Therapeutics which accelerate degradation BoNT/A LC within neurons

ARTICLE IN PRESS

/ Toxicon 51 (2008) 1–54 17

48. Design of fluorogenic substrates for botulinum

Tanja Ouimet, Herve Pores, Sou-Vinh Orng,Sophie Duquesnoy, Marie-Claude Fournie-Zaluski,P. Roques BernardPharmaleads, Paris-France

Botulinum toxin A is one of the most toxic substancesknown to man. It is constituted by a heavy chain whichcarries the receptor binding and membrane translocationdomain, and a light chain, a metalloprotease responsiblefor the inhibition of neurotransmitter release and paraly-sis by the specific cleavage of SNAP-25, part of theexocytosis complex. Based on this protease activity, usingquenched fluorigenic peptides, Pharmaleads developed ahigh-affinity peptide substrate used in a specific, rapid,sensitive and reliable quantification designated EzyBot.This technique, useful for the detection of bioterroristcontamination and for the quantification of therapeutictoxins, is based on the introduction of a highly fluorescentpyrenylalanine (Pya) with a nitro-phenylalanine (Nop)repressor around the cleavage site. Using the naturalsequence of the BoNT/A substrate, peptides of variouslengths encompassing the cleavage site flanked with Pyaand Nop were designed. More specifically, a minimal 17-mer and a longer 48-mer peptide reaching the previouslyidentified a-exosite were synthesized, with the lattershowing significantly higher affinity for BoNT/A. Peptidemapping of the 48-mer peptide reveals that this increasedaffinity is also due to an intermediate peptide sequencelocated between the N-terminal end of the 17-mer and thea-exosite sequence, revealing a new cooperative exositefor BoNT/A substrate binding.

Keywords: Metalloprotease; Exosite; Fluorimetric quantification; EzyBot

10.1016/j.toxicon.2008.04.050

49. Therapeutics which accelerate degradation BoNT/ALC within neurons

George A. Oyler a,b, C.L. Kuo a, J. Sepulveda a, Y.C. Tsai c,R. Kincaid d, S. Tzipori a, Charles B. Shoemaker a

a Tufts Cummings School of Vet. Med., MA, USAb Synaptic Research, Baltimore, MD, USAc NCI, Maryland, USAd Veritas, MD, USA

Shortening persistence of BoNT intoxication is a thera-peutic goal since no treatment exists after paralysis ensues.The ubiquitin–proteasome system degrades BoNT/E LCfaster than BoNT/A; thus, preferential ubiquitination ofE or deubiquitination of A contributes to persistence. Wehave produced ‘‘designer’’ E3 ubiquitin ligases whichspecifically ubiquitinate BoNT/A LC. E3 ligases are modular:one module binds to substrate target protein and a secondcatalyzes ubiquitination. Our BoNT/A LC designer E3 ligasesutilize an A-LC-directed domain: SNAP25nc or anti-BoNT/ALC camelid antibody (B8-VHH) and an E3 functionaldomain: Fbox, RING, HECT, R-IBR-R, or U-box. B8-VHH

Abstracts Toxins 2008

binds to A-LC with 2 nM kDa and inhibits LC proteaseactivity. Intracellular B8-VHH protects co-transfectedSNAP25 from LC cleavage. SNAP25nc and B8-VHH designerligases protect SNAP25 from LC cleavage primarily byaccelerating LC degradation. The optimal designer ligaseconsists of B8-VHH and Fbox domains. We are pursuingprotein delivery systems for the B8-VHH/Fbox designerligase and truncating the F-box domain to the minimaleffective region.

Support: NO1-AI30050 from NIAID.

Keywords: Persistence; Ubiquitin; Camelid antibody

10.1016/j.toxicon.2008.04.051

50. Botulinum toxin injection into the trapezius muscleof cynomolgus monkeys: Comparison of serotype Awith B

Eric Pappert a, Joseph Arezzo b

a Univ. of Texas Health Sci. Ctr./Solstice Neurosciences, Inc., San Antonio, USAb Albert Einstein College of Medicine, Bronx, USA

Objective: To compare the magnitude and duration ofmuscle paralysis induced by botulinum toxin (BoNT) typeA and type B in cynomolgus monkeys.

Methods: Twenty-two monkeys were injected with oneof five doses of BoNT-A, BoNT-B or a placebo in each oftheir trapezius muscles. Compound muscle action poten-tials (CMAP) were measured at baseline and 2, 4, 8, 12 and16 weeks post-injection in all animals and at 20 and 24weeks post-injection in the two highest dose groups.Toxins were injected and data scored with the investiga-tors ‘‘blinded’’ to the dose and serotype used in eachmuscle.

Results: At 2 weeks, the degree of paralysis for eachdose pair was approximately equivalent, with a rangeacross doses of 60–95%. A clear dose–recovery relation-ship was observed, with longer paralysis associated withhigher BoNT doses. At the lowest doses tested (i.e., 0.5 U ofBoNT-A and 16 U of BoNT-B), recovery, defined as adecrement of less than 20%, was evident by 12–16 weeksin all monkeys; at the highest doses (i.e., 10 U of BoNT-Aand 500 U of BoNT-B), the same degree of recovery wasdocumented only after 24 weeks.

Conclusions: The duration of induced paralysis isrelated to the BoNT dose and the initial degree of paralysisfor both serotypes. When BoNT doses that produceapproximately equivalent initial paralytic effects arecompared, the duration of action is similar acrossserotypes.

Keywords: Botulinum toxin; Duration of action

10.1016/j.toxicon.2008.04.052