Investigations into small molecule non-peptidic inhibitors of the botulinum neurotoxins

Abstract

Botulinum neurotoxins (BoNTs), proteins secreted by the bacteria genus Clostridium, represent a group of extremely lethal toxins and a potential bioterrorism threat. As the current therapeutic options are of a predominantly prophylactic nature and cannot be used en masse, new strategies and ultimately potential treatments are desperately needed to combat any widespread release of these neurotoxins. In these regards, our laboratory has been working on developing new alternatives to treat botulinum intoxication through the development of inhibitors of the light chain proteases, the etiological agent which causes BoNT intoxication. Such a strategy has required the construction of two high-throughput screens and small molecule non-peptidic libraries; excitingly, inhibitors of the BoNT/A protease have been uncovered and are being optimized via structure activity relationship studies.

Fig. 1

BoNT/A holotoxin (reprinted with permission from Trends Biochem. Sci. 2002, 57, 552.) The light chain endopeptidase (catalytic domain) is depicted in red color.

Fig. 2

Overview of the SNARE proteins targeted by the 7 different serotypes of botulinum neurotoxin.

Fig. 3

Structures of ‘hit’ compounds from the initial in situ screen. With an IC₅₀ of 15 μM, 4-chloro-cinnamic hydroxamate (1) was the most potent one.

Fig. 4

Structure-activity relationship (SAR) study sectors on the original ‘hit’ (1) and the structure of the new lead structure with improved potency (6).

Fig. 5

Crystal structures of 1 (A) and 6 (B) in the active site of BoNT/A LC protease (adapted with permission from Chem. Biol. 2007, 14, 533).

Fig. 6

Second-generation SAR of BoNT/A protease hydroxamate inhibitors.

Fig. 7

Structure of adamantane acetic acid hydroxamate (10), the most potent inhibitor of BoNT/A LC protease to date (K_I = 460 nM) and a new lead structure for inhibitor development. 11: ⁶⁰LSELDRADALQAGAS(K(Dnp))⁷⁶FE(Dpa(Mca))⁷⁹SAAKLKRKYWWKNLK⁹⁴ 12: ⁶⁰LSELDDRADALQAG(Pya)⁷⁴SQ(Nop)⁷⁷ESSAAKLKRKYWWKNLK⁹⁴ Native: ⁶⁰LSELDRADALQAGASQFETSAAKLKRKYWWKNLK⁹⁴

Fig. 8

Peptides synthesized and incorporated into high throughput assays for the detection of inhibitors of BoNT/B LC. (11) Developed in our laboratory: K(DNP) = 2,4-dinitrophenyl (DNP) attached to lysine, Dpa(MCA)= diaminopropionic acid (Dpa) attached to 7-methoxycoumarin acetic acid (MCA); (12) Developed by the Roques laboratory (Anne, et al., 2004): Pya = pyrenylalanine, Nop = p-nitrophenylalanine; Native: A fragment of the native substrate containing the core central region (syb 60–94) of synaptobrevin (the substrate of the BoNT/B LC).