Phthalazinone-based lactams and cyclic ureas as ROCK2 selective inhibitors.

Derivatives of lactam, cyclic urea and carbamate were explored as aniline amide replacements in a series of phthalazinone-based ROCK inhibitors. Potent ROCK2 inhibitors such as 22 were identified with excellent overall kinase selectivity as well as good isoform selectivity over ROCK1.

The influence of a solvent environment on direct non-covalent interactions between two molecules: A symmetry-adapted perturbation theory study of polarization tuning of π-π interactions by water.

High-level quantum chemical computations have provided significant insight into the fundamental physical nature of non-covalent interactions. These studies have focused primarily on gas-phase computations of small van der Waals dimers; however, these interactions frequently take place in complex chemical environments, such as proteins, solutions, or solids. To better understand how the chemical environment affects non-covalent interactions, we have undertaken a quantum chemical study of π-π interactions in an aqueous solution, as exemplified by T-shaped benzene dimers surrounded by 28 or 50 explicit water molecules. We report interaction energies (IEs) using second-order Møller-Plesset perturbation theory, and we apply the intramolecular and functional-group partitioning extensions of symmetry-adapted perturbation theory (ISAPT and F-SAPT, respectively) to analyze how the solvent molecules tune the π-π interactions of the solute. For complexes containing neutral monomers, even 50 explicit waters (constituting a first and partial second solvation shell) change total SAPT IEs between the two solute molecules by only tenths of a kcal mol-1, while significant changes of up to 3 kcal mol-1 of the electrostatic component are seen for the cationic pyridinium-benzene dimer. This difference between charged and neutral solutes is attributed to large non-additive three-body interactions within solvated ion-containing complexes. Overall, except for charged solutes, our quantum computations indicate that nearby solvent molecules cause very little "tuning" of the direct solute-solute interactions. This indicates that differences in binding energies between the gas phase and solution phase are primarily indirect effects of the competition between solute-solute and solute-solvent interactions.

Optimized damping parameters for empirical dispersion corrections to symmetry-adapted perturbation theory.

Symmetry-adapted perturbation theory (SAPT) has become an invaluable tool for studying the fundamental nature of non-covalent interactions by directly computing the electrostatics, exchange (steric) repulsion, induction (polarization), and London dispersion contributions to the interaction energy using quantum mechanics. Further application of SAPT is primarily limited by its computational expense, where even its most affordable variant (SAPT0) scales as the fifth power of system size [O(N5)] due to the dispersion terms. The algorithmic scaling of SAPT0 is reduced from O(N5)→O(N4) by replacing these terms with the empirical D3 dispersion correction of Grimme and co-workers, forming a method that may be termed SAPT0-D3. Here, we optimize the damping parameters for the -D3 terms in SAPT0-D3 using a much larger training set than has previously been considered, namely, 8299 interaction energies computed at the complete-basis-set limit of coupled cluster through perturbative triples [CCSD(T)/CBS]. Perhaps surprisingly, with only three fitted parameters, SAPT0-D3 improves on the accuracy of SAPT0, reducing mean absolute errors from 0.61 to 0.49 kcal mol-1 over the full set of complexes. Additionally, SAPT0-D3 exhibits a nearly 2.5× speedup over conventional SAPT0 for systems with ∼300 atoms and is applied here to systems with up to 459 atoms. Finally, we have also implemented a functional group partitioning of the approach (F-SAPT0-D3) and applied it to determine important contacts in the binding of salbutamol to G-protein coupled ß1-adrenergic receptor in both active and inactive forms. SAPT0-D3 capabilities have been added to the open-source Psi4 software.

Identification of 5H-chromeno[3,4-c]pyridine and 6H-isochromeno[3,4-c]pyridine derivatives as potent and selective dual ROCK inhibitors.

A novel series of 5H-chromeno[3,4-c]pyridine, 6H-isochromeno[3,4-c]pyridine and 6H-isochromeno[4,3-d]pyrimidine derivatives as dual ROCK1 and ROCK2 inhibitors is described. Optimization led to compounds with sub-nanomolar inhibitory affinity for both kinases and excellent kinome selectivity. Compound 19 exhibited ROCK1 and ROCK2 IC50 of 0.67 nM and 0.18 nM respectively.

