The Novel, Clinical-Stage Soluble Guanylate Cyclase Activator BI 685509 Protects from Disease Progression in Models of Renal Injury and Disease.

Activation of soluble guanylate cyclase (sGC) to restore cyclic guanosine monophosphate (cGMP) and improve functionality of nitric oxide (NO) pathways impaired by oxidative stress is a potential treatment of diabetic and chronic kidney disease. We report the pharmacology of BI 685509, a novel, orally active small molecule sGC activator with disease-modifying potential. BI 685509 and human sGC α1/ß1 heterodimer containing a reduced heme group produced concentration-dependent increases in cGMP that were elevated modestly by NO, whereas heme-free sGC and BI 685509 greatly enhanced cGMP with no effect of NO. BI 685509 increased cGMP in human and rat platelet-rich plasma treated with the heme-oxidant ODQ; respective EC50 values were 467 nM and 304 nM. In conscious telemetry-instrumented rats, BI 685509 did not affect mean arterial pressure (MAP) or heart rate (HR) at 3 and 10 mg/kg (p.o.), whereas 30 mg/kg decreased MAP and increased HR. Ten days of BI 685509 at supratherapeutic doses (60 or 100 mg/kg p.o., daily) attenuated MAP and HR responses to a single 100 mg/kg challenge. In the ZSF1 rat model, BI 685509 (1, 3, 10, and 30 mg/kg per day, daily) coadministered with enalapril (3 mg/kg per day) dose-dependently reduced proteinuria and incidence of glomerular sclerosis; MAP was modestly reduced at the higher doses versus enalapril. In the 7-day rat unilateral ureteral obstruction model, BI 685509 dose-dependently reduced tubulointerstitial fibrosis (P < 0.05 at 30 mg/kg). In conclusion, BI 685509 is a potent, orally bioavailable sGC activator with clear renal protection and antifibrotic activity in preclinical models of kidney injury and disease. SIGNIFICANCE STATEMENT: BI 685509 is a novel small soluble guanylate cyclase (sGC) molecule activator that exhibits an in vitro profile consistent with that of an sGC activator. BI 685509 reduced proteinuria and glomerulosclerosis in the ZSF1 rat, a model of diabetic kidney disease (DKD), and reduced tubulointerstitial fibrosis in a rat 7-day unilateral ureteral obstruction model. Thus, BI 685509 is a promising new therapeutic agent and is currently in phase II clinical trials for chronic kidney disease and DKD.

In vivo selective inhibition of TRPC6 by antagonist BI 749327 ameliorates fibrosis and dysfunction in cardiac and renal disease.

Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.

A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat.

Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR.

Hepatocyte Smoothened Activity Controls Susceptibility to Insulin Resistance and Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH), a leading cause of cirrhosis, strongly associates with the metabolic syndrome, an insulin-resistant proinflammatory state that disrupts energy balance and promotes progressive liver degeneration. We aimed to define the role of Smoothened (Smo), an obligatory component of the Hedgehog signaling pathway, in controlling hepatocyte metabolic homeostasis and, thereby, susceptibility to NASH. METHODS: We conditionally deleted Smo in hepatocytes of healthy chow-fed mice and performed metabolic phenotyping, coupled with single-cell RNA sequencing (RNA-seq), to characterize the role of hepatocyte Smo in regulating basal hepatic and systemic metabolic homeostasis. Liver RNA-seq datasets from 2 large human cohorts were also analyzed to define the relationship between Smo and NASH susceptibility in people. RESULTS: Hepatocyte Smo deletion inhibited the Hedgehog pathway and promoted fatty liver, hyperinsulinemia, and insulin resistance. We identified a plausible mechanism whereby inactivation of Smo stimulated the mTORC1-SREBP1c signaling axis, which promoted lipogenesis while inhibiting the hepatic insulin cascade. Transcriptomics of bulk and single Smo-deficient hepatocytes supported suppression of insulin signaling and also revealed molecular abnormalities associated with oxidative stress and mitochondrial dysfunction. Analysis of human bulk RNA-seq data revealed that Smo expression was (1) highest in healthy livers, (2) lower in livers with NASH than in those with simple steatosis, (3) negatively correlated with markers of insulin resistance and liver injury, and (4) declined progressively as fibrosis severity worsened. CONCLUSIONS: The Hedgehog pathway controls insulin sensitivity and energy homeostasis in adult livers. Loss of hepatocyte Hedgehog activity induces hepatic and systemic metabolic stress and enhances susceptibility to NASH by promoting hepatic lipoxicity and insulin resistance.

Deep learning-based quantification of NAFLD/NASH progression in human liver biopsies.

