Small molecule inhibitors of anthrax edema factor.

Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production.

Virtual medicinal chemistry: in silico pre-docking functional group transformation for discovery of novel inhibitors of botulinum toxin serotype A light chain.

A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed.

Antidotes to anthrax lethal factor intoxication. Part 3: Evaluation of core structures and further modifications to the C2-side chain.

Four core structures capable of providing sub-nanomolar inhibitors of anthrax lethal factor (LF) were evaluated by comparing the potential for toxicity, physicochemical properties, in vitro ADME profiles, and relative efficacy in a rat lethal toxin (LT) model of LF intoxication. Poor efficacy in the rat LT model exhibited by the phenoxyacetic acid series (3) correlated with low rat microsome and plasma stability. Specific molecular interactions contributing to the high affinity of inhibitors with a secondary amine in the C2-side chain were revealed by X-ray crystallography.

Small molecule pan-dengue and West Nile virus NS3 protease inhibitors.

BACKGROUND: Dengue fever, dengue haemorrhagic fever, and dengue shock syndrome are caused by infections with any of the four serotypes of the dengue virus (DENV), and are an increasing global health risk. The related West Nile virus (WNV) causes significant morbidity and mortality as well, and continues to be a threat in endemic areas. Currently no FDA-approved vaccines or therapeutics are available to prevent or treat any of these infections. Like the other members of Flaviviridae, DENV and WNV encode a protease (NS3) which is essential for viral replication and therefore is a promising target for developing therapies to treat dengue and West Nile infections. METHODS: Flaviviral protease inhibitors were identified and biologically characterized for mechanism of inhibition and DENV antiviral activity. RESULTS: A guanidinylated 2,5-dideoxystreptamine class of compounds was identified that competitively inhibited the NS3 protease from DENV(1-4) and WNV with 50% inhibitory concentration values in the 1-70 µM range. Cytotoxicity was low; however, antiviral activity versus DENV-2 on VERO cells was not detectable. CONCLUSIONS: This class of compounds is the first to demonstrate competitive pan-dengue and WNV NS3 protease inhibition and, given the sequence conservation among flavivirus NS3 proteins, suggests that developing a pan-dengue or possibly pan-flavivirus therapeutic is feasible.

Structural characterization of three novel hydroxamate-based zinc chelating inhibitors of the Clostridium botulinum serotype A neurotoxin light chain metalloprotease reveals a compact binding site resulting from 60/70 loop flexibility.

Neurotoxins synthesized by Clostridium botulinum bacteria (BoNT), the etiological agent of human botulism, are extremely toxic proteins making them high-risk agents for bioterrorism. Small molecule inhibitor development has been focused on the light chain zinc-dependent metalloprotease domain of the neurotoxin, an effort that has been hampered by its relatively flexible active site. Developed in concert with structure--activity relationship studies, the X-ray crystal structures of the complex of BoNT serotype A light chain (BoNT/A LC) with three different micromolar-potency hydroxamate-based inhibitors are reported here. Comparison with an unliganded BoNT/A LC structure reveals significant changes in the active site as a result of binding by the unique inhibitor scaffolds. The 60/70 loop at the opening of the active site pocket undergoes the largest conformational change, presumably through an induced-fit mechanism, resulting in the most compact catalytic pocket observed in all known BoNT/A LC structures.

Antidotes to anthrax lethal factor intoxication. Part 2: structural modifications leading to improved in vivo efficacy.

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model.

Antidotes to anthrax lethal factor intoxication. Part 1: Discovery of potent lethal factor inhibitors with in vivo efficacy.

Sub-nanomolar small molecule inhibitors of anthrax lethal factor have been identified using SAR and Merck L915 (4) as a model compound. One of these compounds (16) provided 100% protection in a rat lethal toxin model of anthrax disease.