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Introduction : Transient receptor potential vanilloid 4 (TRPV4) is an ion channel that is widely expressed and is activated by numerous chemical, osmotic and mechanical stimuli. By modulating Ca 2+ entry, TRPV4 regulates cellular signaling associated with a variety of (patho)physiological processes and is a target of interest for treatment of human diseases including heart failure, respiratory diseases, gastrointestinal disorders, dermatological conditions, pain and cancer, among others. Areas covered : This article reviews small molecule TRPV4 antagonists and new therapeutic use claims disclosed in the patent literature from 2015 to 2020, including applications covering the first potent and selective TRPV4 clinical candidate and other advanced chemotypes. Expert opinion : TRPV4 has proven to be a tractable target and significant progress in discovery of TRPV4 antagonists has been realized in recent years. Several unique chemical templates with drug-like properties inhibit the channel and show efficacy in models that suggest their potential for treatment of a variety of diseases. While compelling clinical efficacy has not yet been seen in the limited early studies conducted with GSK2798745, evaluation of TRPV4 antagonists in larger trials across several indications is warranted given the availability of high-quality candidates and the promise of therapeutic benefit based on pre-clinical evidence.

GSK2798745, an antagonist of the transient receptor potential vanilloid 4 (TRPV4) ion channel, was recently investigated in clinical trials for the treatment of cardiac and respiratory diseases. Human plasma and urine samples collected from healthy volunteers following oral administration were analyzed to identify circulating and excreted metabolites of the parent drug. One major circulating metabolite ( 1 ) was found in pooled human plasma samples, accounting for approximately half of the observed drug-related material. Isolation of metabolite 1 from urine samples followed by MS and NMR studies led to a putative structural assignment of 1 where hydroxylation of GSK2798745 occurred on the central ring, producing a penta-substituted cyclohexane structure containing three stereocenters. Two unique chemical syntheses of the proposed structure were developed to confirm the identity of metabolite 1 and provide access to gram quantities for biological characterization., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

A two-step metal-halogen exchange and diastereoselective copper-mediated Michael addition onto a complex α,β-unsaturated system has been developed and applied toward the synthesis of bisaryl Nrf2 activators. Optimization of metal-halogen exchange using ( n -Bu) 3 MgLi allowed for the preparation of custom aryl-functionalized magnesiate reagents at noncryogenic temperatures. Following transmetalation, these reagents were used in highly diastereoselective Michael addition reactions.

Investigation of TRPV4 as a potential target for the treatment of pulmonary edema associated with heart failure generated a novel series of acyclic amine inhibitors displaying exceptional potency and PK properties. The series arose through a scaffold hopping approach, which relied on use of an internal H-bond to replace a saturated heterocyclic ring. Optimization of the lead through investigation of both aryl regions revealed approaches to increase potency through substituents believed to enhance separate intramolecular and intermolecular H-bond interactions. A proposed internal H-bond between the amine and neighboring benzenesulfonamide was stabilized by electronically modulating the benzenesulfonamide. In the aryl ether moiety, substituents para to the nitrile demonstrated an electronic effect on TRPV4 recognition. Finally, the acyclic amines inactivated CYP3A4 and this liability was addressed by modifications that sterically preclude formation of a putative metabolic intermediate complex to deliver advanced TRPV4 antagonists as leads for discovery of novel medicines.

TRPV4 is a ubiquitously expressed, non-selective cation channel activated by a range of stimuli including hypotonicity, temperature, pH, stretch and endogenous ligands. Agents that modulate TRPV4 are sought as potential therapeutics for the treatment of many diseases including osteoarthritis, respiratory illnesses, gastrointestinal disorders, pain and congestive heart failure. In recent years, significant advances in TRPV4 drug discovery have been realized as at least seven novel TRPV4 agonist or antagonist templates were reported and the first selective TRPV4 antagonist was evaluated in early clinical trials., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Authors affiliated with GlaxoSmithKline have received compensation in the form of salary and stock., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

GSK3527497, a preclinical candidate for the inhibition of TRPV4, was identified starting from the previously reported pyrrolidine sulfonamide TRPV4 inhibitors 1 and 2 . Optimization of projected human dose was accomplished by specifically focusing on in vivo pharmacokinetic parameters CL u , Vdss u , and MRT. We highlight the use of conformational changes as a novel approach to modulate Vdss u and present results that suggest that molecular-shape-dependent binding to tissue components governs Vdss u in addition to bulk physicochemical properties. Optimization of CL u within the series was guided by in vitro metabolite identification, and the poor FaSSIF solubility imparted by the crystalline properties of the pyrrolidine diol scaffold was improved by the introduction of a charged moiety to enable excellent exposure from high crystalline doses. GSK3527497 is a preclinical candidate suitable for oral and iv administration that is projected to inhibit TRPV4 effectively in patients from a low daily clinical dose.

