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

Abstract

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.

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

“…Although much smaller tetrapeptide inhibitors of LcA with nM k i have been described (30), the significance of the 32-mer peptide as an inhibitor lies in the fact that it is a part of the LcA itself, not resembling the true substrate. Recently, three hydroxamate-containing small molecule inhibitors having 5-6 M k i values were found to induce the most compact catalytic pocket in LcA (34).…”

Identification of small molecule inhibitors of botulinum neurotoxin serotype E via footprint similarity

“…20 Silvaggi et al 21 determined the crystal structure of ortho-para-chloro-cinnamic hydroxamate revealing the hydroxamic acid chelates the active site zinc. Building on this finding, Thompson et al 22 an experimental K i of 1.29 μM using an HPLC-based assay and represents the first reported small molecule inhibitor identified that targets the BoNT/E catalytic site. 26 Recent follow up work by the same group, 27 employing pharmacophore modeling, led to the identification of four roughly equipotent analogs.…”

Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening

“…Hydroxamates have been shown to have efficacy in mice, but have relatively short in vivo half-lives [18]. Developing tight binding non-chelating inhibitors of BoNT/A has proven to be a difficult task in part due to the high conformational plasticity of the binding pocket and induced conformational changes in adjacent loops upon substrate or inhibitor binding [19]. The exceptionally large substrate binding surface of BoNT/A poses an extremely challenging problem to design effective small molecule inhibitors that are capable of disrupting the extensive protein-protein interactions within the substrate binding interface.…”