Adventures in public data.

This article contains the slides and transcript of a talk given by Dan Zaharevitz at the "Visions of a Semantic Molecular Future" symposium held at the University of Cambridge Department of Chemistry on 2011-01-19. A recording of the talk is available on the University Computing Service's Streaming Media Service archive at http://sms.cam.

Identification of small-molecule inhibitors of the colorectal cancer oncogene Krüppel-like factor 5 expression by ultrahigh-throughput screening.

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library.

RSK2 Binding Models Delineate Key Features for Activity.

Due to its overexpression and activation in human cancer cells and tissues, an emerging molecular target in cancer therapeutics is p90 ribosomal s6 kinase 2 (RSK2). While a growing number of RSK2 inhibitors have been reported in the literature, only the crystal structure of RSK2 in complex with an AMP analogue provides a structural basis for understanding RSK2 inhibition. To remedy this, we used our fluorescence polarization assay to determine the RSK2 activity for a set of structurally diverse compounds, and followed this by modeling their binding modes in an all-atom, energy refined crystal structure of RSK2.

Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex.

A large number of hormones and local agonists activating guanine-binding protein-coupled receptors (GPCR) play a major role in cancer progression. Here, we characterize the new imidazo-pyrazine derivative BIM-46174, which acts as a selective inhibitor of heterotrimeric G-protein complex. BIM-46174 prevents the heterotrimeric G-protein signaling linked to several GPCRs mediating (a) cyclic AMP generation (Galphas), (b) calcium release (Galphaq), and (c) cancer cell invasion by Wnt-2 frizzled receptors and high-affinity neurotensin receptors (Galphao/i and Galphaq).

Homology model of RSK2 N-terminal kinase domain, structure-based identification of novel RSK2 inhibitors, and preliminary common pharmacophore.

Ribosomal S6 kinase 2 (RSK2) is a serine/threonine kinase that plays a role in human cancer and Coffin-Lowry syndrome and is comprised of two nonidentical kinase domains, each domain with its own ATP-binding site. RSK2 can be inactivated by different types of small organic molecules. Potent RSK2 inhibitors include the two classic bisindole maleimide PKC inhibitors, Ro31-8220 and GF109203X, and the natural product SL0101 that was shown to bind specifically to the ATP pocket of the N-terminal domain (NTD).

A common pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach.

Modulating the structure and function of tubulin and microtubules is an important route to anticancer therapeutics, and therefore, small molecules that bind to tubulin and cause mitotic arrest are of immense interest. A large number of synthetic and natural compounds with diverse structures have been shown to bind at the colchicine site, one of the major binding sites on tubulin, and inhibit tubulin assembly. Using the recently determined X-ray structure of the tubulin:colchicinoid complex as the template, we employed docking studies to determine the binding modes of a set of structurally diverse colchicine site inhibitors.

Epoxide-containing side chains enhance antiproliferative activity of paullones.

The introduction of side chains bearing epoxide motifs into the molecular scaffold of kenpaullone and 9-trifluoromethylpaullone led to improved antiproliferative activity of the novel derivatives for human tumor cell lines. The syntheses were accomplished applying Stille coupling for the introduction of unsaturated side chains into the 2-position of the paullones and subsequently employing a hydrogen peroxide/nitrile mixture for the epoxidation of C,C-double bonds.

An all-atom model of the pore-like structure of hexameric VP40 from Ebola: structural insights into the monomer-hexamer transition.

The matrix protein VP40 is an indispensable component of viral assembly and budding by the Ebola virus. VP40 is a monomer in solution, but can fold into hexameric and octameric states, two oligomeric conformations that play central roles in the Ebola viral life cycle. While the X-ray structures of monomeric and octameric VP40 have been determined, the structure of hexameric VP40 has only been solved by three-dimensional electron microscopy (EM) to a resolution of approximately 30 A.

Homology model of the CDK1/cyclin B complex.

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches.

Structure-aided optimization of kinase inhibitors derived from alsterpaullone.

In order to perform computer-aided design of novel alsterpaullone derivatives, the vicinity of the entrance to the ATP-binding site was scanned for areas that could be useful as anchoring points for additional protein-ligand interactions. Based on the alignment of alsterpaullone in a CDK1/cyclin B homology model, substituents were attached to the 2-position of the parent scaffold to enable contacts within the identified areas. Synthesis of the designed structures revealed three derivatives (3-5) with kinase-inhibitory activity similar to alsterpaullone.

Conformational sampling of the botulinum neurotoxin serotype A light chain: implications for inhibitor binding.

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes--post exposure.

Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003.

To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries.

Poly(ethylene glycol) prodrugs of the CDK inhibitor, alsterpaullone (NSC 705701): synthesis and pharmacokinetic studies.

