Linda N. Casillas

N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a Novel and Potent Transient Receptor Potential Vanilloid 4 Channel Agonist Induces Urinary Bladder Contraction and Hyperactivity: Part I

Abstract The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2+ influx in mouse and human TRPV4 expressing HEK cells (EC 50 values of 18 and 2.1 nM, respectively), and evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α−PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4 +/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 -/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4−/− bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4−/− bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4α-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4−/− mice revealed an enhanced bladder capacity in the TRPV4−/− mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4−/− mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

GSK1016790A, a Novel and Potent TRPV4 Channel Agonist Induces Urinary Bladder Contraction and Hyperactivity: Part I

Abstract The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2+ influx in mouse and human TRPV4 expressing HEK cells (EC 50 values of 18 and 2.1 nM, respectively), and evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α−PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4 +/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 -/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4−/− bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4−/− bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4α-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4−/− mice revealed an enhanced bladder capacity in the TRPV4−/− mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4−/− mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Systemic Activation of the Transient Receptor Potential Vanilloid Subtype 4 Channel Causes Endothelial Failure and Circulatory Collapse: Part 2

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4−/− null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-l-arginine methyl ester; l-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (l-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

Catalytic in vivo protein knockdown by small-molecule PROTACs

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the targets ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

An Orally Active TRPV4 Channel Blocker Prevents and Resolves Pulmonary Edema Induced by Heart Failure

Transient receptor potential vanilloid 4 (TRPV4) channels are expressed in human heart failure lungs, which can be blocked to prevent and resolve heart failure–induced pulmonary edema. Ion Channel Blockade Prevents Pulmonary Edema Heart failure affects not only the heart and vessels but also the lungs. As blood pressure builds up in the lung’s vessels, fluid leaks into the lungs. Treatment options are limited for these patients, mostly because the mechanism underlying pulmonary edema is unclear. Here, Thorneloe and colleagues implicate the activation of the transient receptor potential vanilloid 4 (TRPV4) ion channel in the onset of edema during heart failure and show that a small-molecule drug can prevent such leakage. Activation of the ion channel TRPV4 results in pulmonary edema in animal lungs. The authors first confirmed that TRPV4 was expressed in normal human lungs and then demonstrated that it was increased in lung tissue from patients with a history of congestive heart failure. Using a small-molecule screen, Thorneloe et al. discovered GSK2193874. In human cells in vitro and mouse lungs ex vivo, the small molecule effectively blocked TRPV4 channels to maintain endothelial (vessel) layer integrity. A related study by Huh et al. (this issue) shows that the drug indeed prevents vascular leakage of human cell cultures in vitro. The GSK2193874 analog GSK2263095 displayed similar activity in canine lungs ex vivo. In vivo in rat models of heart failure, the authors found that the drug was effective in both preventing and reversing pulmonary edema. The molecule only protected against lung permeability at high (pathological) pulmonary venous pressure. Thorneloe and colleagues showed that GSK2193874 blocked TRPV4 activity across species, including in human cells, without adversely affecting heart rate or arterial pressure. This suggests that TRPV4 blockers might be used therapeutically to treat patients with heart failure–induced pulmonary edema. Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca2+ influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

OR3-001 – RIP2 kinase is activated in Blau Syndrome and IBD

Blau Syndrome (Blau) is a granulomatous auto-inflammatory disease caused by mutations in NOD2 that have been proposed to result in phosphorylation of RIP2 kinase and the production of pro-inflammatory cytokines. Such monogenic diseases can bring to light pathways that are also likely to be involved in more genetically complex diseases. For example, increased RIP2 phosphorylation has been observed in inflammatory bowel disease (IBD), although the role of RIP2 in IBD has not been determined. We are developing a first-in-class, highly potent and selective inhibitor of RIP2 kinase, which may provide therapeutic benefit in both Blau and IBD.

Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 μM).