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

Lin, L;Olson, ME;Sugane, T;Turner, LD;Tararina, MA;Nielsen, AL;Kurbanov, EK;Pellett, S;Johnson, EA;Cohen, SM;Allen, KN;Janda, KD;

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.