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). In this paper, we present an atomic model of the RSK2 NTD (residues 68-323), which was built to simultaneously bind the distinctive molecular scaffolds of SL0101, Ro31-8220, and GF109203X. The RSK2 NTD model was used to identify two novel RSK2 inhibitors from the National Cancer Institute open chemical repository and to develop a preliminary structure-based pharmacophore model.

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. These binding models were subsequently used to construct a comprehensive, structure-based pharmacophore that in combination with molecular dynamics simulations confirms and extends our understanding of binding interactions at the colchicine site.

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.

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. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.

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. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes=3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including alpha-helices and beta-sheets, remained relatively unchanged during 1 ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC.

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. Among these new derivatives, 2-methoxy-9-methylsulfonylpaullone proved to be superior to the standard alsterpaullone with respect to CDK1 inhibition.

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. Both compounds were highly water soluble, were stable in buffer, and released alsterpaullone in vitro and in vivo. Studies were conducted in mice to investigate the influence of PEGylation on the plasma pharmacokinetics of alsterpaullone. The total plasma clearance rate was decreased up to 32-fold, and the biological halflife lengthened up to 8-fold when alsterpaullone was injected i.v. as a PEG-conjugate and compared to injection of the unconjugated compound. The most pronounced effect on the pharmacokinetics of alsterpaullone was produced by a 40-kDa PEG urea-linked conjugate. When the 40- and 20-kDa urea-linked conjugates were administered by i.p. injection, high relative bioavailability (46% and 99%, respectively) of alsterpaullone was observed.

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. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have K(i) values in the 0.5-5 microM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

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). All inhibitors were further verified using a HPLC-based assay. Conformational analyses of these compounds, in conjunction with molecular docking studies, were used to predict structural features that contribute to inhibitor binding and potency. Based on these results, a common pharmacophore for BoNT/A LC inhibitors is proposed. This is the first study to report small molecules (non-peptidics) that inhibit BoNT/A LC metalloprotease activity in the low microM range.

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. Twenty-one compounds had mean inhibitory concentrations of <1 microM; >75% were quinones and >40% were of the para-naphthoquinone structural type. Most notable was NSC 95397 (2,3-bis-[2-hydroxyethylsulfanyl]-[1,4]naphthoquinone), which displayed mixed inhibition kinetics with in vitro K(i) values for Cdc25A, -B, and -C of 32, 96, and 40 nM, respectively. NSC 95397 was more potent than any inhibitor of dual specificity phosphatases described previously and 125- to 180-fold more selective for Cdc25A than VH1-related dual-specificity phosphatase or protein tyrosine phosphatase 1b, respectively. Modification of the bis-thioethanol moiety markedly decreased enzyme inhibitory activity, indicating its importance for bioactivity. NSC 95397 showed significant growth inhibition against human and murine carcinoma cells and blocked G(2)/M phase transition. A potential Cdc25 site of interaction was postulated based on molecular modeling with these quinones. We propose that inhibitors based on this chemical structure could serve as useful tools to probe the biological function of Cdc25.

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. Cancer Inst. 81 (1989) 1088] demonstrated that compounds with similar mechanism of cell growth inhibition show similar patterns of activity in the NCI screen. This observation was developed into an algorithm called COMPARE and has been successfully used to predict mechanisms for a wide variety of compounds. More recently, this method has been extended to associate patterns of cell growth inhibition by compounds with measurements of molecular entities (such as gene expression) in the cell lines in the NCI screen. The COMPARE method and associated data are freely available on the Developmental Therapeutics Program (DTP) web site (http://dtp.nci.nih.gov/). Examples of the use of COMPARE on these web pages will be explained and demonstrated. Published by Elsevier Science Inc.