Discovery of a phenylpyrazole amide ROCK inhibitor as a tool molecule for in vivo studies.

Structure-activity relationship optimization on a series of phenylpyrazole amides led to the identification of a dual ROCK1 and ROCK2 inhibitor (25) which demonstrated good potency, kinome selectivity and favorable pharmacokinetic profiles. Compound 25 was selected as a tool molecule for in vivo studies including evaluating hemodynamic effects in telemeterized mice, from which moderate decreases in blood pressure were observed.

The Surprising Importance of Peptide Bond Contacts in Drug-Protein Interactions.

The study of noncovalent interactions, notably including drug-protein binding, relies heavily on the language of localized functional group contacts: hydrogen bonding, π-π interactions, CH-π contacts, halogen bonding, etc. Applying the state-of-the-art functional group symmetry-adapted perturbation theory (F-SAPT) to an important question of chloro versus methyl aryl substitution in factor Xa inhibitor drugs, we find that a localized contact model provides an incorrect picture for the origin of the enhancement of chloro-containing ligands. Instead, the enhancement is found to originate from many intermediate-range contacts distributed throughout the binding pocket, particularly including the peptide bonds in the protein backbone. The contributions from these contacts are primarily electrostatic in nature, but require ab initio computations involving nearly the full drug-protein pocket system to be accurately quantified.

Pharmacologic profile of the Adnectin BMS-962476, a small protein biologic alternative to PCSK9 antibodies for low-density lipoprotein lowering.

Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low-density lipoprotein (LDL) in cardiovascular disease, although seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type III-domain and engineered for high-affinity target binding. BMS-962476, an ∼11-kDa polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with subnanomolar affinity to human. The X-ray cocrystal structure of PCSK9 with a progenitor Adnectin shows ∼910 Å(2) of PCSK9 surface covered next to the LDL receptor binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED50 ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 >99% and LDL-cholesterol ∼55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently downregulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.

Discovery of BMS-986339, a Pharmacologically Differentiated Farnesoid X Receptor Agonist for the Treatment of Nonalcoholic Steatohepatitis.

While several farnesoid X receptor (FXR) agonists under clinical investigation for the treatment of nonalcoholic steatohepatitis (NASH) have shown beneficial effects, adverse effects such as pruritus and elevation of plasma lipids have limited their clinical efficacy and approvability. Herein, we report the discovery and preclinical evaluation of compound 32 (BMS-986339), a nonbile acid FXR agonist with a pharmacologically distinct profile relative to our previously reported agonist BMS-986318. Compound 32 exhibited potent in vitro and in vivo activation of FXR, albeit with a context-dependent profile that resulted in tissue-selective effects in vivo. To our knowledge, this is the first report that demonstrates differential induction of Fgf15 in the liver and ileum by FXR agonists in vivo. Compound 32 demonstrated robust antifibrotic efficacy despite reduced activation of certain genes in the liver, suggesting that the additional pharmacology of BMS-986318 does not further benefit efficacy, possibly presenting an opportunity for reduced adverse effects. Further evaluation in humans is warranted to validate this hypothesis.

Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors.

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide-valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC(50) of 7 nM and EC(2×PT) of 1.7 µM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide-valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.

Discovery of BMS-986318, a Potent Nonbile Acid FXR Agonist for the Treatment of Nonalcoholic Steatohepatitis.

Herein we report the discovery and preclinical biological evaluation of 6-(2-(5-cyclopropyl-3-(3,5-dichloropyridin-4-yl)isoxazol-4-yl)-7-azaspiro[3.5]non-1-en-7-yl)-4-(trifluoromethyl)quinoline-2-carboxylic acid, compound 1 (BMS-986318), a nonbile acid farnesoid X receptor (FXR) agonist. Compound 1 exhibits potent in vitro and in vivo activation of FXR, has a suitable ADME profile, and demonstrates efficacy in the mouse bile duct ligation model of liver cholestasis and fibrosis. The overall profile of compound 1 supports its continued evaluation.

