Keith A. Koch

A novel kinase inhibitor establishes a predominant role for protein kinase D as a cardiac class IIa histone deacetylase kinase

Class IIa histone deacetylases (HDACs) repress genes involved in pathological cardiac hypertrophy. The anti‐hypertrophic action of class IIa HDACs is overcome by signals that promote their phosphorylation‐dependent nuclear export. Several kinases have been shown to phosphorylate class IIa HDACs, including calcium/calmodulin‐dependent protein kinase (CaMK), protein kinase D (PKD) and G protein‐coupled receptor kinase (GRK). However, the identity of the kinase(s) responsible for phosphorylating class IIa HDACs during cardiac hypertrophy has remained controversial. We describe a novel and selective small molecule inhibitor of PKD, bipyridyl PKD inhibitor (BPKDi). BPKDi blocks signal‐dependent phosphorylation and nuclear export of class IIa HDACs in cardiomyocytes and concomitantly suppresses hypertrophy of these cells. These studies define PKD as a principal cardiac class IIa HDAC kinase.

Identification of Orally Available Naphthyridine Protein Kinase D Inhibitors

A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D (PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic mechanism with application as a heart failure therapy. As PKC was previously identified as the immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral administration. Their effects on heart morphology and function are discussed herein.

Identification of Potent and Selective Amidobipyridyl Inhibitors of Protein Kinase D

The synthesis and biological evaluation of potent and selective PKD inhibitors are described herein. The compounds described in the present study selectively inhibit PKD among other putative HDAC kinases. The PKD inhibitors of the present study blunt phosphorylation and subsequent nuclear export of HDAC4/5 in response to diverse agonists. These compounds further establish the central role of PKD as an HDAC4/5 kinase and enhance the current understanding of cardiac myocyte signal transduction. The in vivo efficacy of a representative example compound on heart morphology is reported herein.

Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel irreversible inhibitors of CRM1.

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.

3,5-Diarylazoles as novel and selective inhibitors of protein kinase D

The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD/HDAC5 signaling.

Screening and Identification of a Novel Class of TGF-β Type 1 Receptor Kinase Inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor–like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420 000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration–response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC50 value of 0.7 µM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC50 value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.

ASK1 contributes to fibrosis and dysfunction in models of kidney disease.

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal–regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

p38α: A Profibrotic Signaling Nexus.

Article, see p 549 Fibrosis of the heart is driven by the reprogramming of resident fibroblasts into contractile myofibroblasts that express high levels of extracellular matrix. Cardiac fibrosis may be beneficial, replenishing regions of myocyte loss with a structural scar after infarction, or maladaptive, involving excess extracellular matrix deposition in response to long-standing stress. Uncontrolled cardiac fibrosis can have dire consequences. For example, fibrosis increases the passive stiffness of the myocardium, contributing to diastolic dysfunction, and disrupts electric conduction in the heart, causing arrhythmias and sudden cardiac death. Clinical trials aimed at treating heart failure with preserved ejection fraction, in part by blocking cardiac fibrosis, have mainly centered on inhibiting components of the renin-angiotensin-aldosterone system and have largely been unsuccessful.1 As such, cardiac fibrosis is a major unmet medical need, and the discovery of new mechanisms that control fibrosis in the heart is required for development of innovative therapies for this prominent and devastating process. Transforming growth factor (TGF)-β is a cytokine that controls fibrosis across organ systems. Binding of TGF-β to its cell surface receptor triggers phosphorylation and nuclear translocation of SMAD transcription factors, which bind regulatory elements in a variety of profibrotic genes. An article published by Molkentin et al in this issue of Circulation 2 defines a crucial role for noncanonical, SMAD-independent TGF-β signaling in the control of cardiac fibrosis. In a tour-de-force of elegant genetic gain- and loss-of-function studies, the authors demonstrate that the mitogen-activated protein kinase p38α is a nodal effector of profibrotic TGF-β signaling in the heart. These findings were reported in this seminal article: 1. p38α ( Mapk14 gene) deletion in cultured fibroblasts blocked differentiation of the cells into α-smooth muscle actin-positive myofibroblasts in response to TGF-β, angiotensin II, or cyclic stretching. 2. The block to myofibroblast differentiation in cultured fibroblasts lacking p38α could be …