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.

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.

Development and characterization of rat duodenal organoids for ADME and toxicology applications.

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.

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.

Identification of novel azaindazole CCR1 antagonist clinical candidates.

Exploring various cyclization strategies, using a submicromolar pyrazole HTS screening hit 6 as a starting point, a novel indazole based CCR1 antagonist core was discovered. This report presents the design and SAR of CCR1 indazole and azaindazole antagonists leading to the identification of three development compounds, including 19e that was advanced to early clinical trials.

Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis.

There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies.

Dephosphorylation of Rb (Thr-821) in response to cell stress.

The retinoblastoma tumor suppressor Rb is regulated by reversible phosphorylation that is dependent upon cyclin-dependent kinase (CDK) and protein phosphatase type 1 (PP1) activity in replicating cells. Hyperphosphorylated Rb allows cells to proliferate, whereas the hypophosphorylated isoform of Rb inhibits proliferation. Of the many phosphorylation sites of Rb, there is functional information available for a very few. In this report, we show that threonine-821 (Thr-821) of Rb is dephosphorylated earlier than other phosphorylation sites when cells are grown under hypoxic conditions which leads to Rb activation and G(1) arrest. This finding is interesting because Thr-821 of Rb remains phosphorylated throughout the cell division cycle in replicating cells. We hypothesized that the phosphorylation state of Thr-821 of Rb may depend on cellular stress. We report in this study that, when nontransformed CV1 epithelial cells and Hs578T breast cancer cells are treated with the chemotherapeutic agent cytosine arabinoside (Ara-C), Thr-821 of Rb is rapidly dephosphorylated concomitant with dissociation of the PP1 regulatory subunit PNUTS (phosphatase nuclear targeting subunit) from PP1 enzyme. These data are consistent with the concept that differential regulation of Rb-directed phosphatase activity exists when cells are progressing through the cell cycle compared to that observed when cells are under stress.