Aroylguanidine-based factor Xa inhibitors: the discovery of BMS-344577.

We report the design and synthesis of a novel class of N,N'-disubstituted aroylguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The structure-activity relationships (SAR) investigation led to the discovery of the nicotinoyl guanidine 22 as a potent FXa inhibitor (FXa IC(50)=4 nM, EC(2xPT)=7 microM). However, the potent CYP3A4 inhibition activity (IC(50)=0.3 microM) of 22 precluded its further development. Detailed analysis of the X-ray crystal structure of compound 22 bound to FXa indicated that the substituent at the 6-position of the nicotinoyl group of 22 would be solvent-exposed, suggesting that efforts to attenuate the unwanted CYP activity could focus at this position without affecting FXa potency significantly. Further SAR studies on the 6-substituted nicotinoyl guanidines resulted in the discovery of 6-(dimethylcarbamoyl) nicotinoyl guanidine 36 (BMS-344577, IC(50)=9 nM, EC(2xPT)=2.5 microM), which was found to be a selective, orally efficacious FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models.

Cyanoguanidine-based lactam derivatives as a novel class of orally bioavailable factor Xa inhibitors.

The N,N'-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K(i)=6.5nM, EC(2xPT)=32muM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand-protein interactions are discussed.

(3R,5S,E)-7-(4-(4-fluorophenyl)-6-isopropyl-2-(methyl(1-methyl-1h-1,2,4-triazol-5-yl)amino)pyrimidin-5-yl)-3,5-dihydroxyhept-6-enoic acid (BMS-644950): a rationally designed orally efficacious 3-hydroxy-3-methylglutaryl coenzyme-a reductase inhibitor with reduced myotoxicity potential.

3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) inhibitors, more commonly known as statins, represent the gold standard in treating hypercholesterolemia. Although statins are regarded as generally safe, they are known to cause myopathy and, in rare cases, rhabdomyolysis. Statin-dependent effects on plasma lipids are mediated through the inhibition of HMGR in the hepatocyte, whereas evidence suggests that myotoxicity is due to inhibition of HMGR within the myocyte. Thus, an inhibitor with increased selectivity for hepatocytes could potentially result in an improved therapeutic window. Implementation of a strategy that focused on in vitro potency, compound polarity, cell selectivity, and oral absorption, followed by extensive efficacy and safety modeling in guinea pig and rat, resulted in the identification of compound 1b (BMS-644950). Using this discovery pathway, we compared 1b to other marketed statins to demonstrate its outstanding efficacy and safety profile. With the potential to generate an excellent therapeutic window, 1b was advanced into clinical development.

Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode.

A novel series of pyrrolidine-containing GPR40 agonists is described as a potential treatment for type 2 diabetes. The initial pyrrolidine hit was modified by moving the position of the carboxylic acid, a key pharmacophore for GPR40. Addition of a 4-cis-CF3 to the pyrrolidine improves the human GPR40 binding Ki and agonist efficacy. After further optimization, the discovery of a minor enantiomeric impurity with agonist activity led to the finding that enantiomers (R,R)-68 and (S,S)-68 have differential effects on the radioligand used for the binding assay, with (R,R)-68 potentiating the radioligand and (S,S)-68 displacing the radioligand. Compound (R,R)-68 activates both Gq-coupled intracellular Ca2+ flux and Gs-coupled cAMP accumulation. This signaling bias results in a dual mechanism of action for compound (R,R)-68, demonstrating glucose-dependent insulin and GLP-1 secretion in vitro. In vivo, compound (R,R)-68 significantly lowers plasma glucose levels in mice during an oral glucose challenge, encouraging further development of the series.

A comparative study of available software for high-accuracy homology modeling: from sequence alignments to structural models.

