James E. Sheppeck

Discovery and Structure–Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P1)

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates a multitude of physiological processes such as lymphocyte trafficking, cardiac function, vascular development, and inflammation. Because of the ability of S1P1 receptor agonists to suppress lymphocyte egress, they have great potential as therapeutic agents in a variety of autoimmune diseases. In this article, the discovery of selective, direct acting S1P1 agonists utilizing an ethanolamine scaffold containing a terminal carboxylic acid is described. Potent S1P1 agonists such as compounds 18a and 19a which have greater than 1000-fold selectivity over S1P3 are described. These compounds efficiently reduce blood lymphocyte counts in rats through 24 h after single doses of 1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds are discussed. Compound 19a was further studied in two preclinical models of disease, exhibiting good efficacy in both the rat adjuvant arthritis model (AA) and the mouse experimental autoimmune encephalomyelitis model (EAE).

Discovery of potent and selective nonsteroidal indazolyl amide glucocorticoid receptor agonists

Modification of a phenolic lead structure based on lessons learned from increasing the potency of steroidal glucocorticoid agonists lead to the discovery of exceptionally potent, nonsteroidal, indazole GR agonists. SAR was developed to achieve good selectivity against other nuclear hormone receptors with the ultimate goal of achieving a dissociated GR agonist as measured by human in vitro assays. The specific interactions by which this class of compounds inhibits GR was elucidated by solving an X-ray co-crystal structure.

Synthesis and structure–activity relationships of novel indazolyl glucocorticoid receptor partial agonists

SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptor agonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors.

Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the β1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

Heterocyclic glucocorticoid receptor modulators with a 2,2-dimethyl-3-phenyl-N-(thiazol or thiadiazol-2-yl)propanamide core.

A series of heterocyclic glucocorticoid receptor (GR) modulators with 2,2-dimethyl-3-phenyl-N-(thiazol or thiadiazol-2-yl)propanamide core are described. Structure-activity relationships suggest a combination of H-bond acceptor and a 4-fluorophenyl moiety as being important structural components contributing to the glucocorticoid receptor binding and functional activity for this series of GR modulators.

Pharmacological Characterization of IW-1973, a Novel Soluble Guanylate Cyclase Stimulator with Extensive Tissue Distribution, Antihypertensive, Anti-Inflammatory, and Antifibrotic Effects in Preclinical Models of Disease

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5′-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1–10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.

Discovery of IWP-051, a novel orally bioavailable soluble guanylate cyclase stimulator with sustained and dose-dependent hemodynamic effects.

Discovery of IWP-051, a novel orally bioavailable soluble guanylate cyclase stimulator with sustained and dose-dependent hemodynamic effects Takashi Nakai, Nicholas R Perl, Rajesh R Iyengar, Ara Mermerian, G-Yoon J Im, Thomas W-H Lee, Glen R Rennie, James Jia, Paul A Renhowe, Timothy C Barden, James E Sheppeck II, Karthik Iyer, Joon Jung, G Todd Milne, Chrissie Segal, Kimberly Long, Joy Miyashiro, Sylvie Bernier, Sarah Jacobson, Jenny Tobin, Courtney Shea, Peter Germano, Yueh-tyng Chien, Daniel Zimmer

Concomitant administration of sGC stimulators with common classes of anti-hypertensive agents results in increased efficacy in spontaneously hypertensive rats

Background Soluble guanylate cyclase (sGC) stimulators demonstrate smooth muscle relaxation and vasodilation via the nitric oxide (NO)-sGC-cyclic guanosine monophosphate (cGMP) pathway. A novel class of sGC stimulators, the pyrazole-pyrimidines, was synthesized with the objective of creating a potent, once-a-day (QD) oral treatment for cardiovascular diseases. Several compounds from this class were identified as potent stimulators of sGC in vitro (EC50 = 40-287 nM). These compounds were evaluated in pharmacokinetic (PK) and blood pressure pharmacodynamics (PD) in vivo rat and dog models and were shown to exhibit sustained compound exposure (Thalf = >7 hours in preclinical species) after oral dosing, predicting QD dosing in humans. Further, they significantly decreased mean arterial blood pressure (MAP (≥ 10mmHg) after oral dosing. The potential for sGC stimulators to work in combination with reference antihypertensive therapies was assessed in an in vivo PD assay in a spontaneous hypertensive rat (SHR) model. Doses of losartan, atenolol, amlodipine, and our sGC stimulators that induced an effect (< 30mmHg) on MAP were chosen. IWP-121, a representative sGC stimulator, was shown to provide additional MAP lowering effects when combined with losartan, atenolol, or amlodipine, resulting in an increase in overall blood pressure effects between 5-50%. By linking compound concentration to blood pressure change for each compound alone and in combination, we were able to assess the PK/PD relationships for the individual and combined effects.

IWP-121: a novel sGC stimulator that reduces blood pressure and exhibits anti-fibrotic and anti-inflammatory activities in the Dahl Salt-Sensitive rat model

Background Soluble guanylate cyclase (sGC) is an intracellular receptor that can be activated by nitric oxide (NO) and sGC stimulators to produce cyclic guanosine monophosphate (cGMP), thereby modulating a number of downstream cellular and physiological responses including phosphorylation of VASP and vasodilation. In the Dahl Salt-Sensitive (DSS) rat model of hypertension, cGMP production by sGC is decreased, most likely due to reactive oxygen species (ROS) converting NO to peroxynitrite, resulting in depleted pools of NO available to bind to sGC. In this study we evaluated the efficacy of a novel sGC stimulator (IWP-121) in the DSS model. Male DSS rats (230-270 grams) received high-salt diet (8% NaCl) for 2 weeks followed by high salt plus compound for 6 additional weeks. IWP-121 was administered at doses of 1, 3, and 10 mg/kg/ day in the chow (n=8/group). Losartan (30 mg/kg/day in the water) was used as a positive control, in addition to both High Salt (HS) and Normal Salt (NS). All groups were compared to HS control group for analyses. IWP-121 dose dependently decreased mean blood pressure (MAP) throughout the study. Additionally, IWP-121 (at all doses tested) and losartan had statistically significant effects on decreasing heart hypertrophy and plasma NT-proBNP but only IWP-121 had an effect on attenuating liver hypertrophy. IWP-121 decreased microalbuminuria (an indicator of kidney end organ damage) as well as attenuated serum biomarkers known to be involved in inflammatory and fibrotic processes. Conclusion In the rat DSS model of hypertension, there is a decrease in cGMP levels most likely due to the inactivation of endogenous NO by ROS. The sGC stimulator IWP-121, when administered in the diet exhibited sustained dosedependent reduction in blood pressure. Additionally, IWP-121 attenuated heart and liver hypertrophy and reduced NT-proBNP, a biomarker of heart failure. The compound reduced levels of biomarkers for inflammation and fibrosis, and demonstrated renal end organ protection. sGC stimulators, like IWP-121 may have broad therapeutic application by modulating multiple relevant therapeutic endpoints including blood pressure, hypertrophy, inflammation, and fibrosis.