Discovery of Potent Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 1 Using a Novel Growth-Based Protocol of in Silico Screening and Optimization in CONTOUR.

With the continuous progress in ultralarge virtual libraries which are readily accessible, it is of great interest to explore this large chemical space for hit identification and lead optimization using reliable structure-based approaches. In this work, a novel growth-based screening protocol has been designed and implemented in the structure-based design platform CONTOUR. The protocol was used to screen the ZINC database in silico and optimize hits to discover 11ß-HSD1 inhibitors. In contrast to molecular docking, the virtual screening process makes significant improvements in computational efficiency without losing chemical equities through partitioning 1.8 million ZINC compounds into fragments, docking fragments to form key hydrogen bonds with anchor residues, reorganizing molecules into molecular fragment trees using matched fragments and common substructures, and then regrowing molecules with the help of developed intelligent growth features inside the protein binding site to find hits. The growth-base screening approach is validated by the high hit rate. A total of 50 compounds have been selected for testing; of these, 15 hits having diverse scaffolds are found to inhibit 11ß-HSD1 with IC50 values of less than 1 µM in a biochemical enzyme assay. The best hit which exhibits an enzyme IC50 of 33 nM is further developed to a novel series of bicyclic 11ß-HSD1 inhibitors with the best inhibition of enzyme IC50 of 3.1 nM. The final lead candidate exhibits IC50 values of 7.2 and 21 nM in enzyme and adipocyte assays, respectively, displayed greater than 1000-fold of selectivity over 11ß-HSD2 and two other related hydroxysteroid dehydrogenases, and can serve as good starting points for further optimization to develop clinical candidates.

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.

Influence of sub-chronic selective 11ß-hydroxysteroid dehydrogenase 1 inhibition on the hypothalamic-pituitary-adrenal axis in female cynomolgus monkeys.

Inhibition of local cortisol regeneration from circulating cortisone by blocking 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. Chronic modulation of glucocorticoid homeostasis may result in hypothalamic-pituitary-adrenal (HPA) axis stimulation. HPA axis over-activation leading androgen excess would be undesirable in a therapeutic intervention designed to treat a chronic condition such as the metabolic syndrome. To address whether 11ß-HSD1 inhibition would lead to excess androgens, we treated female cynomolgus monkeys with a selective inhibitor, BI 135558, for 4 weeks. Continual action of the compound over the dosing period was confirmed by constant plasma exposure, and a maintained change in urinary glucocorticoid metabolites consistent with 11ß-HSD1 inhibition. No significant changes in adrenal function, as evidenced by an adrenocorticotropic hormone (ATCH) challenge, were observed. An examination of androgenic hormones revealed a slight increase in dehydroepiandrosterone sulfate (DHEA-S), while other hormones such as testosterone remained within reference values. Overall, treatment with BI 135558 in monkeys did not result in obvious over-activation of the HPA axis.

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.

Pharmacological characterization of the selective 11ß-hydroxysteroid dehydrogenase 1 inhibitor, BI 135585, a clinical candidate for the treatment of type 2 diabetes.

To combat the increased morbidity and mortality associated with the developing diabetes epidemic new therapeutic interventions are desirable. Inhibition of intracellular cortisol generation from cortisone by blocking 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. A challenge in developing 11ß-HSD1 inhibitors has been the species selectivity of small molecules, as many compounds are primate specific. Here we describe our strategy to identify potent selective 11ß-HSD1 inhibitors while ensuring target engagement in key metabolic tissues, liver and fat. This strategy enabled the identification of the clinical candidate, BI 135585.

An electronic environment and contact direction sensitive scoring function for predicting affinities of protein-ligand complexes in Contour(®).

Contour(®) is a computational structure-based drug design technology that grows drug-like molecules by assembling context sensitive fragments in well-defined binding pockets. The grown molecules are scored by a novel empirical scoring function developed using high-resolution crystal structures of diverse classes of protein-ligand complexes and associated experimental binding affinities. An atomic model bearing features of the valence bond and VSEPR theories embodying their molecular electronic environment has been developed for non-covalent intermolecular interactions. On the basis of atomic hybridization and polarization states, each atom is modeled by features representing electron lone pairs, p-orbitals, and polar and non-polar hydrogens. A simple formal charge model was used to differentiate between polar and non-polar atoms. The interaction energy and the desolvation contribution of the protein-ligand association energy is computed as a linear sum of pair-wise interactions and desolvation terms. The pair-wise interaction energy captures short-range positive electrostatic interactions via hydrogen bonds, electrostatic repulsion of like charges, and non-bond contacts. The desolvation energy is estimated by calculating the energy required to desolvate interaction surfaces of the protein and the ligand in the complex. The scoring function predicts binding energies of a diverse set of protein-ligand complexes used for training with a correlation coefficient of 0.61. It also performs equally well in predicting association energies of a diverse validation set of protein-ligand complexes with a correlation coefficient of 0.57, which is equivalent to or better than 12 other scoring functions tested against this set including X-Score, GOLD, and DrugScore.