Two methods were devised to conjugate PEG to alsterpaullone (NSC 705701) via the N of the indole ring portion of the molecule. In the first approach, activation of the indole was accomplished by reaction with p-nitrophenyl chloroformate to produce a reactive carbamate that was then condensed with a mono blocked diamine to form a urea bond followed by deblocking and conjugation to PEG. The second route used the anion of the indole and produced a carbamate bond.

CDK1-inhibitory activity of paullones depends on electronic properties of 9-substituents.

Multiple linear regression analysis was employed in an effort to establish a quantitative structure-activity relationship model for the CDK1-inhibitory activity of a series of 9-substituted paullones. While the electronic properties of the 9-substituents proved to be of high relevance for CDK1 inhibition, both lipophilic and a steric parameters could not be included in a meaningful equation for the calculation of biological properties. The equation solely based on the electronic parameter was successfully used for the prediction of the CDK1-inhibitory activity of a small test set comprising novel paullones with sulfur-containing 9-substituents.

Identification of small molecule inhibitors of anthrax lethal factor.

The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF.

Evaluation and comparison of 3D-QSAR CoMSIA models for CDK1, CDK5, and GSK-3 inhibition by paullones.

With a view to the rational design of selective GSK-3beta inhibitors, 3D-QSAR CoMSIA models were developed for the inhibition of the three serine/threonine kinases CDK1/cyclin B, CDK5/p25, and GSK-3beta by compounds from the paullone inhibitor family. The models are based on the kinase inhibition data of 52 paullone entities, which were aligned by a docking routine into the ATP-binding cleft of a CDK1/cyclin B homology model. Variation of grid spacing and column filtering were used during the optimization of the models.

Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity.

Botulinum neurotoxins (BoNTs) are among the most lethal biological substances to have been weaponized and are listed as biodefense category A agents. Currently, no small molecule (non-peptidic) therapeutics exist to counter this threat; hence, identifying and developing compounds that inhibit BoNTs is a high priority. In the present study, a high-throughput assay was used to identify small molecules that inhibit the metalloprotease activity of BoNT serotype A light chain (BoNT/A LC).

Correlation of nucleoside and nucleobase transporter gene expression with antimetabolite drug cytotoxicity.

Antimetabolite drugs that inhibit nucleic acid metabolism are widely used in cancer chemotherapy. Nucleoside and nucleobase transporters are important for the cellular uptake of nucleic acids and their corresponding anticancer analogue drugs. Thus, these transporters may play a role both in antimetabolite drug sensitivity, by mediating the uptake of nucleoside analogues, and in antimetabolite drug resistance, by mediating the uptake of endogenous nucleosides that may rescue cells from toxicity.

Synthesis of paullones with aminoalkyl side chains.

Paullones 3 and 4 with aminoalkyl side chains in 2- or 3-position were synthesized as derivatives of kenpaullone 1. Both 3 and 4 showed the characteristic CDK1-inhibitory activity of the paullones and a modest antiproliferative activity on cultured human tumor cell lines. Hence, 3 and 4 appear to be suitable tools for affinity studies directed to find additional intracellular paullone targets.

Identification of a potent and selective pharmacophore for Cdc25 dual specificity phosphatase inhibitors.

Small molecules provide powerful tools to interrogate biological pathways but many important pathway participants remain refractory to inhibitors. For example, Cdc25 dual-specificity phosphatases regulate mammalian cell cycle progression and are implicated in oncogenesis, but potent and selective inhibitors are lacking for this enzyme class. Thus, we evaluated 10,070 compounds in a publicly available chemical repository of the National Cancer Institute for in vitro inhibitory activity against oncogenic, full-length, recombinant human Cdc25B.

COMPARE: a web accessible tool for investigating mechanisms of cell growth inhibition.

For more than 10 years the National Cancer Institute (NCI) has tested compounds for their ability to inhibit the growth of human tumor cell lines in culture (NCI screen). Work of Ken Paull [J. Natl.

Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.

An analysis of the activity of compounds tested in pre-clinical in vivo and in vitro assays by the National Cancer Institute's Developmental Therapeutics Program was performed. For 39 agents with both xenograft data and Phase II clinical trials results available, in vivo activity in a particular histology in a tumour model did not closely correlate with activity in the same human cancer histology, casting doubt on the correspondence of the pre-clinical models to clinical results. However, for compounds with in vivo activity in at least one-third of tested xenograft models, there was correlation with ultimate activity in at least some Phase II trials.

Chemical synthesis and biological properties of pyridine epothilones.

Background: Numerous analogs of the antitumor agents epothilones A and B have been synthesized in search of better pharmacological profiles. Insights into the structure-activity relationships within the epothilone family are still needed and more potent and selective analogs of these compounds are in demand, both as biological tools and as chemotherapeutic agents, especially against drug-resistant tumors.

Results: A series of pyridine epothilone B analogs were designed, synthesized and screened.

Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3beta (GSK-3beta) (IC50: 4-80 nM) and the neuronal CDK5/p25 (IC50: 20-200 nM).