ABSTRACT
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.
Subject(s)
Anthrax/drug therapy , Bacillus anthracis/drug effects , Bacterial Toxins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Antigens, Bacterial/pharmacology , Bacterial Toxins/pharmacology , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/biosynthesis , Dose-Response Relationship, Drug , Humans , Mice , Molecular Structure , Protein Kinases/metabolism , RAW 264.7 Cells , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Botulinum Toxins, Type A/antagonists & inhibitors , Protease Inhibitors/chemistry , Binding Sites , Botulinum Toxins, Type A/metabolism , Carboxylic Acids/chemistry , Catalytic Domain , Chemistry, Pharmaceutical , Drug Design , Drug Evaluation, Preclinical , Hydroxamic Acids/chemistry , Molecular Docking Simulation , Protease Inhibitors/chemical synthesis , Protease Inhibitors/metabolism , Protein Binding , Serotyping , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Acetates/chemical synthesis , Anthrax/drug therapy , Antidotes/chemical synthesis , Bacillus anthracis/drug effects , Bacterial Toxins/antagonists & inhibitors , Acetates/pharmacokinetics , Acetates/pharmacology , Animals , Antidotes/pharmacokinetics , Antidotes/pharmacology , Antigens, Bacterial , Bacillus anthracis/physiology , Crystallography, X-Ray , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP2D6 Inhibitors , Microsomes, Liver/enzymology , Models, Molecular , Rabbits , RatsABSTRACT
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.