An open question in protein homology modeling is, how well do current modeling packages satisfy the dual criteria of quality of results and practical ease of use? To address this question objectively, we examined homology-built models of a variety of therapeutically relevant proteins. The sequence identities across these proteins range from 19% to 76%. A novel metric, the difference alignment index (DAI), is developed to aid in quantifying the quality of local sequence alignments. The DAI is also used to construct the relative sequence alignment (RSA), a new representation of global sequence alignment that facilitates comparison of sequence alignments from different methods. Comparisons of the sequence alignments in terms of the RSA and alignment methodologies are made to better understand the advantages and caveats of each method. All sequence alignments and corresponding 3D models are compared to their respective structure-based alignments and crystal structures. A variety of protein modeling software was used. We find that at sequence identities >40%, all packages give similar (and satisfactory) results; at lower sequence identities (<25%), the sequence alignments generated by Profit and Prime, which incorporate structural information in their sequence alignment, stand out from the rest. Moreover, the model generated by Prime in this low sequence identity region is noted to be superior to the rest. Additionally, we note that DSModeler and MOE, which generate reasonable models for sequence identities >25%, are significantly more functional and easier to use when compared with the other structure-building software.

Synthesis of novel potent dipeptidyl peptidase IV inhibitors with enhanced chemical stability: interplay between the N-terminal amino acid alkyl side chain and the cyclopropyl group of alpha-aminoacyl-l-cis-4,5-methanoprolinenitrile-based inhibitors.

A series of methanoprolinenitrile-containing dipeptide mimetics were synthesized and assayed as inhibitors of the N-terminal sequence-specific serine protease dipeptidyl peptidase IV (DPP-IV). The catalytic action of DPP-IV is the principle means of degradation of glucagon-like peptide-1, a key mediator of glucose-stimulated insulin secretion, and DPP-IV inhibition shows clinical benefit as a novel mechanism for treatment of type 2 diabetes. However, many of the reversible inhibitors to date suffer from chemical instability stemming from an amine to nitrile intramolecular cyclization. Installation of a cyclopropyl moiety at either the 3,4- or 4,5-position of traditional 2-cyanopyrrolidide proline mimetics led to compounds with potent inhibitory activity against the enzyme. Additionally, cis-4,5-methanoprolinenitriles with beta-branching in the N-terminal amino acid provided enhanced chemical stability and high inhibitory potency. This class of inhibitors also exhibited the ability to suppress prandial glucose elevations after an oral glucose challenge in male Zucker rats.

Analysis and optimization of structure-based virtual screening protocols. 2. Examination of docked ligand orientation sampling methodology: mapping a pharmacophore for success.

An important element of any structure-based virtual screening (SVS) technique is the method used to orient the ligands in the target active site. This has been a somewhat overlooked issue in recent SVS validation studies, with the assumption being made that the performance of an algorithm for a given set of orientation sampling settings will be representative for the general behavior of said technique. Here, we analyze five different SVS targets using a variety of sampling paradigms within the DOCK, GOLD and PROMETHEUS programs over a data set of approximately 10,000 noise compounds, combined with data sets containing multiple active compounds. These sets have been broken down by chemotype, with chemotype hit rate used to provide a measure of enrichment with a potentially improved relevance to real world SVS experiments. The variability in enrichment results produced by different sampling paradigms is illustrated, as is the utility of using pharmacophores to constrain sampling to regions that reflect known structural biology. The difference in results when comparing chemotype with compound hit rates is also highlighted.

Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties.

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

Diphenylpyridylethanamine (DPPE) derivatives as cholesteryl ester transfer protein (CETP) inhibitors.

A series of diphenylpyridylethanamine (DPPE) derivatives was identified exhibiting potent CETP inhibition. Replacing the labile ester functionality in the initial lead 7 generated a series of amides and ureas. Further optimization of the DPPE series for potency resulted in the discovery of cyclopentylurea 15d, which demonstrated a reduction in cholesterol ester transfer activity (48% of predose level) in hCETP/apoB-100 dual transgenic mice. The PK profile of 15d was suboptimal, and further optimization of the N-terminus resulted in the discovery of amide 20 with an improved PK profile and robust efficacy in transgenic hCETP/apoB-100 mice and in hamsters. Compound 20 demonstrated no significant changes in either mean arterial blood pressure or heart rate in telemeterized rats despite sustained high exposures.

Cannabinoid CB(1) receptor ligand binding and function examined through mutagenesis studies of F200 and S383.

The cannabinoid CB(1) G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB(1) receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB(1) receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB(1) receptor ligands.