T-type calcium channel blockers: spiro-piperidine azetidines and azetidinones-optimization, design and synthesis.

A series of spiro-azetidines and azetidinones has been evaluated as novel blockers of the T-type calcium channel (Ca(V)3.2) which is a new therapeutic target for the potential treatment of both inflammatory and neuropathic pain. Confirmation and optimization of the potency, selectivity and DMPK properties of leads will be described.

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.

Spiro-piperidine azetidinones as potent TRPV1 antagonists.

A series of spiro-piperidine azetidinone were synthesized and evaluated as potential TRPV1 antagonists. An important issue of plasma stability was investigated and resolved. Further focused SAR study lead to the discovery of a potent antagonist with good oral pharmacokinetic profile in rat.

Characterization of adenosine receptors in the human bladder carcinoma T24 cell line.

The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Ca2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A1, A2A and A2B receptors but not for the adenosine A3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium ([Ca2+]i) with the following order of potency and EC50 values: 5'-N-Ethylcarboxamidoadenosine (NECA) (1153+/-214)>5'-(N-Cyclopropyl)carboxamidoadenosine (CPCA) (1436+/-186)>adenosine (4823+/-932). This rank order of potency is typical of adenosine A2B receptors. In addition, select adenosine receptor antagonists N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706), 8-[4-[((4-Cyano[2,6-]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A2B receptors. The select adenosine A2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that [Ca2+]i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A2B receptor in the T24 cell line.

Inhibition of RORγT Skews TCRα Gene Rearrangement and Limits T Cell Repertoire Diversity.

Recent studies have elucidated the molecular mechanism of RORγT transcriptional regulation of Th17 differentiation and function. RORγT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP) thymocytes. While RORγ antagonists are currently being developed to treat autoimmunity, it remains unclear how RORγT inhibition may impact thymocyte development. In this study, we show that in addition to regulating DP thymocytes survival, RORγT also controls genes that regulate thymocyte migration, proliferation, and T cell receptor (TCR)α selection. Strikingly, pharmacological inhibition of RORγ skews TCRα gene rearrangement, limits T cell repertoire diversity, and inhibits development of autoimmune encephalomyelitis. Thus, targeting RORγT not only inhibits Th17 cell development and function but also fundamentally alters thymic-emigrant recognition of self and foreign antigens. The analysis of RORγ inhibitors has allowed us to gain a broader perspective of the diverse function of RORγT and its impact on T cell biology.

Cloning and functional characterization of dog transient receptor potential vanilloid receptor-1 (TRPV1).

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.

Identification of an allosteric binding site for RORγt inhibition.

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

Cloning and pharmacological characterization of mouse TRPV1.

The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX) = arvanil > capsaicin = olvanil > OLDA > PPAHV) (EC50 values of 0.15+/-0.04 nM, 0.27+/-0.07 nM, 9.1+/-1.2 nM, 3.7+/-0.3 nM, 258+/-105 nM, and 667+/-151 nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC50 (I-RTX) = 0.35+/-0.12 nM, 1.9+/-0.7 nM, and 0.80+/-0.68 nM, IC50 (BCTC) = 1.3+/-0.36 nM, 0.59+/-0.16 nM, and 0.37+/-0.15 nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC50 of 1426+/-316 nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.

Biased ligand modulation of seven transmembrane receptors (7TMRs): functional implications for drug discovery.

Seven transmembrane receptors (7TMRs), also known as G-protein-coupled receptors (GPCRs), have proven to be valuable targets for the development of therapeutics. The expansion of our understanding of 7TMR downstream signaling pathways beyond G-proteins has broadened our appreciation of the versatility of these cell surface receptors. In particular, the increased awareness of 7TMR engagement of ß-arrestin signaling has opened up additional avenues for drug discovery. 7TMRs can adopt different conformations and in response to various ligands can lead to a bias in downstream signaling mechanisms when comparing the overall efficacy between G-protein and ß-arrestin dependent pathways. In 2012, we organized a session at the Spring National Meeting of the American Chemical Society on biased signaling in 7TMRs.1-4 Building on that experience, we provide in this Miniperspective some examples that exemplify developments in the area of biased 7TMR signaling and highlight some cautionary notes as well as some of the exciting opportunities for drug discovery.

