Inhibitor Bound Dengue NS2B-NS3pro Reveals Multiple Dynamic Binding Modes.

Dengue virus poses a significant global health threat as the source of increasingly deleterious dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. As no specific antiviral treatment exists for dengue infection, considerable effort is being applied to discover therapies and drugs for maintenance and prevention of these afflictions. The virus is primarily transmitted by mosquitoes, and infection occurs following viral endocytosis by host cells. Upon entering the cell, viral RNA is translated into a large multisubunit polyprotein which is post-translationally cleaved into mature, structural and nonstructural (NS) proteins. The viral genome encodes the enzyme to carry out cleavage of the large polyprotein, specifically the NS2B-NS3pro cofactor-protease complex-a target of high interest for drug design. One class of recently discovered NS2B-NS3pro inhibitors is the substrate-based trifluoromethyl ketone containing peptides. These compounds interact covalently with the active site Ser135 via a hemiketal adduct. A detailed picture of the intermolecular protease/inhibitor interactions of the hemiketal adduct is crucial for rational drug design. We demonstrate, through the use of protein- and ligand-detected solution-state 19F and 1H NMR methods, an unanticipated multibinding mode behavior of a representative of this class of inhibitors to dengue NS2B-NS3pro. Our results illustrate the highly dynamic nature of both the covalently bound ligand and protease protein structure, and the need to consider these dynamics when designing future inhibitors in this class.

Discovery of novel potent imidazo[1,2-b]pyridazine PDE10a inhibitors.

Design and optimization of a novel series of imidazo[1,2-b]pyridazine PDE10a inhibitors are described. Compound 31 displays excellent pharmacokinetic properties and was also evaluated as an insulin secretagogue in vitro and in vivo.

Fusion to a highly stable consensus albumin binding domain allows for tunable pharmacokinetics.

A number of classes of proteins have been engineered for high stability using consensus sequence design methods. Here we describe the engineering of a novel albumin binding domain (ABD) three-helix bundle protein. The resulting engineered ABD molecule, called ABDCon, is expressed at high levels in the soluble fraction of Escherichia coli and is highly stable, with a melting temperature of 81.5°C. ABDCon binds human, monkey and mouse serum albumins with affinity as high as 61 pM. The solution structure of ABDCon is consistent with the three-helix bundle design and epitope mapping studies enabled a precise definition of the albumin binding interface. Fusion of a 10 kDa scaffold protein to ABDCon results in a long terminal half-life of 60 h in mice and 182 h in cynomolgus monkeys. To explore the link between albumin affinity and in vivo exposure, mutations were designed at the albumin binding interface of ABDCon yielding variants that span an 11 000-fold range in affinity. The PK properties of five such variants were determined in mice in order to demonstrate the tunable nature of serum half-life, exposure and clearance with variations in albumin binding affinity.

Elements and modulation of functional dynamics.

The existing structure-function paradigm of drug discovery has been evolving toward the essential incorporation of dynamics data. This new functional dynamics paradigm emphasizes conformational entropy as a driving force of protein function and intermolecular recognition. Conformational dynamics (a proxy of conformational entropy) impacts the degree of protein (dis)order and the constitution of the conformational ensemble, the mechanisms of allostery and drug resistance, and the free energy of ligand binding. Specific protein and ligand conformations facilitate favorable, reciprocal interactions. The number of protein and ligand conformers that exhibit favorable binding interactions will vary from system to system. All binding scenarios can modulate protein dynamics by various levels of enthalpic and entropic contribution, with significant influence on the functional dynamics of the system. Analysis and consideration of resulting changes of activity, signaling, catalysis, and subsequent phenotypic outcome are powerful motivations in the drug design process.

Pyrimidinopyrimidine inhibitors of ketohexokinase: exploring the ring C2 group that interacts with Asp-27B in the ligand binding pocket.

Inhibitors of ketohexokinase (KHK) have potential for the treatment of diabetes and obesity. We have continued studies on a pyrimidinopyrimidine series of potent KHK inhibitors by exploring the 2-position substituent (R(3)) that interacts with Asp-27B in the ATP-binding region of KHK (viz. 1, 2; Table 1). We found that increased spacing between the terminal ammonium group and the heterocyclic scaffold (viz. 16-20), such that interaction with Asp-27B is not possible, still results in potent KHK inhibition (IC(50)=15-50 nM). We propose a new interaction with Asp-194, which serves to expand the pyrimidinopyrimidine pharmacophore.

Optimization of a pyrazole hit from FBDD into a novel series of indazoles as ketohexokinase inhibitors.

A series of indazoles have been discovered as KHK inhibitors from a pyrazole hit identified through fragment-based drug discovery (FBDD). The optimization process guided by both X-ray crystallography and solution activity resulted in lead-like compounds with good pharmaceutical properties.

Electron density guided fragment-based drug design--a lead generation example.

