Discovery and Characterization of Inhibitors of Pseudomonas aeruginosa Type III Secretion▿ †

The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates up to four protein toxin effectors into human cells, facilitating the establishment and dissemination of infections. To discover inhibitors of this important virulence mechanism, we developed two cellular reporter assays and applied them to a library of 80,000 compounds. The primary screen was based on the dependence of the transcription of T3SS operons on the T3SS-mediated secretion of a negative regulator and consisted of a transcriptional fusion of the Photorhabdus luminescens luxCDABE operon to the P. aeruginosa exoT effector gene. Secondary assays included direct measurements of the T3SS-mediated secretion of a P. aeruginosa ExoS effector-β-lactamase fusion protein as well as the detection of the secretion of native ExoS by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of culture supernatants. Five inhibitors in three chemical classes were demonstrated to inhibit type III secretion selectively with minimal cytotoxicity and with no effects on bacterial growth or on the type II-mediated secretion of elastase. These inhibitors also block the T3SS-mediated secretion of a YopE effector-β-lactamase fusion protein from an attenuated Yersinia pestis strain. The most promising of the inhibitors is a phenoxyacetamide that also blocks the T3SS-mediated translocation of effectors into mammalian cells in culture. Preliminary studies of structure-activity relationships in this phenoxyacetamide series demonstrated a strict requirement for the R-enantiomer at its stereocenter and indicated tolerance for a variety of substituents on one of its two aromatic rings

Concise Synthesis of 6-Cyanobenzo[<i>b</i>]furan, a Useful Building Block

<div><p></p><p>A new three-step synthesis of 6-cyanobenzo[b]furan (<b>6</b>) was developed, starting from commercially available 6-hydroxybenzo[b]furan-3-one (<b>18</b>). Key steps in this process were the first step, which was the reductive dehydration of <b>18</b> to produce 6-hydroxybenzo[b]furan (<b>19</b>), and the last step, which converted the aryl triflate <b>20</b> to the aryl cyanide <b>6</b> in a palladium-catalyzed cross-coupling protocol. Overall yield for this new synthesis was 49%.</p> </div

Identification of a Small-Molecule Entry Inhibitor for Filoviruses▿ †

Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC50s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection