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New FRET Substrate For Botulinum Neurotoxin Type A

Shine, N.; Suryadi, K.

Purpose of Study: A new FRET peptide substrate for botulinum toxin type A (BoNT/A), marketed as SNAPtide flP6 (DABCYL/5-IAF) Prod #523, U.S. Patent pending #61/252,675, was evaluated and compared to SNAPtide (FITC/DABCYL) Prod #521. Methods Used: For kinetic studies, initial rates of cleavage were measured at room temperature (RT) for both SNAPtides after addition of 5 nM BoNT/A light chain (LC), Prod #610A, in Assay Buffer: 50 mM HEPES, pH 7.4 containing 0.05% TWEEN 20. The cleavage product was analyzed using HPLC with fluorescence detection. A standard curve was generated to convert the peak areas to nanomolar of cleaved product. The kinetic data was obtained from plots of initial rates expressed as nM/min versus concentration of FRET substrate. Results were calculated from nonlinear regression analysis using the Michaelis-Menton equation and Kaleidagraph software. Summary of Results: The kinetic data indicate that at RT the new SNAPtide flP6, Prod #523 has a considerably lower Km value, 3.8 M for #523 versus >200 M for #521, and that SNAPtide flP6, Prod #523 is cleaved significantly more efficiently than SNAPtide, Prod #521. The rate of cleavage for SNAPtide flP6, Prod #523, was 13.5 RFU/sec versus 4.8 RFU/sec for SNAPtide, Prod #521 using 8M substrate, with 10 nM BoNT/A LC at 37C. Both substrates show a linear fluorescence response up to approximately 8 M substrate. SNAPtide, Prod #521 is more sensitive using BoNT/A LC for cleavage. At 37C, 12.5 ng/ml can be detected after 2.5 hrs and 1.56 ng/ml can be detected after overnight digest with SNAPtide flP6, Prod #523. With SNAPtide, Prod #521, 0.6 ng/ml is detected at 2.5 hrs and 0.26 ng/ml after overnight digestion. Conclusions: The data indicate that SNAPtide flP6, Prod #523 is an ideal substrate for kinetic studies and for rapid inhibitor screening.