We describe here a method using protein crystallography as the sole detection tool for fragment-based lead discovery. The methodology consists of iterative design, synthesis, and X-ray crystallographic screening of three libraries of compounds. Target-specific compound design, by way of active site electron density in the presence of a bound fragment hit and the intentional lack of solution activity bias form the basis of our approach. We provide an example of this alternative fragment-based drug design (FBDD) method, detailing results from a campaign using ketohexokinase to generate a unique lead series with promising drug-like properties.

Inhibitors of Ketohexokinase: Discovery of Pyrimidinopyrimidines with Specific Substitution that Complements the ATP-Binding Site.

Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket.

Electron density guided fragment-based lead discovery of ketohexokinase inhibitors.

A fragment-based drug design paradigm has been successfully applied in the discovery of lead series of ketohexokinase inhibitors. The paradigm consists of three iterations of design, synthesis, and X-ray crystallographic screening to progress low molecular weight fragments to leadlike compounds. Applying electron density of fragments within the protein binding site as defined by X-ray crystallography, one can generate target specific leads without the use of affinity data. Our approach contrasts with most fragment-based drug design methodology where solution activity is a main design guide. Herein we describe the discovery of submicromolar ketohexokinase inhibitors with promising druglike properties.

Quantum mechanical pairwise decomposition analysis of protein kinase B inhibitors: validating a new tool for guiding drug design.

Quantum mechanical semiempirical comparative binding energy analysis calculations have been carried out for a series of protein kinase B (PKB) inhibitors derived from fragment- and structure-based drug design. These protein-ligand complexes were selected because they represent a consistent set of experimental data that includes both crystal structures and affinities. Seven scoring functions were evaluated based on both the PM3 and the AM1 Hamiltonians. The optimal models obtained by partial least-squares analysis of the aligned poses are predictive as measured by a number of standard statistical criteria and by validation with an external data set. An algorithm has been developed that provides residue-based contributions to the overall binding affinity. These residue-based binding contributions can be plotted in heat maps so as to highlight the most important residues for ligand binding. In the case of these PKB inhibitors, the maps show that Met166, Thr97, Gly43, Glu114, Ala116, and Val50, among other residues, play an important role in determining binding affinity. The interaction energy map makes it easy to identify the residues that have the largest absolute effect on ligand binding. The structure-activity relationship (SAR) map highlights residues that are most critical to discriminating between more and less potent ligands. Taken together the interaction energy and the SAR maps provide useful insights into drug design that would be difficult to garner in any other way.

Design, synthesis, and biological evaluation of (2R,alphaS)-3,4-dihydro-2-[3-(1,1,2,2-tetrafluoroethoxy)phenyl]-5-[3-(trifluoromethoxy)-phenyl]-alpha-(trifluoromethyl)-1(2H)-quinolineethanol as potent and orally active cholesteryl ester transfer protein inhibitor.

With the goal of identifying a CETP inhibitor with high in vitro potency and optimal in vivo efficacy, a conformationally constrained molecule was designed based on the highly potent and flexible 13. The synthetic chemistry efforts led to the discovery of the potent and selective 12. In high-fat fed hamsters, human CETP transgenic mice, and cynomolgus monkeys, the in vivo efficacy of 12 for raising HDL-C was demonstrated to be comparable to torcetrapib.

Synthesis and structure-activity relationships of thiadiazole-derivatives as potent and orally active peroxisome proliferator-activated receptors alpha/delta dual agonists.

Replacement of the methyl-thiazole moiety of GW501516 (a PPARdelta selective agonist) with [1,2,4]thiadiazole gave compound 21 which unexpectedly displayed submicromolar potency as a partial agonist at PPARalpha in addition to the high potency at PPARdelta. A structure-activity relationships study of 21 resulted in the identification of 40 as a potent and selective PPARalpha/delta dual agonist. Compound 40 and its close analogs represent a new series of PPARalpha/delta dual agonists. The high potency, high selectivity, significant gene induction, excellent PK profiles, low P450 inhibition or induction, and good in vivo efficacy in four animal models support 40 being selected as a pre-clinical study candidate, and may render 40 as a valuable pharmacological tool in elucidating the complex roles of PPARalpha/delta dual agonists, and the potential usage for the treatment of metabolic syndrome.

Cycloaddition reactions of thiazolium azomethine ylides: application to pyrrolo[2,1-b]thiazoles.

Thiazolium azomethine ylides, equipped with a C-2 methanethiol group, participate in an efficient [3 + 2] cycloaddition reaction with acetylene derivatives to yield unique pyrrolo[2,1-b]thiazoles. The elimination of the methanethiol leaving group from the cycloadduct has replaced the need for a separate oxidation step and suppresses ring-opening side reactions. Products were obtained in short synthetic sequences to demonstrate their use as a scaffold for compound libraries.

Synthesis and identification of [1,2,4]thiadiazole derivatives as a new series of potent and orally active dual agonists of peroxisome proliferator-activated receptors alpha and delta.

