Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A
Lucy Lin, Margaret E. Olson, Takashi Sugane, Lewis D. Turner, Margarita A. Tararina, Alexander L. Nielsen, Elbek K. Kurbanov, Sabine Pellett, Eric A. Johnson, Seth M. Cohen, Karen N. Allen, Kim D. Janda
Abstract
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.