eCounterscreening: using QSAR predictions to prioritize testing for off-target activities and setting the balance between benefit and risk.

During drug development, compounds are tested against counterscreens, a panel of off-target activities that would be undesirable for a drug to have. Testing every compound against every counterscreen is generally too costly in terms of time and money, and we need to find a rational way of prioritizing counterscreen testing. Here we present the eCounterscreening paradigm, wherein predictions from QSAR models for counterscreen activity are used to generate a recommendation as to whether a specific compound in a specific project should be tested against a specific counterscreen. The rules behind the recommendations, which can be summarized in a risk-benefit plot specific for a counterscreen/project combination, are based on a previously assembled database of prospective QSAR predictions. The recommendations require two user-defined cutoffs: the level of activity in a specific counterscreen that is considered undesirable and the level of risk the chemist is willing to accept that an undesired counterscreen activity will go undetected. We demonstrate in a simulated prospective experiment that eCounterscreening can be used to postpone a large fraction of counterscreen testing and still have an acceptably low risk of undetected counterscreen activity.

Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

Comparison of random forest and Pipeline Pilot Naïve Bayes in prospective QSAR predictions.

Random forest is currently considered one of the best QSAR methods available in terms of accuracy of prediction. However, it is computationally intensive. Naïve Bayes is a simple, robust classification method. The Laplacian-modified Naïve Bayes implementation is the preferred QSAR method in the widely used commercial chemoinformatics platform Pipeline Pilot. We made a comparison of the ability of Pipeline Pilot Naïve Bayes (PLPNB) and random forest to make accurate predictions on 18 large, diverse in-house QSAR data sets. These include on-target and ADME-related activities. These data sets were set up as classification problems with either binary or multicategory activities. We used a time-split method of dividing training and test sets, as we feel this is a realistic way of simulating prospective prediction. PLPNB is computationally efficient. However, random forest predictions are at least as good and in many cases significantly better than those of PLPNB on our data sets. PLPNB performs better with ECFP4 and ECFP6 descriptors, which are native to Pipeline Pilot, and more poorly with other descriptors we tried.

Discovery of novel chemotypes to a G-protein-coupled receptor through ligand-steered homology modeling and structure-based virtual screening.

Melanin-concentrating hormone receptor 1 (MCH-R1) is a G-protein-coupled receptor (GPCR) and a target for the development of therapeutics for obesity. The structure-based development of MCH-R1 and other GPCR antagonists is hampered by the lack of an available experimentally determined atomic structure. A ligand-steered homology modeling approach has been developed (where information about existing ligands is used explicitly to shape and optimize the binding site) followed by docking-based virtual screening. Top scoring compounds identified virtually were tested experimentally in an MCH-R1 competitive binding assay, and six novel chemotypes as low micromolar affinity antagonist "hits" were identified. This success rate is more than a 10-fold improvement over random high-throughput screening, which supports our ligand-steered method. Clearly, the ligand-steered homology modeling method reduces the uncertainty of structure modeling for difficult targets like GPCRs.

Identification of inhibitors to papillomavirus type 16 E6 protein based on three-dimensional structures of interacting proteins.

Human papillomaviruses (HPV) cause cutaneous and genital warts. A subset of HPV types is associated with a high-risk for progression to malignancy. The E6 protein from the high-risk HPV types represents an attractive target for intervention because of its roles in viral propagation and cellular transformation. E6 functions in part by interaction with human cellular proteins, several of which possess a helical E6-binding motif. The role for each amino acid in this motif for binding E6 has been tested through structure determination and site-directed mutagenesis. These structural and molecular biological approaches defined the spatial geometry of functional groups necessary for binding to E6. This E6-binding information (the E6-binding pharmacophore) was transferred into a three-dimensional query format suitable for computational screening of large chemical databases. Compounds were identified and tested using in vitro and cell culture-based assays. Several compounds selectively inhibited E6 interaction with the E6-binding protein E6AP and interfered with the ability of E6 to promote p53 degradation. Such compounds provide leads for the development of new pharmacologic agents to treat papillomavirus infections and their associated cancers.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Prosit, an online service to calculate pseudorotational parameters of nucleosides and nucleotides.

The online service PROSIT (Pseudo-Rotational Online Service and Interactive Tool) is a free service available at http://cactus.nci.nci.gov/prosit/ that performs pseudorotational analysis of nucleosides(tides). PROSIT reads the 3D coordinates of nucleosides and returns the pseudorotational phase angle P, puckering amplitude vmax and other related information. As examples, the sugar conformations in a parallel-stranded guanine tetraplex and a four-way Holliday junction are presented here.

Metal-dependent inhibition of HIV-1 integrase.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202-3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN-DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg(2+) at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg(2+)-based assays, while IN inhibition by salicylhydrazides is strictly Mn(2+)-dependent. Additionally, in target and cell-based assays, the MSHs have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerases I and II at concentrations that effectively inhibit IN. These data suggest that MSHs are selective inhibitors of HIV-1 IN and may serve as leads for antiviral therapeutics.

Discovery of an Orally Available, Brain Penetrant BACE1 Inhibitor that Affords Robust CNS Aß Reduction.

Inhibition of BACE1 to prevent brain Aß peptide formation is a potential disease-modifying approach to the treatment of Alzheimer's disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aß levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aß40 levels when administered orally to rats.

Application of fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design to identify novel microM leads for the development of nM BACE-1 (beta-site APP cleaving enzyme 1) inhibitors.

Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper

Cross-docking of inhibitors into CDK2 structures. 1.