GSK2798745, a clinical candidate, was identified as an inhibitor of the transient receptor potential vanilloid 4 (TRPV4) ion channel for the treatment of pulmonary edema associated with congestive heart failure. We discuss the lead optimization of this novel spirocarbamate series and specifically focus on our strategies and solutions for achieving desirable potency, rat pharmacokinetics, and physicochemical properties. We highlight the use of conformational bias to deliver potency and optimization of volume of distribution and unbound clearance to enable desirable in vivo mean residence times., Competing Interests: The authors declare the following competing financial interest(s): All authors are current or past employees of GlaxoSmithKline and/or stockholders of GlaxoSmithKline.

Pulmonary edema is a common ailment of heart failure patients and has remained an unmet medical need due to dose-limiting side effects associated with current treatments. Preclinical studies in rodents have suggested that inhibition of transient receptor potential vanilloid-4 (TRPV4) cation channels may offer an alternative-and potentially superior-therapy. Efforts directed toward small-molecule antagonists of the TRPV4 receptor have led to the discovery of a novel sulfone pyrrolidine sulfonamide chemotype exemplified by lead compound 6. Design elements toward the optimization of TRPV4 activity, selectivity, and pharmacokinetic properties are described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential is highlighted herein.

A novel series of pyrrolidine sulfonamide transient receptor potential vanilloid-4 (TRPV4) antagonists was developed by modification of a previously reported TRPV4 inhibitor (1). Several core-structure modifications were identified that improved TRPV4 activity by increasing structural rigidity and reducing the entropic energy penalty upon binding to the target protein. The new template was initially discovered as a minor regio-isomeric side product formed during routine structure-activity relationship (SAR) studies, and further optimization resulted in highly potent compounds with a novel pyrrolidine diol core. Further improvements in potency and pharmacokinetic properties were achieved through SAR studies on the sulfonamide substituent to give an optimized lead compound GSK3395879 (52) that demonstrated the ability to inhibit TRPV4-mediated pulmonary edema in an in vivo rat model. GSK3395879 is a tool for studying the biology of TRPV4 and an advanced lead for identifying new heart failure medicines.

Bone Morphogenetic Protein 1 (BMP1) inhibition is a potential method for treating fibrosis because BMP1, a member of the zinc metalloprotease family, is required to convert pro-collagen to collagen. A novel class of reverse hydroxamate BMP1 inhibitors was discovered, and cocrystal structures with BMP1 were obtained. The observed binding mode is unique in that the small molecule occupies the nonprime side of the metalloprotease pocket providing an opportunity to build in metalloprotease selectivity. Structure-guided modification of the initial hit led to the identification of an oral in vivo tool compound with selectivity over other metalloproteases. Due to irreversible inhibition of cytochrome P450 3A4 for this chemical class, the risk of potential drug-drug interactions was managed by optimizing the series for subcutaneous injection., Competing Interests: The authors declare no competing financial interest.

Structure-guided progression of a purine-derived series of TNNI3K inhibitors directed design efforts that produced a novel series of 4,6-diaminopyrimidine inhibitors, an emerging kinase binding motif. Herein, we report a detailed understanding of the intrinsic conformational preferences of the scaffold, which impart high specificity for TNNI3K. Further manipulation of the template based on the conformational analysis and additional structure-activity relationship studies provided enhancements in kinase selectivity and pharmacokinetics that furnished an advanced series of potent inhibitors. The optimized compounds (e.g., GSK854) are suitable leads for identifying new cardiac medicines and have been employed as in vivo tools in investigational studies aimed at defining the role of TNNI3K within heart failure.

Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Investigation of troponin I-interacting kinase (TNNI3K) as a potential target for the treatment of heart failure has produced a series of substituted N-methyl-3-(pyrimidin-4-ylamino)benzenesulfonamide inhibitors that display excellent potency and selectivity against a broad spectrum of protein kinases. Crystal structures of prototypical members bound to the ATP-binding site of TNNI3K reveal two anchoring hydrogen bond contacts: (1) from the hinge region amide N-H to the pyrimidine nitrogen and (2) from the sulfonamide N-H to the gatekeeper threonine. Evaluation of various para-substituted benzenesulfonamides defined a substituent effect on binding affinity resulting from modulation of the sulfonamide H-bond donor strength. An opposite electronic effect emerged for the hinge NH-pyrimidine H-bond interaction, which is further illuminated in the correlation of calculated H-bond acceptor strength and TNNI3K affinity for a variety of hinge binding heterocycles. These fundamental correlations on drug-receptor H-bond interactions may be generally useful tools for the optimization of potency and selectivity in the design of kinase inhibitors.

