No abstract

The increasing prevalence of drug-resistant bacterial infections demands the development of new antibacterials that are not subject to existing mechanisms of resistance. Previously, we described coumarin-based inhibitors of an underexploited bacterial target, namely, the replicative helicase. Here we report the synthesis and evaluation of optimized coumarin-based inhibitors with 9–18-fold increased potency against S. aureus (Sa) and B. anthracis (Ba) helicases. Compounds 20 and 22 provided the best potency, with IC50 values of 3 and 1 µM, respectively, against the DNA duplex strand-unwinding activities of both B. anthracis and S. aureus helicases without affecting the single strand DNA-stimulated ATPase activity. Selectivity index (SI = CC50/MIC) values against S. aureus and B. anthracis for compound 20 were 33 and 66 and for compound 22 were 20 and 40, respectively. In addition, compounds 20 and 22 demonstrated potent antibacterial activity against multiple ciprofloxacin-resistant MRSA strains with MIC values ranging between 0.5–4.2 µg/mL.

NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogs have been synthesized and shown to inhibit BoNT/A LC in a FRET-based enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in these two series is compound 12 (IC 50 = 2.5 µM, FRET assay), which is 4.4-fold more potent than the lead structure, and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase. Structure-activity relationship studies have revealed structural features important to potency and enzyme specificity.

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure-activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.

Botulinum Neurotoxins (BoNTs) are used therapeutically and in cosmetics, providing potential for bioterrorist activity, thus driving the search for small-molecule BoNT inhibitors. This report describes a 70,000-compound screen for inhibition of BoNT/A using a FRET assay to detect proteolysis of a peptide substrate. Hits were confirmed, followed by evaluation to determine compound specificity. Inhibitors fell into three main chemical classes, and on the basis of potency and specificity of inhibition, the activities of two chemotypes were examined further. Compounds exhibited specificity for BoNT/A LC inhibition with respect to other metalloproteases and displayed activity in a neuronal assay for botulinum intoxication.