Anti-inflammatory actions of Chemoattractant Receptor-homologous molecule expressed on Th2 by the antagonist MK-7246 in a novel rat model of Alternaria alternata elicited pulmonary inflammation.

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats. Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats. We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.

TRPV1 antagonists as potential antitussive agents.

Cough is an important defensive pulmonary reflex that removes irritants, fluids, or foreign materials from the airways. However, when cough is exceptionally intense or when it is chronic and/or nonproductive it may require pharmacologic suppression. For many patients, antitussive therapies consist of OTC products with inconsequential efficacies. On the other hand, the prescription antitussive market is dominated by older opioid drugs such as codeine. Unfortunately, "codeine-like" drugs suppress cough at equivalent doses that also often produce significant ancillary liabilities such as GI constipation, sedation, and respiratory depression. Thus, the discovery of a novel and effective antitussive drug with an improved side effect profile relative to codeine would fulfill an unmet clinical need in the treatment of cough. Afferent pulmonary nerves are endowed with a multitude of potential receptor targets, including TRPV1, that could act to attenuate cough. The evidence linking TRPV1 to cough is convincing. TRPV1 receptors are found on sensory respiratory nerves that are important in the generation of the cough reflex. Isolated pulmonary vagal afferent nerves are responsive to TRPV1 stimulation. In vivo, TRPV1 agonists such as capsaicin elicit cough when aerosolized and delivered to the lungs. Pertinent to the debate on the potential use of TRPV1 antagonist as antitussive agents are the observations that airway afferent nerves become hypersensitive in diseased and inflamed lungs. For example, the sensitivity of capsaicin-induced cough responses following upper respiratory tract infection and in airway inflammatory diseases such as asthma and COPD is increased relative to that of control responses. Indeed, we have demonstrated that TRPV1 antagonism can attenuate antigen-induced cough in the allergic guinea pig. However, it remains to be determined if the emerging pharmacologic profile of TRPV1 antagonists will translate into a novel human antitussive drug. Current efforts in clinical validation of TRPV1 antagonists revolve around various pain indications; therefore, clinical evaluation of TRPV1 antagonists as antitussive agents will have to await those outcomes.

TRPV1 antagonists attenuate antigen-provoked cough in ovalbumin sensitized guinea pigs.

We examined the molecular pharmacology and in vivo effects of a TRPV1 receptor antagonist, N-(4-Tertiarybutylphenyl)-4(3-cholorphyridin-2-yl)-tetrahydro-pyrazine1(2H) - carboxamide (BCTC) on the guinea pig TRPV1 cation channel. BCTC antagonized capsaicin-induced activation and PMA-mediated activation of guinea pig TRPV1 with IC50 values of 12.2 +/- 5.2 nM, and 0.85 +/- 0.10 nM, respectively. In addition, BCTC (100 nM) completely blocked the ability of heterologously expressed gpTRPV1 to respond to decreases in pH. Thus, BCTC is able to block polymodal activation of gpTRPV1. Furthermore, in nodose ganglia cells, capsaicin induced Ca2+ influx through TRPV1 channel was inhibited via BCTC in a concentration dependent manner. In in vivo studies capsaicin (10 - 300 muM) delivered by aerosol to the pulmonary system of non-sensitized guinea pigs produced an increase in cough frequency. In these studies, the tussigenic effects of capsaicin (300 muM) were blocked in a dose dependent fashion when BCTC (0.01-3.0 mg/kg, i.p.) was administered 30 minutes before challenge. The high dose of BCTC (3.0 mg/kg, i.p) produced a maximum inhibition of capsaicin-induced cough of 65%. We also studied the effects of BCTC (0.03 and 3.0) when administered 60 minutes before capsaicin. Under these conditions, BCTC (3.0 mg/kg, i.p) produced a maximum decrease in capsaicin-induced cough of 31%. In ovalbumin passively sensitized guinea pigs, we found that BCTC (1 and 3 mg/kg, i.p.) attenuated antigen ovalbumin (0.3%) cough responses by 27% and 60%, respectively. We conclude that TRPV1 channel activation may play role in cough mediated by antigen in sensitized guinea pigs. Our results supports increasing evidence that TRPV1 may play a role in the generation of the cough response.

Functional TRPV4 channels are expressed in human airway smooth muscle cells.

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.