Non-alcoholic fatty liver disease (NAFLD) affects about 24% of the world's population. Progression of early stages of NAFLD can lead to the more advanced form non-alcoholic steatohepatitis (NASH), and ultimately to cirrhosis or liver cancer. The current gold standard for diagnosis and assessment of NAFLD/NASH is liver biopsy followed by microscopic analysis by a pathologist. The Kleiner score is frequently used for a semi-quantitative assessment of disease progression. In this scoring system the features of active injury (steatosis, inflammation, and ballooning) and a separated fibrosis score are quantified. The procedure is time consuming for pathologists, scores have limited resolution and are subject to variation. We developed an automated deep learning method that provides full reproducibility and higher resolution. The system was established with 296 human liver biopsies and tested on 171 human liver biopsies with pathologist ground truth scores. The method is inspired by the way pathologist's analyze liver biopsies. First, the biopsies are analyzed microscopically for the relevant histopathological features. Subsequently, histopathological features are aggregated to a per-biopsy score. Scores are in the identical numeric range as the pathologist's ballooning, inflammation, steatosis, and fibrosis scores, but on a continuous scale. Resulting scores followed a pathologist's ground truth (quadratic weighted Cohen's κ on the test set: for steatosis 0.66, for inflammation 0.24, for ballooning 0.43, for fibrosis 0.62, and for the NAFLD activity score (NAS) 0.52. Mean absolute errors on a test set: for steatosis 0.29, for inflammation 0.53, for ballooning 0.61, for fibrosis 0.78, and for the NAS 0.77).

Pharmacological TRPC6 inhibition improves survival and muscle function in mice with Duchenne muscular dystrophy.

Gene mutations causing loss of dystrophin result in the severe muscle disease known as Duchenne muscular dystrophy (DMD). Despite efforts at genetic repair, DMD therapy remains largely palliative. Loss of dystrophin destabilizes the sarcolemmal membrane, inducing mechanosensitive cation channels to increase calcium entry and promote cell damage and, eventually, muscle dysfunction. One putative channel is transient receptor potential canonical 6 (TRPC6); we have shown that TRPC6 contributed to abnormal force and calcium stress-responses in cardiomyocytes from mice lacking dystrophin that were haplodeficient for utrophin (mdx/utrn+/- [HET] mice). Here, we show in both the HET mouse and the far more severe homozygous mdx/utrn-/- mouse that TRPC6 gene deletion or its selective pharmacologic inhibition (by BI 749327) prolonged survival 2- to 3-fold, improving skeletal and cardiac muscle and bone defects. Gene pathways reduced by BI 749327 treatment most prominently regulated fat metabolism and TGF-ß1 signaling. These results support the testing of TRPC6 inhibitors in human trials for other diseases as a novel DMD therapy.

Benzimidazolone as potent chymase inhibitor: modulation of reactive metabolite formation in the hydrophobic (P1) region.

A new class of chymase inhibitor featuring a benzimidazolone core with an acid side chain and a P(1) hydrophobic moiety is described. Incubation of the lead compound with GSH resulted in the formation of a GSH conjugate on the benzothiophene P(1) moiety. Replacement of the benzothiophene with different heterocyclic systems such as indoles and benzoisothiazole is feasible. Among the P(1) replacements, benzoisothiazole prevents the formation of GSH conjugate and an in silico analysis of oxidative potentials agreed with the experimental outcome.

Modulation of vascular contraction via soluble guanylate cyclase signaling in a novel ex vivo method using rat precision-cut liver slices.

Fibrotic processes in the liver of non-alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre-clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin-1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin-1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin-induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non-responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.

The key role of NLRP3 and STING in APOL1-associated podocytopathy.

Coding variants in apolipoprotein L1 (APOL1), termed G1 and G2, can explain most excess kidney disease risk in African Americans; however, the molecular pathways of APOL1-induced kidney dysfunction remain poorly understood. Here, we report that expression of G2 APOL1 in the podocytes of Nphs1rtTA/TRE-G2APOL1 (G2APOL1) mice leads to early activation of the cytosolic nucleotide sensor, stimulator of interferon genes (STING), and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. STING and NLRP3 expression was increased in podocytes from patients with high-risk APOL1 genotypes, and expression of APOL1 correlated with caspase-1 and gasdermin D (GSDMD) levels. To demonstrate the role of NLRP3 and STING in APOL1-associated kidney disease, we generated transgenic mice with the G2 APOL1 risk variant and genetic deletion of Nlrp3 (G2APOL1/Nlrp3 KO), Gsdmd (G2APOL1/Gsdmd KO), and STING (G2APOL1/STING KO). Knockout mice displayed marked reduction in albuminuria, azotemia, and kidney fibrosis compared with G2APOL1 mice. To evaluate the therapeutic potential of targeting NLRP3, GSDMD, and STING, we treated mice with MCC950, disulfiram, and C176, potent and selective inhibitors of NLRP3, GSDMD, and STING, respectively. G2APOL1 mice treated with MCC950, disulfiram, and C176 showed lower albuminuria and improved kidney function even when inhibitor treatment was initiated after the development of albuminuria.

