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A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with an IC 50 of 4.3nM and in primary human adipose tissue with an IC 80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11β 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) 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 Aβ levels was detected., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor β (LXRβ) 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 LXRβ and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour was used to design agonists containing a piperazine core. Compound 17 binds to LXRβ 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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Liver X receptor (LXR) α and LXRβ 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.

Structure based design led directly to 1,3-oxazinan-2-one 9a with an IC(50) of 42 nM against 11β-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 11β-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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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 11β-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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

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.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved. Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding. Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits. The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base. The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure., (Copyright 1999 Academic Press.)

When the membrane fraction of Saccharomyces carlsbergensis was incubated with radiolabeled isopentenyl diphosphate in the presence of farnesyl diphosphate and Mg2+, phosphorylated and free long-chain polyprenols were formed. The reaction was inhibited by EDTA and heavy metal cations. A series of non-ionic detergents were studied for their efficacy to solubilize the prenyltransferase. The enzyme completely lost its activity in the presence of 0.1% of Triton X-100. n-Octyl-beta-(D)glucopyranoside at the concentration of 0.25-0.5% (10-15 mM) was used to solubilize the prenyltransferase. Both the membrane-bound enzyme and the solubilizate possessed a broad pH optimum shifted to alkaline pH values. The temperature optimum of the solubilizate was somewhat lower than that of the membrane preparation, owing to the significantly lower thermostability of the solubilized enzyme in comparison with the membrane-bound one. The phosphorylated reaction products formed in the presence of the membrane preparation had the same composition as the yeast dolichol synthesized in vivo. Non-phosphorylated polyprenols were formed during the incubation with membranes but not the solubilized enzyme. The composition of the polyprenols was also coincident with that of yeast dolichol, and the individual C80-homolog of the mixture was polyprenol but not dolichol as judged by adsorption HPLC. The results are discussed in relation to the terminal stages in the biosynthesis of dolichol derivatives.