A series of selective TNNI3K inhibitors were developed by modifying the hinge-binding heterocycle of a previously reported dual TNNI3K/B-Raf inhibitor. The resulting quinazoline-containing compounds exhibit a large preference (up to 250-fold) for binding to TNNI3K versus B-Raf, are useful probes for elucidating the biological pathways associated with TNNI3K, and are leads for discovering novel cardiac medicines. GSK114 emerged as a leading inhibitor, displaying significant bias (40-fold) for TNNI3K over B-Raf, exceptional broad spectrum kinase selectivity, and adequate oral exposure to enable its use in cellular and in vivo studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A series of cardiac troponin I-interacting kinase (TNNI3K) inhibitors arising from 3-((9H-purin-6-yl)amino)-N-methyl-benzenesulfonamide (1) is disclosed along with fundamental structure-function relationships that delineate the role of each element of 1 for TNNI3K recognition. An X-ray structure of 1 bound to TNNI3K confirmed its Type I binding mode and is used to rationalize the structure-activity relationship and employed to design potent, selective, and orally bioavailable TNNI3K inhibitors. Identification of the 7-deazapurine heterocycle as a superior template (vs purine) and its elaboration by introduction of C4-benzenesulfonamide and C7- and C8-7-deazapurine substituents produced compounds with substantial improvements in potency (>1000-fold), general kinase selectivity (10-fold improvement), and pharmacokinetic properties (>10-fold increase in poDNAUC). Optimal members of the series have properties suitable for use in in vitro and in vivo experiments aimed at elucidating the role of TNNI3K in cardiac biology and serve as leads for developing novel heart failure medicines.

Percutaneous coronary intervention is first-line therapy for acute coronary syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via reperfusion injury, a phenomenon driven in large part by oxidative stress. Therapies to limit this progression have proven elusive, with no major classes of new agents since the development of anti-platelets/anti-thrombotics. We report that cardiac troponin I-interacting kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes ischemia/reperfusion injury, oxidative stress, and myocyte death. TNNI3K-mediated injury occurs through increased mitochondrial superoxide production and impaired mitochondrial function and is largely dependent on p38 mitogen-activated protein kinase (MAPK) activation. We developed a series of small-molecule TNNI3K inhibitors that reduce mitochondrial-derived superoxide generation, p38 activation, and infarct size when delivered at reperfusion to mimic clinical intervention. TNNI3K inhibition also preserves cardiac function and limits chronic adverse remodeling. Our findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic heart and is a tractable therapeutic target for ACS. Pharmacologic TNNI3K inhibition would be cardiac-selective, preventing potential adverse effects of systemic kinase inhibition.

Fostriecin and cytostatin are structurally related natural inhibitors of serine/threonine phosphatases, with promising antitumor activity. The total synthesis of these antitumor agents has enabled the production of structural analogs, which are useful to explore the biological significance of features contained in the parent compounds. Here, the inhibitory activity of fostriecin, cytostatin, and 10 key structural analogs were tested in side-by-side phosphatase assays to further characterize their inhibitory activity against PP1c (Ser/Thr protein phosphatase 1 catalytic subunit), PP2Ac (Ser/Thr protein phosphatase 2A catalytic subunit), PP5c (Ser/Thr protein phosphatase 5 catalytic subunit), and chimeras of PP1 (Ser/Thr protein phosphatase 1) and PP5 (Ser/Thr protein phosphatase 5), in which key residues predicted for inhibitor contact with PP2A (Ser/Thr protein phosphatase 2A) were introduced into PP1 and PP5 using site-directed mutagenesis. The data confirm the importance of the C9-phosphate and C11-alcohol for general inhibition and further demonstrate the importance of a predicted C3 interaction with a unique cysteine (Cys(269)) in the beta12-beta13 loop of PP2A. The data also indicate that additional features beyond the unsaturated lactone contribute to inhibitory potency and selectivity. Notably, a derivative of fostriecin lacking the entire lactone subunit demonstrated marked potency and selectivity for PP2A, while having substantially reduced and similar activity against PP1 and PP1/PP2A- PP5/PP2A-chimeras that have greatly increased sensitivity to both fostriecin and cytostatin. This suggests that other features [e.g., the (Z,Z,E)-triene] also contribute to inhibitory selectivity. When considered together with previous data, these studies suggest that, despite the high structural conservation of the catalytic site in PP1, PP2A and PP5, the development of highly selective catalytic inhibitors should be feasible.

