A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site.

The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150-180° orients the side chain ß-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a > 2000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.

Evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through short sequence duplications.

Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≥ 2 µg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.

Evidence for a distinct ligand binding site on tubulin discovered through inhibition by GDP of paclitaxel-induced tubulin assembly in the absence of exogenous GTP.

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg(2+), which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on alpha-tubulin, based on molecular modeling studies.

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.

Specific RITA Modification Produces Hyperselective Cytotoxicity While Maintaining In Vivo Antitumor Efficacy.

The preclinical antitumor agent RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan, NSC 652287), an analog of the natural product α-terthiophene, failed during the development phase due to acute pulmonary toxicity in animal models. A series of synthetic modifications to RITA's heterocyclic scaffold resulted in activity ranging from broadly cytotoxic to highly selective. In the NCI 60-cell line screen, these "hyperselective" agents (e.g., imatinib) are rare. A selectivity index (SI) was developed to quantify this desirable feature, which is 20 for imatinib, whereas RITA's SI is only 0.10. One of the described hyperselective RITA analogs (SI = 7.9) completely lost activity in the presence of a known SULT1A1 inhibitor. These results, coupled with previous evidence that RITA is a SULT1A1 substrate, suggest that carbinol modification by a sulfate leaving group and subsequent formation of a reactive carbocation may explain RITA's broad cytotoxicity. Although SULT1A1 expression is required for susceptibility, hyperselective analogs exhibited reduced association of activity with SULT1A1 mRNA expression compared with RITA, apparently requiring some additional target(s). In support of this hypothesis, there is a strong correlation (P < 0.01, r = 0.95) between quantum mechanically calculated energy barriers for carbocation formation from sulfonated analogs and SI, indicating that hyperselective RITA analogs generate reactive carbocations less readily after sulfate activation. Importantly, narrowing the cytotoxicity profile of RITA did not eliminate its analogs' in vivo antitumor activity, as several new hyperselective agents, NSC 773097 (1), 773392 (2), and 782846 (6), displayed impressive activity against A498 xenografts in mice.

A refined pharmacophore identifies potent 4-amino-7-chloroquinoline-based inhibitors of the botulinum neurotoxin serotype A metalloprotease.

We previously identified structurally diverse small molecule (non-peptidic) inhibitors (SMNPIs) of the botulinum neurotoxin serotype A (BoNT/A) light chain (LC). Of these, several (including antimalarial drugs) contained a 4-amino-7-chloroquinoline (ACQ) substructure and a separate positive ionizable amine component. The same antimalarials have also been found to interfere with BoNT/A translocation into neurons, via pH elevation of the toxin-mediated endosome. Thus, this structural class of small molecules may serve as dual-function BoNT/A inhibitors. In this study, we used a refined pharmacophore for BoNT/A LC inhibition to identify four new, potent inhibitors of this structural class (IC50's ranged from 3.2 to 17 muM). Molecular docking indicated that the binding modes for the new SMNPIs are consistent with those of other inhibitors that we have identified, further supporting our structure-based pharmacophore. Finally, structural motifs of the new SMNPIs, as well as two structure-based derivatives, were examined for activity, providing valuable information about pharmacophore component contributions to inhibition.

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.

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.

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture.

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.

Human T-cell leukemia virus type 1 integration target sites in the human genome: comparison with those of other retroviruses.

Retroviral integration into the host genome is not entirely random, and integration site preferences vary among different retroviruses. Human immunodeficiency virus (HIV) prefers to integrate within active genes, whereas murine leukemia virus (MLV) prefers to integrate near transcription start sites and CpG islands. On the other hand, integration of avian sarcoma-leukosis virus (ASLV) shows little preference either for genes, transcription start sites, or CpG islands. While host cellular factors play important roles in target site selection, the viral integrase is probably the major viral determinant. It is reasonable to hypothesize that retroviruses with similar integrases have similar preferences for target site selection. Although integration profiles are well defined for members of the lentivirus, spumaretrovirus, alpharetrovirus, and gammaretrovirus genera, no members of the deltaretroviruses, for example, human T-cell leukemia virus type 1 (HTLV-1), have been evaluated. We have mapped 541 HTLV-1 integration sites in human HeLa cells and show that HTLV-1, like ASLV, does not specifically target transcription units and transcription start sites. Comparing the integration sites of HTLV-1 with those of ASLV, HIV, simian immunodeficiency virus, MLV, and foamy virus, we show that global and local integration site preferences correlate with the sequence/structure of virus-encoded integrases, supporting the idea that integrase is the major determinant of retroviral integration site selection. Our results suggest that the global integration profiles of other retroviruses could be predicted from phylogenetic comparisons of the integrase proteins. Our results show that retroviruses that engender different insertional mutagenesis risks can have similar integration profiles.

Inhibition of metalloprotease botulinum serotype A from a pseudo-peptide binding mode to a small molecule that is active in primary neurons.

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K(i) = 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K(i) values ranging from 3.0 to 10.0 microM. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 microM.

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.

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. In this paper, we present the refinement of the EM reconstruction of truncated hexameric VP40 to approximately 20 A and the construction of an all-atom model (residues 44-212) using the EM model at approximately 20 A and the X-ray structure of monomeric VP40 as templates. The hexamer model suggests that the monomer-hexamer transition involves a conformational change in the N-terminal domain that is not evident during octamerization and therefore, may provide the basis for elucidating the biological function of VP40.

Human cellular nucleic acid-binding protein Zn2+ fingers support replication of human immunodeficiency virus type 1 when they are substituted in the nucleocapsid protein.

A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn(2+) fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn(2+) fingers. The sequence of the NH(2)-terminal NC Zn(2+) finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn(2+) finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn(2+) fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn(2+) finger compared to mutant and wild-type Zn(2+) fingers in NC that support replication. The present study shows that six of seven of the Zn(2+) fingers from the CNBP protein can be used as substitutes for the Zn(2+) finger in the NH(2)-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn(2+) finger mutants.

Charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination.

We have identified a region near the C terminus of capsid (CA) of murine leukemia virus (MLV) that contains many charged residues. This motif is conserved in various lengths in most MLV-like viruses. One exception is that spleen necrosis virus (SNV) does not contain a well-defined domain of charged residues. When 33 amino acids of the MLV motif were deleted to mimic SNV CA, the resulting mutant produced drastically reduced amounts of virions and the virions were noninfectious. Furthermore, these viruses had abnormal sizes, often contained punctate structures resembling those in the cell cytoplasm, and packaged both ribosomal and viral RNA. When 11 or 15 amino acids were deleted to modify the MLV CA to resemble those from other gammaretroviruses, the deletion mutants produced virions at levels comparable to those of the wild-type virus and were able to complete one round of virus replication without detectable defects. We generated 10 more mutants that displayed either the wild-type or mutant phenotype. The distribution of the wild-type or mutant phenotype did not directly correlate with the number of amino acids deleted, suggesting that the function of the motif is determined not simply by its length but also by its structure. Structural modeling of the wild-type and mutant proteins suggested that this region forms alpha-helices; thus, we termed this motif the "charged assembly helix." This is the first description of the charged assembly helix motif in MLV CA and demonstration of its role in virus budding and assembly.

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.