Predicting protein/ligand binding affinity is one of the most challenging computational chemistry tasks. Numerous methods have been developed to address this challenge, but they all have limitations. Failure to account for protein flexibility has been a shortcoming of many methods. In this cross-docking study the data set comprised 150 inhibitor complexes of the protein kinase CDK2. Gold and Glide performed well in terms of docking accuracy. The chance of cross-docking a ligand within a 2 A RMSD of its experimental pose was found to be 50%. Relative binding potency was not properly predicted from scoring functions, even though cross-docking of each inhibitor into each protein structure was performed and only scores of correctly docked ligands were considered. An accompanying paper (Voigt, J. H.; Elkin, C.; Madison, V. S. Duca, J. S. J. Chem. Inf. Model. 2008, 48, 669-678) covers cross-docking and docking accuracy from the perspective of using multiple protein structures.

Cross-docking of inhibitors into CDK2 structures. 2.

In the preceding paper (Duca, J. S.; Madison, V. S.; Voigt, J. H. J. Chem. Inf. Model. 2008, 48, 659-668), the accuracy of docking and affinity predictions of the Gold and Glide programs were investigated using single protein conformations spanning 150 CDK2/inhibitor crystallographic complexes. High docking accuracy was observed with both methods; furthermore, Glide showed modest log(IC50)/score correlations. In this part of the study, the effect of combining docking results from multiple protein conformations in a consensus fashion was probed. This approach enhanced docking accuracy only for Glide, which was attributed to the nature of its scoring function. For log(IC50)/score correlations, particular emphasis was placed on considering only scores from correctly docked poses. Using multiple instead of single protein structures showed an improvement in the correlations. Validation sets and scrambling experiments were used to examine the statistical significance and predictivity of these correlations. Rather than actual improvements in scoring accuracy, docking to multiple protein conformations produced overfitting artifacts.

2,6-Disubstituted N-arylsulfonyl piperidines as gamma-secretase inhibitors.

A novel piperidine series of gamma-secretase inhibitors, potentially useful for the treatment of Alzheimer's disease, is disclosed. SAR investigation revealed the requirement for cis-stereochemistry of the substituents attached to the core, which resulted in the chair-like diaxial conformation of the piperidine ring. The series was optimized to provide inhibitors with IC(50)'s in the single-digit nanomolar range. Absolute stereochemistry of the active enantiomer was assigned.

Enhanced CACTVS browser of the Open NCI Database.

A Web-based, graphical user interface has been developed to conduct rapid searches by numerous criteria in the more than 250,000 structures of the Open NCI Database. It is based on the chemistry information toolkit CACTVS. Nearly all structures and anticancer and anti-HIV screening data provided by NCI's Developmental Therapeutics Program have been included. This data set has been augmented by a large amount of additional, mostly computed, data, such as calculated log P values, predicted biological activities, systematically determined names, and others. Complex boolean searches are possible. Flexible substructure searches have been implemented. The user can conduct 3D pharmacophore queries in up to 25 conformations precalculated for each compound. Numerous output formats as well as 2D and 3D visualization options are provided. It is possible to export search results in various forms and with choices for data contents in the exported files, for structure sets ranging in size from a single compound to the entire database. Only a Web browser is needed to use this service, with a few plug-ins being useful but optional.

PROSIT: pseudo-rotational online service and interactive tool, applied to a conformational survey of nucleosides and nucleotides.

A Pseudo-Rotational Online Service and Interactive Tool (PROSIT) designed to perform complete pseudorotational analysis of nucleosides and nucleotides is described. This service is freely available at http://cactus.nci.nih.gov/prosit/. Files containing nucleosides/nucleotides or DNA/RNA segments, isolated or bound to other molecules (e.g., a protein) can be uploaded to be processed by PROSIT. The service outputs the pseudorotational phase angle P, puckering amplitude numax, and other related information for each nucleoside/nucleotide detected. The service was implemented using the chemoinformatics toolkit CACTVS. PROSIT was used for a survey of nucleosides contained in the Cambridge Structural Database and nucleotides in high-resolution crystal structures from the Nucleic Acid Database. Special cases discussed include nucleosides having constrained sugar moieties with extreme puckering amplitudes, and several specific DNA/RNA helices and protein-bound DNA oligonucleotides (Dickerson-Drew dodecamer, RNA/DNA hybrid viral polypurine tract, Z-DNA enantiomers, B-DNA containing (L)-alpha-threofuranosyl nucleotides, TATA-box binding protein/TATA-box complex, and DNA (cytosine C5)-methyltransferase complexed with an oligodeoxyribonucleotide containing transition state analogue 5,6-dihydro-5-azacytosine). When the puckering amplitude decreases to a small value, the sugar becomes increasingly planar, thus reducing the significance of the phase angle P. We introduce the term "central conformation" to describe this part of the pseudorotational hyperspace in contrast to the conventional north and south conformations.

Acylsulfonamide-containing PTP1B inhibitors designed to mimic an enzyme-bound water of hydration.

Previously, it had been reported that 6-(phosphonodifluoromethyl)-2-naphthoic acid binds to the protein-tyrosine phosphatase PTP1B with its 2-carboxyl group interacting only indirectly through a bridging water molecule. Reported herein is a family of new analogues that utilize acylsulfonamido functionality both to mimic this water of hydration and to provide an additional new site for elaboration not found in the parent carboxyl-containing analogue. Target acylsulfonamides were prepared in two steps from commercially available primary sulfonamides, which were selected based on in silico screening for their potential ability to interact with one of three binding surfaces proximal to the PTP1B catalytic site. In general, modest potency enhancements were observed. Arylacylsulfonamides represent a structure-based extension of inhibitor design that may have broader utility in the development of PTP1B inhibitors.