Imidazopyridyl compounds as aldosterone synthase inhibitors.

The inhibition of aldosterone synthase (CYP11B2) may be an effective treatment of hypertension and heart failure, among other ailments. Previously reported benzimidazole CYP11B2 inhibitors led the way for bioisosteric imidazopyridines that are both potent and selective over CYP11B1.

Discovery of indazole aldosterone synthase (CYP11B2) inhibitors as potential treatments for hypertension.

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.

Plasma Levels of Risk-Variant APOL1 Do Not Associate with Renal Disease in a Population-Based Cohort.

Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1­HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1­HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1­HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1­HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.

Rapid detection and quantification of apolipoprotein L1 genetic variants and total levels in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Human genetics studies in African Americans have shown a strong correlation between polymorphisms in the ApoL1 gene and chronic kidney disease (CKD). To gain further insight into the etiology of ApoL1-associated kidney diseases, the determination of circulating levels of both wild type as well as ApoL1 variants could be of significant use. To date, antibodies that discriminate between all three ApoL1 variant forms (wild type, G1 and G2) are not available. We aimed to develop a rapid method for detecting and quantifying ApoL1 variants and total levels in plasma. METHODS: Ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) in multiple-reaction monitoring acquisition mode was used to quantify ApoL1. RESULTS: We demonstrated that it is feasible to detect and quantify ApoL1 variants (wild type, G1 and G2), and total ApoL1 concentrations in plasma. ApoL1 genotypes determined by LC/MS agreed perfectly with the traditional method DNA sequencing for 74 human subjects. The method exhibited at least three orders of linearity with a lower limit of quantification of 10 nM. Moreover, the method can readily be multiplexed for the quantification of a panel of protein markers in a single sample. CONCLUSIONS: The method reported herein obviates the need to perform DNA genotyping of ApoL1 variants, which is of significant value in cases where stored samples are unsuitable for DNA analysis. More importantly, the method could potentially be of use in the early identification of individuals at risk of developing CKD, and for the stratification of patients for treatment with future ApoL1-modifying therapies.

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase.

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.

Platensimycin is a selective FabF inhibitor with potent antibiotic properties.

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Discovery of a potent and selective small molecule hGPR91 antagonist.

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 µM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.

Transcriptome profiling and network analysis of genetically hypertensive mice identifies potential pharmacological targets of hypertension.

Hypertension is a condition with major cardiovascular and renal complications, affecting nearly a billion patients worldwide. Few validated gene targets are available for pharmacological intervention, so there is a need to identify new biological pathways regulating blood pressure and containing novel targets for treatment. The genetically hypertensive "blood pressure high" (BPH), normotensive "blood pressure normal" (BPN), and hypotensive "blood pressure low" (BPL) inbred mouse strains are an ideal system to study differences in gene expression patterns that may represent such biological pathways. We profiled gene expression in liver, heart, kidney, and aorta from BPH, BPN, and BPL mice and determined which biological processes are enriched in observed organ-specific signatures. As a result, we identified multiple biological pathways linked to blood pressure phenotype that could serve as a source of candidate genes causal for hypertension. To distinguish in the kidney signature genes whose differential expression pattern may cause changes in blood pressure from those genes whose differential expression pattern results from changes in blood pressure, we integrated phenotype-associated genes into Genetic Bayesian networks. The integration of data from gene expression profiling and genetics networks is a valuable approach to identify novel potential targets for the pharmacological treatment of hypertension.

Discovery of Spirocyclic Aldosterone Synthase Inhibitors as Potential Treatments for Resistant Hypertension.

Herein we report the discovery and hit-to-lead optimization of a series of spirocyclic piperidine aldosterone synthase (CYP11B2) inhibitors. Compounds from this series display potent CYP11B2 inhibition, good selectivity versus related CYP enzymes, and lead-like physical and pharmacokinetic properties.

Discovery and Clinical Evaluation of MK-8150, A Novel Nitric Oxide Donor With a Unique Mechanism of Nitric Oxide Release.

BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.