Issues in therapeutic development against botulinum neurotoxins (BONT)

Abstract

Studies of the BoNT holotoxin are limited by its intrinsic toxicity. The N-terminal light chain (LC) catalytic domain is a target for therapeutic drug development. But investigators have experienced large variations in its solubility, stability, and specific activity. Efforts to develop inhibitors are likely to be adversely affected by such variations. Thus, a thorough understanding of the factors behind these variations, and finding ways in obtaining a consistently soluble, stable, and active preparation of the LC are very important. In addition, LC has been reported to undergo post-translocation modification in neurons. The most logical targets for drug development should be such modified LC. Investigation of the properties of these modified species is therefore also very important. We have investigated several factors that lead to physical and catalytic instability of the LC. They include an autocatalytic reaction whereby the LC is fragmented into two major peptides with largely diminished activity that was prevented by adding polyols. We also found that inadvertent, gentle agitation rapidly precipitated the LC that is catalytically inactive, but did not affect BSA, ovalbumin, and thermolysin. Because the precipitation of BoNT domains must preclude some partly denaturation step(s), the resulting denatured species may display varied activity, stability, and solubility. Protection from precipitation and inactivation was attained by including low concentrations of nonionic detergents. The research was supported by JSTO-CBD Project#3.10011_06_RD_B. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.