, down-regulation of Th17-, Th2- and Th1-type cell responses. The results provide novel evidence that daphnetin has therapeutic effects on autoimmune arthritis through modulating the balance of Tregs and Th17.