The DOCK program explores possible orientations of a molecule within a macromolecular active site by superimposing atoms onto precomputed site points. Here we compare a number of different search methods, including an exhaustive matching algorithm based on a single docking graph. We evaluate the performance of each method by screening a small database of molecules to a variety of macromolecular targets. By varying the amount of sampling, we can monitor the time convergence of scores and rankings. We not only show that the site point᎐directed search is tenfold faster than a random search, but that the single graph matching algorithm boosts the speed of database screening up to 60-fold. The new algorithm, in fact, outperforms the bipartite graph matching algorithm currently used in DOCK. The results indicate that a critical issue for rapid database screening is the extent to which a search method biases run time toward the highest-ranking molecules. The single docking graph matching algorithm will be incorporated into DOCK version 4.0.

In this paper we describe the search strategies developed for docking flexible molecules to macomolecular sites that are incorporated into the widely distributed DOCK software, version 4.0. The search strategies include incremental construction and random conformation search and utilize the existing Coulombic and Lennard-Jones grid-based scoring function. The incremental construction strategy is tested with a panel of 15 crystallographic testcases, created from 12 unique complexes whose ligands vary in size and flexibility. For all testcases, at least one docked position is generated within 2 A of the crystallographic position. For 7 of 15 testcases, the top scoring position is also within 2 A of the crystallographic position. The algorithm is fast enough to successfully dock a few testcases within seconds and most within 100 s. The incremental construction and the random search strategy are evaluated as database docking techniques with a database of 51 molecules docked to two of the crystallographic testcases. Incremental construction outperforms random search and is fast enough to reliably rank the database of compounds within 15 s per molecule on an SGI R10000 cpu.

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.

Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel and pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers. To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. .

A number of single-digit nanomolar, low-molecular-weight plasmepsin II aspartyl protease inhibitors have been identified using combinatorial chemistry and structure-based design. By identifying multiple, small-molecule inhibitors using the parallel synthesis of several focused libraries, it was possible to select for compounds with desirable characteristics including enzyme specificity and minimal binding to serum proteins. The best inhibitors identified have Ki's of 2-10 nM, molecular weights between 594 and 650 Da, between 3- and 15-fold selectivity toward plasmepsin II over cathepsin D, the most closely related human protease, good calculated log P values (2.86-4.56), and no apparent binding to human serum albumin at 1 mg/mL in an in vitro assay. These compounds represent the most potent non-peptide plasmepsin II inhibitors reported to date.

The success of these studies clearly demonstrates the power of coupling the complementary methods of combinatorial chemistry and structure-based design. We anticipate that the general approaches described here will be successful for other members of the aspartyl protease class and for many other enzyme classes.

This paper describes the development of a set of new 2D fingerprints for the purposes of virtual screening in a pharmaceutical environment. The new fingerprints are based on established ones: the changes in their design included the introduction of overlapping pharmacophore feature types, feature counts for pharmacophore and structural fingerprints, as well as changes in the resolution in property description for property fingerprints. The effects of each of these changes on virtual screening performance were monitored using two types of training sets, emulating different stages in the drug discovery process. The results demonstrate that these changes all lead to an improvement in virtual screening performance.

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