Virtual fragment screening: an exploration of various docking and scoring protocols for fragments using Glide

Fragment-based drug discovery approaches allow for a greater coverage of chemical space and generally produce high efficiency ligands. As such, virtual and experimental fragment screening are increasingly being coupled in an effort to identify new leads for specific therapeutic targets. Fragment docking is employed to create target-focussed subset of compounds for testing along side generic fragment libraries. The utility of the program Glide with various scoring schemes for fragment docking is discussed. Fragment docking results for two test cases, prostaglandin D2 synthase and DNA ligase, are presented and compared to experimental screening data. Self-docking, cross-docking, and enrichment studies are performed. For the enrichment runs, experimental data exists indicating that the docking decoys in fact do not inhibit the corresponding enzyme being examined. Results indicate that even for difficult test cases fragment docking can yield enrichments significantly better than random. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Virtual target screening: validation using kinase inhibitors

Computational methods involving virtual screening could potentially be employed to discover new biomolecular targets for an individual molecule of interest (MOI). However, existing scoring functions may not accurately differentiate proteins to which the MOI binds from a larger set of macromolecules in a protein structural database. An MOI will most likely have varying degrees of predicted binding affinities to many protein targets. However, correctly interpreting a docking score as a hit for the MOI docked to any individual protein can be problematic. In our method, which we term "Virtual Target Screening (VTS)", a set of small drug-like molecules are docked against each structure in the protein library to produce benchmark statistics. This calibration provides a reference for each protein so that hits can be identified for an MOI. VTS can then be used as tool for: drug repositioning (repurposing), specificity and toxicity testing, identifying potential metabolites, probing protein structures for allosteric sites, and testing focused libraries (collection of MOIs with similar chemotypes) for selectivity. To validate our VTS method, twenty kinase inhibitors were docked to a collection of calibrated protein structures. Here, we report our results where VTS predicted protein kinases as hits in preference to other proteins in our database. Concurrently, a graphical interface for VTS was developed.     

Design of compound libraries for fragment screening

Approaches to the design of libraries for fragment screening are illustrated with reference to a 20 k generic fragment screening library and a 1.2 k generic NMR screening library. Tools and methods for library design that have been developed within AstraZeneca are described, including Foyfi fingerprints and the Flush program for neighborhood characterization. It will be shown how Flush and the BigPicker, which selects maximally diverse sets of compounds, are used to apply the Core and Layer method for library design. Approaches to partitioning libraries into cocktails are also described.     

Investigation of MM-PBSA rescoring of docking poses

Target-based virtual screening is increasingly used to generate leads for targets for which high quality three-dimensional (3D) structures are available. To allow large molecular databases to be screened rapidly, a tiered scoring scheme is often employed whereby a simple scoring function is used as a fast filter of the entire database and a more rigorous and time-consuming scoring function is used to rescore the top hits to produce the final list of ranked compounds. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approaches are currently thought to be quite effective at incorporating implicit solvation into the estimation of ligand binding free energies. In this paper, the ability of a high-throughput MM-PBSA rescoring function to discriminate between correct and incorrect docking poses is investigated in detail. Various initial scoring functions are used to generate docked poses for a subset of the CCDC/Astex test set and to dock one set of actives/inactives from the DUD data set. The effectiveness of each of these initial scoring functions is discussed. Overall, the ability of the MM-PBSA rescoring function to (i) regenerate the set of X-ray complexes when docking the bound conformation of the ligand, (ii) regenerate the X-ray complexes when docking conformationally expanded databases for each ligand which include "conformation decoys" of the ligand, and (iii) enrich known actives in a virtual screen for the mineralocorticoid receptor in the presence of "ligand decoys" is assessed. While a pharmacophore-based molecular docking approach, PhDock, is used to carry out the docking, the results are expected to be general to use with any docking method.     

Virtual screening using protein-ligand docking: avoiding artificial enrichment

This study addresses a number of topical issues around the use of protein-ligand docking in virtual screening. We show that, for the validation of such methods, it is key to use focused libraries (containing compounds with one-dimensional properties, similar to the actives), rather than "random" or "drug-like" libraries to test the actives against. We also show that, to obtain good enrichments, the docking program needs to produce reliable binding modes. We demonstrate how pharmacophores can be used to guide the dockings and improve enrichments, and we compare the performance of three consensus-ranking protocols against ranking based on individual scoring functions. Finally, we show that protein-ligand docking can be an effective aid in the screening for weak, fragment-like binders, which has rapidly become a popular strategy for hit identification. All results presented are based on carefully constructed virtual screening experiments against four targets, using the protein-ligand docking program GOLD.     

Virtual screening methods that complement HTS

In this review, we discuss a number of computational methods that have been developed or adapted for molecule classification and virtual screening (VS) of compound databases. In particular, we focus on approaches that are complementary to high-throughput screening (HTS). The discussion is limited to VS methods that operate at the small molecular level, which is often called ligand-based VS (LBVS), and does not take into account docking algorithms or other structure-based screening tools. We describe areas that greatly benefit from combining virtual and biological screening and discuss computational methods that are most suitable to contribute to the integration of screening technologies. Relevant approaches range from established methods such as clustering or similarity searching to techniques that have only recently been introduced for LBVS applications such as statistical methods or support vector machines. Finally, we discuss a number of representative applications at the interface between VS and HTS.     

