New FRET substrate for botulinum neurotoxin type A

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

“…This series of putative, bifunctional inhibitors was evaluated using a time-dependent assay using the fluorescence resonance energy transfer (FRET) substrate SNAPtide . Enzyme and inhibitor were preincubated for 0.5–30 min, followed by 100-fold dilution into substrate solutions to initiate the enzymatic reaction, while effectively diluting the inhibitor and halting further covalent inhibition.…”

Use of Crystallography and Molecular Modeling for the Inhibition of the Botulinum Neurotoxin A Protease

“…Having established solid synthetic routes, hydroxamates 7-28 were evaluated for BoNT/A LC inhibition (Table 1) using a previously described 26 fluorescence resonance energy transfer (FRET)based assay that uses a truncated form of the BoNT/A LC (amino acids 1-425) and a SNAPtide substrate. 30,31 Compounds were pre-incubated for 30 minutes at various concentrations with 10 nM BoNT/A LC followed by addition of 4 µM substrate with reaction progress fluorescently monitored at ex/em 490/523 nm. The initial target hydroxamate 7 had an IC50 value of 4.0 µM, thus showing no real improvement when compared to the alkyl-containing counterpart 6 (IC50 = 5.7 µM).…”

“…Notably, amide coupling conditions for 30 using less-reactive/orthosubstituted aniline reagents resulted in no formation of the desired product. Fortunately, a change in order of steps (route 2) reacting aniline reagents with 29 directly to give carboxylic acids (32) was successful. Furthermore, optimization of workup procedures allowed both the amide coupling and deprotection step to be combined, allowing rapid access to final compounds 7−28.…”