The total synthesis of cytostatin, an antitumor agent belonging to the fostriecin family of natural products, is described in full detail. The convergent approach relied on a key epoxide-opening reaction to join the two stereotriad units and a single-step late-stage stereoselective installation of the sensitive (Z,Z,E)-triene through a beta-chelation-controlled nucleophilic addition. The synthetic route provided rapid access to the C4-C6 stereoisomers of the cytostatin lactone, which were prepared and used to define the C4-C6 relative stereochemistry of the natural product. In addition to the natural product, each of the C10-C11 diastereomers of cytostatin was divergently prepared (11 steps from key convergence step) by this route and used to unequivocally confirm the relative and absolute stereochemistry of cytostatin. Each of the cytostatin diastereomers exhibited a reduced activity toward inhibition of PP2A (>100-fold), demonstrating the importance of the presence and stereochemistry of the C10-methyl and C11-hydroxy groups for potent PP2A inhibition. Extensions of the studies provided dephosphocytostatin, sulfocytostatin (a key analogue related to the natural product sultriecin), 11-deshydroxycytostatin, and an analogue lacking the entire C12-C18 (Z,Z,E)-triene segment, which were used to define the magnitude of the C9-phosphate (>4000-fold), C11-alcohol (250-fold), and triene (220-fold) contribution to PP2A inhibition. A model of cytostatin bound to the active site of PP2A is presented, compared to that of fostriecin, which is also presented in detail for the first time, and used to provide insights into the role of the key substituents. Notably, the alpha,beta unsaturated lactone of cytostatin, like that of fostriecin, is projected to serve as a key electrophile, providing a covalent adduct with Cys269 unique to PP2A, contributing to its potency (> or =200-fold for fostriecin) and accounting for its selectivity.

A genetic screen was developed for the identification of genes related to thiamin biosynthesis and degradation. Genes conferring resistance to bacimethrin or 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine were selected from Escherichia coli and Bacillus subtilis genomic libraries. Hits from the selection included the known thiamin biosynthetic genes thiC, thiE, and dxs as well as five genes of previously unknown function (E. coli yjjX, yajO, ymfB, and cof and B. subtilis yveN). The gene products YmfB and Cof catalyze the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate. YmfB also converts thiamin pyrophosphate into thiamin phosphate.

The conversion of 5-aminoimidazole ribonucleotide (AIR) into 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) is a fascinating reaction on the thiamin biosynthetic pathway in bacteria and is probably the most complex unresolved rearrangement in primary metabolism. We have successfully reconstituted this reaction in a cell-free system. The E. coli thiC gene product and an additional unidentified E. coli protein are required for the reaction. In addition, SAM and nicotinamide cofactors are required for full activity. Labeling studies to determine the origin of most of the atoms in the pyrimidine are described. Based on these studies, a new mechanism for HMP formation is proposed., (Copyright 2004 The Royal Society of Chemistry)

Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 +/- 3, and <2%, respectively; "top-down" MS/MS of these ions show oxidation at the sites R(28)-V(31), E(95)-F(107), and K(216) of 22%, 28%, and 34%, respectively, but with a possible (approximately 4%) fourth site at V(125)-D(150). However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable approximately 13% oxygen at the V(125)-D(150) site. For the "bottom-up" approach, detection of the approximately 4% oxidation at the V(125)-D(150) site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just approximately 4% of this, but the uniqueness of its mass value (approximately 1-2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V(125)-D(150) until K(216) is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K(216), H(137), and D(139) are conserved residues.

The structural characterization of proteins expressed from the genome is a major problem in proteomics. The solution to this problem requires the separation of the protein of interest from a complex mixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errors and posttranslational modifications. For this, the "top down" mass spectrometry (MS) approach, extended by the greatly increased protein fragmentation from electron capture dissociation (ECD), has been applied to characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation of proline residues in proteins. With Fourier transform (FT) MS, electrospray ionization (ESI) of a complex mixture from an E. coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but none corresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS, ThiG, and ThiF). MS/MS of one ion species (representing approximately 1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different. Further purification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneously as ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminal Met and Ser from the 27 kDa ThiG. For a second system, the molecular weight of the 45 kDa phosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme A biosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this is due to the removal of the N-terminal Met. For a third system, viral prolyl 4-hydroxylase (26 kDa), ECD showed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, and MS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) and to formation of disulfide bonds (2 x 2 Da) at C32-C49 and C143-C147, although 10 S-S positions were possible. In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins was relatively unsuccessful. The addition of ECD substantially increases the capabilities of top down FTMS for the detailed structural characterization of large proteins.