Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11ß hydroxysteroid dehydrogenase type 1 inhibitor.

A potent, in vivo efficacious 11ß hydroxysteroid dehydrogenase type 1 (11ß HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11ß HSD1 activity in human adipocytes with an IC50 of 4.3nM and in primary human adipose tissue with an IC80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11ß HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

Brain penetrant liver X receptor (LXR) modulators based on a 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole core.

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aß) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aß levels was detected.

Discovery of a Novel, Orally Efficacious Liver X Receptor (LXR) ß Agonist.

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor ß (LXRß) agonist (17). Contour technology is a structure-based drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRß and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour was used to design agonists containing a piperazine core. Compound 17 binds to LXRß with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes in vitro and in vivo. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis.

Identification of spirooxindole and dibenzoxazepine motifs as potent mineralocorticoid receptor antagonists.

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

Regulation of sphingomyelin phosphodiesterase acid-like 3A gene (SMPDL3A) by liver X receptors.

Liver X receptor (LXR) α and LXRß function as physiological sensors of cholesterol metabolites (oxysterols), regulating key genes involved in cholesterol and lipid metabolism. LXRs have been extensively studied in both human and rodent cell systems, revealing their potential therapeutic value in the contexts of atherosclerosis and inflammatory diseases. The LXR genome landscape has been investigated in murine macrophages but not in human THP-1 cells, which represent one of the frequently used monocyte/macrophage cell systems to study immune responses. We used a whole-genome screen to detect direct LXR target genes in THP-1 cells treated with two widely used LXR ligands [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317) and 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy] phenylacetic acid hydrochloride (GW3965)]. This screen identified the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene as a novel LXR-regulated gene, with an LXR response element within its promoter. We investigated the regulation of SMPDL3A gene expression by LXRs across several human and mouse cell types. These studies indicate that the induction of SMPDL3A is LXR-dependent and is restricted to human blood cells with no induction observed in mouse cellular systems.

Biphenyl/diphenyl ether renin inhibitors: filling the S1 pocket of renin via the S3 pocket.

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.

Structure-based design and synthesis of 1,3-oxazinan-2-one inhibitors of 11ß-hydroxysteroid dehydrogenase type 1.

Structure based design led directly to 1,3-oxazinan-2-one 9a with an IC(50) of 42 nM against 11ß-HSD1 in vitro. Optimization of 9a for improved in vitro enzymatic and cellular potency afforded 25f with IC(50) values of 0.8 nM for the enzyme and 2.5 nM in adipocytes. In addition, 25f has 94% oral bioavailability in rat and >1000× selectivity over 11ß-HSD2. In mice, 25f was distributed to the target tissues, liver, and adipose, and in cynomolgus monkeys a 10 mg/kg oral dose reduced cortisol production by 85% following a cortisone challenge.

Discovery of VTP-27999, an Alkyl Amine Renin Inhibitor with Potential for Clinical Utility.

Structure guided optimization of a series of nonpeptidic alkyl amine renin inhibitors allowed the rational incorporation of additional polar functionality. Replacement of the cyclohexylmethyl group occupying the S1 pocket with a (R)-(tetrahydropyran-3-yl)methyl group and utilization of a different attachment point led to the identification of clinical candidate 9. This compound demonstrated excellent selectivity over related and unrelated off-targets, >15% oral bioavailability in three species, oral efficacy in a double transgenic rat model of hypertension, and good exposure in humans.

Discovery and optimization of adamantyl carbamate inhibitors of 11ß-HSD1.

Synthesis of 2-adamantyl carbamate derivatives of piperidines and pyrrolidines led to the discovery of 9a with an IC(50) of 15.2 nM against human 11ß-HSD1 in adipocytes. Optimization for increased adipocyte potency, metabolic stability and selectivity afforded 11k and 11l, both of which were >25% orally bioavailable in rat.

Spirocyclic ureas: orally bioavailable 11 beta-HSD1 inhibitors identified by computer-aided drug design.

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11beta-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.

Design and optimization of renin inhibitors: Orally bioavailable alkyl amines.

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

Purification and characterization of recombinant human renin for X-ray crystallization studies.

BACKGROUND: The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies. RESULTS: A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained. CONCLUSION: This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.

Cloning, characterization and site-directed mutagenesis of canine renin.

Inhibition of renin has been shown to be successful in managing hypertension and maintaining cardiac health. Canine models have played a key role in preclinical assessment of renin inhibitors. Here we report the cloning of canine prorenin gene. The amino acid sequence of mature canine renin was approximately 70% identical to that of human renin. The full-length prorenin was expressed in HEK 293 cells, purified and converted to its active form by trypsin-mediated cleavage of the 43 residue propeptide. The mature enzyme was characterized by steady-state kinetics using a peptide corresponding to the canine angiotensinogen sequence, Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-OH (cleavage between Leu(10)-Leu(11)). The reaction followed Michaelis-Menten kinetics with a K(M) of 120 microM and a second-order rate constant (k(cat)/K(M)) of 1.7 x 10(5) M(-)(1)s(-)(1). The enzyme was inhibited by various human renin inhibitors, but at reduced potency compared to the human renin. The basis of the species specificity was investigated by mutagenesis. Based on primary sequence and structural alignments, three mutants were prepared (G149S-S150T, V286L, G149S-S150T-V286L). Each mutant yielded catalytically active enzymes with lower specific activities than native canine renin. V286L had the greatest effect on substrate specificity, while G149S, S150T mutations produced enzymes with inhibitor profiles similar to human renin.