Mapping the genetic architecture of human traits to cell types in the kidney identifies mechanisms of disease and potential treatments.

The functional interpretation of genome-wide association studies (GWAS) is challenging due to the cell-type-dependent influences of genetic variants. Here, we generated comprehensive maps of expression quantitative trait loci (eQTLs) for 659 microdissected human kidney samples and identified cell-type-eQTLs by mapping interactions between cell type abundances and genotypes. By partitioning heritability using stratified linkage disequilibrium score regression to integrate GWAS with single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing data, we prioritized proximal tubules for kidney function and endothelial cells and distal tubule segments for blood pressure pathogenesis. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of commonly used antihypertensive and renal-protective drugs and identifies drug repurposing opportunities for kidney disease.

Discovery of potent inhibitors of interleukin-2 inducible T-cell kinase (ITK) through structure-based drug design.

Interleukin-2 inducible T-cell kinase (ITK) is a member of the Tec kinase family and is involved with T-cell activation and proliferation. Due to its critical role in acting as a modulator of T-cells, ITK inhibitors could provide a novel route to anti-inflammatory therapy. This work describes the discovery of ITK inhibitors through structure-based design where high-resolution crystal structural information was used to optimize interactions within the kinase specificity pocket of the enzyme to improve both potency and selectivity.

5-Aminomethylbenzimidazoles as potent ITK antagonists.

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.

2-Aminobenzimidazoles as potent ITK antagonists: trans-stilbene-like moieties targeting the kinase specificity pocket.

Based on the information from molecular modeling and X-ray crystal structures, the kinase specificity pocket of ITK could be occupied upon extension of the right-hand-side of the 2-benzimidazole core of the inhibitors. 2-Aminobenzimidazoles with a trans-stilbene-like extension were designed and synthesized as novel ITK antagonists. Significant improvement on binding affinity and cellular activity were obtained through the trans-stilbene-like antagonists. Several compounds showed inhibitory activity in an IL-2 functional assay.

Itk kinase inhibitors: initial efforts to improve the metabolical stability and the cell activity of the benzimidazole lead.

Previously, we reported a series of novel benzimidazole based Itk inhibitors that exhibited excellent enzymatic potency and selectivity but low microsomal stability. Employing a structure based approach a new series of inhibitors with comparable potency and selectivity to the original series and with a potential for improved microsome stability was identified.

5-Aminomethyl-1H-benzimidazoles as orally active inhibitors of inducible T-cell kinase (Itk).

A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure-activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.

Discovery, SAR and X-ray structure of 1H-benzimidazole-5-carboxylic acid cyclohexyl-methyl-amides as inhibitors of inducible T-cell kinase (Itk).

A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.

Renal compartment-specific genetic variation analyses identify new pathways in chronic kidney disease.

Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-ß pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.

Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors.

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.

Transgenic expression of human APOL1 risk variants in podocytes induces kidney disease in mice.

African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.

Urinary Exosomal miRNA Signature in Type II Diabetic Nephropathy Patients.

MicroRNAs (miRNAs) are short non-coding RNA species which are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of diabetic nephropathy. miRNAs are present in urine in a remarkably stable form packaged in extracellular vesicles, predominantly exosomes. In the present study, urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays. In total, the expression of 16 miRNA species was deregulated (>2-fold) in DN patients compared to healthy donors and T2D patients: the expression of 14 miRNAs (miR-320c, miR-6068, miR-1234-5p, miR-6133, miR-4270, miR-4739, miR-371b-5p, miR-638, miR-572, miR-1227-5p, miR-6126, miR-1915-5p, miR-4778-5p and miR-2861) was up-regulated whereas the expression of 2 miRNAs (miR-30d-5p and miR-30e-5p) was down-regulated. Most of the deregulated miRNAs are involved in progression of renal diseases. Deregulation of urinary exosomal miRNAs occurred in micro-albuminuric DN patients but not in normo-albuminuric DN patients. We used qRT-PCR based analysis of the most strongly up-regulated miRNAs in urinary exosomes from DN patients, miRNAs miR-320c and miR-6068. The correlation of miRNA expression and micro-albuminuria levels could be replicated in a confirmation cohort. In conclusion, urinary exosomal miRNA content is altered in type II diabetic patients with DN. Deregulated miR-320c, which might have an impact on the TGF-ß-signaling pathway via targeting thrombospondin 1 (TSP-1) shows promise as a novel candidate marker for disease progression in type II DN that should be evaluated in future studies.