Cardiovascular disease is the most common cause of morbidity and mortality in developed nations. To effectively target dyslipidemia to reduce the risk of cardiovascular disease, it may be beneficial to activate the peroxisome proliferator-activated receptors (PPARs) PPARalpha and PPARdelta simultaneously through a single molecule. Replacement of the methylthiazole of 5 (the PPARdelta selective agonist) with [1,2,4]thiadiazole gave compound 13, which unexpectedly displayed submicromolar potency as a partial agonist at PPARalpha in addition to the high potency at PPARdelta. Optimization of 13 led to the identification of 24 as a potent and selective PPARalpha/delta dual agonist. Compound 24 and its close analogs represent a new series of PPARalpha/delta dual agonists. The high potency, significant gene induction, excellent PK profiles, and good in vivo efficacies in three animal models may render compound 24 as a valuable pharmacological tool in elucidating the complex roles of PPARalpha/delta dual agonists and as a potential treatment of the metabolic syndrome.

Conformational sampling of bioactive molecules: a comparative study.

The necessity to generate conformations that sample the entire conformational space accessible to a given molecule is ubiquitous in the field of computer-aided drug design. Protein-ligand docking, 3D database searching, and 3D QSAR are three commonly used techniques that depend critically upon the quality and diversity of the generated conformers. Although there are a wide range of conformational search algorithms available, the extent to which they sample conformational space is often unclear. To address this question, we conducted a robust comparison of the search algorithms implemented in several widely used molecular modeling packages, including Catalyst, Macromodel, Omega, MOE, and Rubicon as well as our own method, stochastic proximity embedding (SPE). We found that SPE used in conjunction with conformational boosting, a heuristic for biasing conformational search toward more extended or compact geometries, along with Catalyst, are significantly more effective in sampling the full range of conformational space compared to the other methods, which show distinct preferences for either more extended or more compact geometries.

Processing of small molecule databases for automated docking.

Virtual screening involves the mining of small molecule databases from various sources. The small molecule databases used in virtual screening are typically processed, from simple 2D representations, to maximize their information content and to optimize them for input to the particular virtual screening technology being used. Processing interprets or adds molecular information related to connectivity, stereochemistry, protonation, tautomers and conformation. For virtual screening with an automated docking protocol, a technique that relies on specific intermolecular atom-atom contacts for ranking molecules, it is expected that the pre-processing protocol can affect the results of the docking experiment. The possible effects of processing on docking results have not been extensively studied, and this topic has only recently emerged as a significant aspect of the docking-based virtual screening process. One recent report highlights significant effects of different processing procedures on docking enrichment, while another outlines a general ligand preparation strategy. Here we survey and comment on recent practice in the field.

A cluster-based strategy for assessing the overlap between large chemical libraries and its application to a recent acquisition.

We report on the structural comparison of the corporate collections of Johnson & Johnson Pharmaceutical Research & Development (JNJPRD) and 3-Dimensional Pharmaceuticals (3DP), performed in the context of the recent acquisition of 3DP by JNJPRD. The main objective of the study was to assess the druglikeness of the 3DP library and the extent to which it enriched the chemical diversity of the JNJPRD corporate collection. The two databases, at the time of acquisition, collectively contained more than 1.1 million compounds with a clearly defined structural description. The analysis was based on a clustering approach and aimed at providing an intuitive quantitative estimate and visual representation of this enrichment. A novel hierarchical clustering algorithm called divisive k-means was employed in combination with Kelley's cluster-level selection method to partition the combined data set into clusters, and the diversity contribution of each library was evaluated as a function of the relative occupancy of these clusters. Typical 3DP chemotypes enriching the diversity of the JNJPRD collection were catalogued and visualized using a modified maximum common substructure algorithm. The joint collection of JNJPRD and 3DP compounds was also compared to other databases of known medicinally active or druglike compounds. The potential of the methodology for the analysis of very large chemical databases is discussed.

Identification of 2-acylaminothiophene-3-carboxamides as potent inhibitors of FLT3.

A series of 2-acylaminothiophene-3-carboxamides has been identified which exhibit potent inhibitory activity against the FLT3 tyrosine kinase. Compound 44 inhibits the isolated enzyme (IC50 = 0.027 microM) and blocks the proliferation of MV4-11 cells (IC50 = 0.41 microM). Structure-activity relationship studies within this series are described in the context of a proposed binding model within the ATP binding site of the enzyme.

Comparison of automated docking programs as virtual screening tools.

The performance of several commercially available docking programs is compared in the context of virtual screening. Five different protein targets are used, each with several known ligands. The simulated screening deck comprised 1000 molecules from a cleansed version of the MDL drug data report and 49 known ligands. For many of the known ligands, crystal structures of the relevant protein-ligand complexes were available. We attempted to run experiments with each docking method that were as similar as possible. For a given docking method, hit rates were improved versus what would be expected for random selection for most protein targets. However, the ability to prioritize known ligands on the basis of docking poses that resemble known crystal structures is both method- and target-dependent.

Docking: successes and challenges.

The state of the art of various computational aspects of docking-based virtual screening of database of small molecules is presented. The review encompasses the different search algorithms and the scoring functions used in docking methods and their applications to protein and nucleic acid drug targets. Recent progress made in the development and application of methods to include target flexibility are summarized. The fundamental issues and challenges involved in comparing various docking methods are discussed. Limitations of current technologies as well as future prospects are presented.