Structure-based design technology contour and its application to the design of renin inhibitors.

It is well-known that the structure-based design approach has had a measurable impact on the drug discovery process in identifying novel and efficacious therapeutic agents for a variety of disease targets. The de novo design approach has inherent potential to generate novel molecules that best fit into a protein binding site when compared to all of the computational methods applied to structure-based design. In its initial attempts, this approach did not achieve much success due to technical hurdles. More recently, the algorithmic advancements in the methodologies and clever strategies developed to design drug-like molecules have improved the success rate. We describe a state-of-the-art structure-based design technology called Contour and provide details of the algorithmic enhancements we have implemented. Contour was designed to create novel drug-like molecules by assembling synthetically viable fragments in the protein binding site using a high-resolution crystal structure of the protein. The technology consists of a sophisticated growth algorithm and a novel scoring function based on a directional model. The growth algorithm generates molecules by dynamically selecting only those fragments from the fragment library that are complementary to the binding site, and assembling them by sampling the conformational space for each attached fragment. The scoring function embodying the essential elements of the binding interactions aids in the rank ordering of grown molecules and helps identify those that have high probability of exhibiting activity against the protein target of interest. The application of Contour to identify inhibitors against human renin enzyme eventually leading to the clinical candidate VTP-27,999 will be discussed here.

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.

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists.

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure-activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K(+) channel. Preferred compounds were subsequently evaluated for selectivity in an alpha(1)-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.

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.

Cyclic benzamidines as orally efficacious NR2B-selective NMDA receptor antagonists.

A novel series of cyclic benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 29 is orally active in a carrageenan-induced rat hyperalgesia model of pain.

Identification and characterization of 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate, an orally bioavailable, brain penetrant NR2B selective N-methyl-D-aspartate receptor antagonist.

The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease.

NR2B-selective N-methyl-D-aspartate antagonists: synthesis and evaluation of 5-substituted benzimidazoles.

Two classes of 5-substituted benzimidazoles were identified as potent antagonists of the NR2B subtype of the N-methyl-d-aspartate (NMDA) receptor. Selected compounds show very good selectivity versus the NR2A, NR2C, and NR2D subtypes of the NMDA receptor as well as versus hERG-channel activity and alpha(1)-adrenergic binding. Benzimidazole 37a shows excellent activity in the carrageenan-induced mechanical hyperalgesia assay in rats as well as good pharmacokinetic behavior in dogs.

In vivo canine cardiac electrophysiologic profile of 1,4-benzodiazepine IKs blockers.

Previous cardiac electrophysiologic studies of blockers of the slowly activating delayed rectifier (IKs) current have focused primarily on ventricular repolarization. This report summarizes an extensive in vivo cardiac electrophysiologic profile of four 1,4-benzodiazepine IKs blocker analogues (L-761334, L-763540, L-761710, and L-768673) in dogs. At 3.0 mg/kg intravenously, all four analogues elicited 14.5%-21.4% increases in ventricular refractoriness and 19.2%-22.6% increases in QTc interval. Concomitant 11.1%-13.5% increases in atrial refractoriness were noted with all four analogues. Decreases in sinus heart rate of 8.4%-17.3% were noted with all four compounds. No effects on atrial, His Purkinje, ventricular conduction or atrial and ventricular excitation were observed. One analogue, L-761710, significantly delayed atrioventricular (AV) nodal conduction (40.7+/-17.4% increase in atrial-to-His interval) and increased the AV conduction system functional refractory period 19.9+/-6.2%. The lack of effect of the other three 1,4-benzodiazepine IKs blockers on AV nodal function at dosages producing comparable effects on atrial and ventricular refractoriness suggest that the AV nodal effects of L-761710 were unrelated to IKs blockade. These findings indicate IKs plays important roles in both atrial and ventricular refractoriness as well as pacemaker function in the dog heart, suggesting potential utility for IKs blockers in the treatment of atrial and ventricular arrhythmias.