Tam Luong Nguyen

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 Ki values in the 0.5–5 μM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

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 (Ki = 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 Institutes Open Repository. Four of the inhibitors possess Ki values ranging from 3.0 to 10.0 μm. 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 μm.

Structural Model of the Pre-pore Ring-like Structure of Panton-Valentine Leukocidin: Providing Dimensionality to Biophysical and Mutational Data

Abstract Panton-Valentine leukocidin (PVL) is a bipartite toxin that plays an important role in the pathogenesis of methicillin-resistant Staphylococcus aureus. Recent clinical data suggest a correlation between PVL and severe cases of S. aureus pneumonia. A clear understanding of the structure and function of PVL is critical to the development of novel, effective treatments. Here, we report an all-atom model of the macromolecular structure of Panton-Valentine leukocidin in its octameric, pre-pore conformation that confirms and extends our understanding of the toxins mechanism of action.

Targeting RSK: An Overview of Small Molecule Inhibitors

Ribosomal S6 kinase (RSK) is a family of serine/threonine kinases that has been identified as a promising anti-cancer target. While a number of protein kinase inhibitors that have potent activity against other serine/threonine kinases were shown to also inactivate RSK, there is keen interest in the three different inhibitor chemotypes that were shown to be RSK specific, since these compounds have tremendous utility as chemical probes in elucidating the biochemistry of the RSK signaling cascade and unraveling the molecular basis of cancer. Because each compound may have therapeutic potential, the nonspecific kinase inhibitors as well as the RSK specific inhibitors will be discussed.

Three-dimensional Model of the Pore Form of Anthrax Protective Antigen. Structure and Biological Implications

Abstract Although pore formation by protective antigen (PA) is critical to cell intoxication by anthrax toxin (AT), the structure of the pore form of PA (the PA63 pore) has not been determined. Hence, in this study, the PA63 pore was modeled using the X-ray structures of monomeric PA and heptameric α-hemolysin (α-HL) as templates. The PA63 pore model consists of two weakly associated domains, namely the cap and stem domains. The ring-like cap domain has a length of 80 Å and an outside diameter of 120 Å, while the cylinder-like stem domain has a length of 100 Å and outside diameter of ∼28 Å. This provides the PA63 pore model with a length of 180 Å. Based on experimental results, the channel in the PA63 pore model was built to have a minimum diameter of ∼12 Å, depending on side chain conformations. Because of its large size and structural complexity, the all-atom model of the PA63 pore is the end-stage construction of four separate modeling projects described herein. The final model is consistent with published experimental results, including mutational analysis and channel conductance experiments. In addition, the model was energetically and hydropathically refined to optimize molecular packing within the protomers and at the protomer-protomer interfaces. By providing atomic detail to biochemical and biophysical data, the PA63 pore model may afford new insights into the binding mode of PA on the membrane surface, the prepore-pore transition, and the mechanism of cell entry by anthrax toxin.

Association of Ebola Virus Matrix Protein VP40 with Microtubules

ABSTRACT Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and Analogues: Synthesis, Cytotoxic Activity and Study of the Mechanism of Action

The synthesis of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and analogues is reported. Their cytotoxic activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of selected compounds was examined for potential inhibition of tubulin assembly in comparison with the potent colchicine site agent combretastatin A-4. The most potent compounds also strongly and selectively inhibited the phosphorylation of the oncoprotein kinase Akt in cancer cells. The effect of the most interesting compounds was examined on the growth of HT-29 colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for inhibitors of tubulin assembly.

Pharmacophore-guided lead optimization: The rational design of a non-zinc coordinating, sub-micromolar inhibitor of the botulinum neurotoxin serotype a metalloprotease

Botulinum neurotoxins, responsible for the neuroparalytic syndrome botulism, are the deadliest of known biological toxins. The work described in this study was based on a three-zone pharmacophore model for botulinum neurotoxin serotype A light chain inhibition. Specifically, the pharmacophore defined a separation between the overlaps of several different, non-zinc(II)-coordinating small molecule chemotypes, enabling the design and synthesis of a new structural hybrid possessing a Ki=600 nM (+/-100 nM).

Interactions of halichondrin B and eribulin with tubulin.

Compounds that modulate microtubule dynamics include highly effective anticancer drugs, leading to continuing efforts to identify new agents and improve the activity of established ones. Here, we demonstrate that [(3)H]-labeled halichondrin B (HB), a complex, sponge-derived natural product, is bound to and dissociated from tubulin rapidly at one binding site per αβ-heterodimer, with an apparent K(d) of 0.31 μM. We found no HB-induced aggregation of tubulin by high-performance liquid chromatography, even following column equilibration with HB. Binding of [(3)H]HB was competitively inhibited by a newly approved clinical agent, the truncated HB analogue eribulin (apparent K(i), 0.80 μM) and noncompetitively by dolastatin 10 and vincristine (apparent K(i)s, 0.35 and 5.4 μM, respectively). Our earlier studies demonstrated that HB inhibits nucleotide exchange on β-tubulin, and this, together with the results presented here, indicated the HB site is located on β-tubulin. Using molecular dynamics simulations, we determined complementary conformations of HB and β-tubulin that delineated in atomic detail binding interactions of HB with only β-tubulin, with no involvement of the α-subunit in the binding interaction. Moreover, the HB model served as a template for an eribulin binding model that furthered our understanding of the properties of eribulin as a drug. Overall, these results established a mechanistic basis for the antimitotic activity of the halichondrin class of compounds.

Novel Broad-Spectrum Bis-(Imidazolinylindole) Derivatives with Potent Antibacterial Activities against Antibiotic-Resistant Strains

ABSTRACT Given the limited number of structural classes of clinically available antimicrobial drugs, the discovery of antibacterials with novel chemical scaffolds is an important strategy in the development of effective therapeutics for both naturally occurring and engineered resistant strains of pathogenic bacteria. In this study, several diarylamidine derivatives were evaluated for their ability to protect macrophages from cell death following infection with Bacillus anthracis, a gram-positive spore-forming bacterium. Four bis-(imidazolinylindole) compounds were identified with potent antibacterial activity as measured by the protection of macrophages and by the inhibition of bacterial growth in vitro. These compounds were effective against a broad range of gram-positive and gram-negative bacterial species, including several antibiotic-resistant strains. Minor structural variations among the four compounds correlated with differences in their effects on bacterial macromolecular synthesis and mechanisms of resistance. In vivo studies revealed protection by two of the compounds of mice lethally infected with B. anthracis, Staphylococcus aureus, or Yersinia pestis. Taken together, these results indicate that the bis-(imidazolinylindole) compounds represent a new chemotype for the development of therapeutics for both gram-positive and gram-negative bacterial species as well as against antibiotic-resistant infections.