Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A

Botulinum neurotoxins have remarkable
persistence (∼weeks
to months in cells), outlasting the small-molecule inhibitors designed
to target them. To address this disconnect, inhibitors bearing two
pharmacophoresa zinc binding group and a Cys-reactive warheadwere
designed to leverage both affinity and reactivity. A series of first-generation
bifunctional inhibitors was achieved through structure-based inhibitor
design. Through X-ray crystallography, engagement of both the catalytic
Zn2+ and Cys165 was confirmed. A second-generation series
improved on affinity by incorporating known reversible inhibitor pharmacophores;
the mechanism was confirmed by exhaustive dialysis, mass spectrometry,
and in vitro evaluation against the C165S mutant.
Finally, a third-generation inhibitor was shown to have good cellular
activity and low toxicity. In addition to our findings, an alternative
method of modeling time-dependent inhibition that simplifies assay
setup and allows comparison of inhibition models is discussed.