Biosensor-based small molecule fragment screening with biolayer interferometry

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein–protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.     

Virtual screening using binary kernel discrimination: analysis of pesticide data

This paper discusses the use of binary kernel discrimination (BKD) for identifying potential active compounds in lead-discovery programs. BKD was compared with established virtual screening methods in a series of experiments using pesticide data from the Syngenta corporate database. It was found to be superior to methods based on similarity searching and substructural analysis but inferior to a support vector machine. Similar conclusions resulted from application of the methods to a pesticide data set for which categorical activity data were available.     

Lessons for fragment library design: analysis of output from multiple screening campaigns

Over the past 8 years, we have developed, refined and applied a fragment based discovery approach to a range of protein targets. Here we report computational analyses of various aspects of our fragment library and the results obtained for fragment screening. We reinforce the finding of others that the experimentally observed hit rate for screening fragments can be related to a computationally defined druggability index for the target. In general, the physicochemical properties of the fragment hits display the same profile as the library, as is expected for a truly diverse library which probes the relevant chemical space. An analysis of the fragment hits against various protein classes has shown that the physicochemical properties of the fragments are complementary to the properties of the target binding site. The effectiveness of some fragments appears to be achieved by an appropriate mix of pharmacophore features and enhanced aromaticity, with hydrophobic interactions playing an important role. The analysis emphasizes that it is possible to identify small fragments that are specific for different binding sites. To conclude, we discuss how the results could inform further development and improvement of our fragment library. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A direct-to-biology high-throughput chemistry approach to reactive fragment screening

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn²⁺ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design–make–test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

A photoreactive fragment screening platform employing direct-to-biology high-throughput chemistry (D2B-HTC) for the rapid iterative synthesis and screening of libraries of photoaffinity bits.     

Virtual high throughput screening in new lead identification

Drug discovery continues to be one of the greatest contemporary challenges and rational application of modelling approaches is the first important step to obtain lead compounds, which can be optimised further. Virtual high throughput screening (VHTS) is one of the efficient approaches to obtain lead structures for a given target. Strategic application of different screening filters like pharmacophore mapping, shape-based, ligand-based, molecular similarity etc., in combination with other drug design protocols provide invaluable insights in lead identification and optimization. Screening of large databases using these computational methods provides potential lead compounds, thus triggering a meaningful interplay between computations and experiments. In this review, we present a critical account on the relevance of molecular modelling approaches in general, lead optimization and virtual screening methods in particular for new lead identification. The importance of developing reliable scoring functions for non-bonded interactions has been highlighted, as it is an extremely important measure for the reliability of scoring function. The lead optimization and new lead design has also been illustrated with examples. The importance of employing a combination of general and target specific screening protocols has also been highlighted.     

Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology

For the detection of the precise and unambiguous binding of fragments to a specific binding site on the target protein, we have developed a novel reporter displacement binding assay technology. The application of this technology for the fragment screening as well as the fragment evolution process with a specific modelling based design strategy is demonstrated for inhibitors of the protein kinase p38alpha. In a fragment screening approach seed fragments were identified which were then used to build compounds from the deep-pocket towards the hinge binding area of the protein kinase p38alpha based on a modelling approach. BIRB796 was used as a blueprint for the alignment of the fragments. The fragment evolution of these deep-pocket binding fragments towards the fully optimized inhibitor BIRB796 included the modulation of the residence time as well as the affinity. The goal of our study was to evaluate the robustness and efficiency of our novel fragment screening technology at high fragment concentrations, compare the screening data with biochemical activity data and to demonstrate the evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors in an in silico approach.     

Virtual screening for compounds that mimic protein-protein interface epitopes

Modulation of protein-protein interactions (PPI) has emerged as a new concept in rational drug design. Here, we present a computational protocol for identifying potential PPI inhibitors. Relevant regions of interfaces (epitopes) are predicted for three-dimensional protein models and serve as queries for virtual compound screening. We present a computational screening protocol that incorporates two different pharmacophore models. One model is based on the mathematical concept of autocorrelation vectors and the other utilizes fuzzy labeled graphs. In a proof-of-concept study, we were able to identify serine protease inhibitors using a predicted trypsin epitope as query. Our virtual screening framework may be suited for rapid identification of PPI inhibitors and suggesting bioactive tool compounds.     

Virtual screening for ligands of the insect molting hormone receptor

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.     

Virtual screening of some heterocyclic structures toward novel antibacterial agents

Infectious diseases and their treatment are among the most important issues in global health and economy. Moreover, increasing prevalence of antibiotic-resistant pathogenic bacteria necessitates the considerable need for discovering new drugs. Some heterocyclic structures with dihydropyridine (DHP) scaffold have been reported as antimicrobial agents. Herein, we report a structure-based virtual screening of a pool of 3,5-disubstituted DHPs and a post-analysis of virtual hits through in vitro antibacterial assessment. Four top-ranked DHP structures (6a–d) were found to interact with the relevant target active sites and exhibited superior stereoelectronic features within their enzyme inhibition. Selected compounds were synthesized and assessed for their antibacterial activity via microdilution method. Results of this study represented a significant application of multi-step virtual screening strategy in identifying privileged DHP structures as good starting points for further developments toward more potent antibacterial agents.