Discovery of N-(Indazol-3-yl)piperidine-4-carboxylic Acids as RORγt Allosteric Inhibitors for Autoimmune Diseases.

The clinical success of anti-IL-17 monoclonal antibodies (i.e., Cosentyx and Taltz) has validated Th17 pathway modulation for the treatment of autoimmune diseases. The nuclear hormone receptor RORγt is a master regulator of Th17 cells and affects the production of a host of cytokines, including IL-17A, IL-17F, IL-22, IL-26, and GM-CSF. Substantial interest has been spurred across both academia and industry to seek small molecules suitable for RORγt inhibition. A variety of RORγt inhibitors have been reported in the past few years, the majority of which are orthosteric binders. Here we disclose the discovery and optimization of a class of inhibitors, which bind differently to an allosteric binding pocket. Starting from a weakly active hit 1, a tool compound 14 was quickly identified that demonstrated superior potency, selectivity, and off-target profile. Further optimization focused on improving metabolic stability. Replacing the benzoic acid moiety with piperidinyl carboxylate, modifying the 4-aza-indazole core in 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing.

Signaling of an allosteric, nanomolar potent, low molecular weight agonist for the follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) activates FSH receptors (FSHR) in granulosa cells to induce follicle differentiation, growth and estradiol production. FSH is used clinically to treat female infertility and is administered by injection. To increase patient convenience and compliance, compound homogeneity and composition, low molecular weight (LMW), orally bioavailable, FSHR agonists are now being developed to replace FSH. In this study, we present the signaling mechanisms of a newly developed LMW dihydropyridine agonist of the FSHR, Org 214444-0. Org 214444-0 is shown to be a stereoselective, nanomolar potent FSHR agonist and selective over the structurally related LHR and TSHR. Org 214444-0 is an allosteric agonist interacting with the transmembrane region of the FSHR. When co-incubated with FSH, Org 214444-0 augments FSH's potency in binding (6.5-fold) and adenylyl cyclase/cAMP activation (3.5-fold) in a concentration-dependent manner. Like FSH, Org 214444-0 induces FSHR internalization and is only marginally effective in stimulating phospholipase C. Moreover, Org 214444-0 stimulates cAMP and estradiol production in human granulosa cells in culture and supports the follicular phase in mature female rats. We conclude that Org 214444-0 is a bonafide FSHR agonist.

Complete inhibition of rhTSH-, Graves' disease IgG-, and M22-induced cAMP production in differentiated orbital fibroblasts by a low-molecular-weight TSHR antagonist.

The TSH receptor (TSHR) on orbital fibroblasts (OF) is a proposed target of the autoimmune attack in Graves' ophthalmopathy. In the present study, we tested whether the novel low-molecular-weight (LMW) TSHR antagonist Org-274179-0 inhibits cAMP production induced by rhTSH, Graves' disease IgG (GD-IgG), or M22 (a potent human monoclonal TSHR stimulating antibody) in cultured and differentiated OF from Graves' ophthalmopathy patients. cAMP production significantly increased after incubation either with 10 mU/ml rhTSH (3-fold; P ≤ 0.05), 1 mg/ml GD-IgG (2-fold; P ≤ 0.05), or 500 ng/ml M22 (5-fold; P ≤ 0.05). Incubation with the LMW TSHR antagonist dose dependently inhibited rhTSH, GD-IgG as well as the M22-induced cAMP production at nanomolar concentrations; complete blockade was affected at 10(-6) M. Our results suggest that GD-IgG- and M22-induced cAMP production in differentiated OF is exclusively mediated via the TSHR because it can be completely blocked by the LMW TSHR antagonist, Org 274179-0.

Mechanism of action of a nanomolar potent, allosteric antagonist of the thyroid-stimulating hormone receptor.

BACKGROUND AND PURPOSE: Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0. EXPERIMENTAL APPROACH: Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants. KEY RESULTS: Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO.

Multidimensional profiling of CSF1R screening hits and inhibitors: assessing cellular activity, target residence time, and selectivity in a higher throughput way.

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit k(off) rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J's pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.

Identification of substituted 6-amino-4-phenyltetrahydroquinoline derivatives: potent antagonists for the follicle-stimulating hormone receptor.

Substituted 6-amino-4-phenyl-tetrahydroquinoline derivatives are described that are antagonists for the G(s)-protein-coupled human follicle-stimulating hormone (FSH) receptor. These compounds show high antagonistic efficacy in vitro using a CHO cell line expressing the human FSH receptor. Antagonist 10 also showed a submicromolar IC(50) in a more physiologically relevant rat granulosa cell assay and was found to significantly inhibit follicle growth and ovulation in an ex vivo mouse model. This compound class may open the way toward a novel, nonsteroidal approach for contraception.