Chirality Holds the Key for Potent Inhibition of the Botulinum Neurotoxin Serotype A Protease

Abstract

Botulinum neurotoxin serotype A (BoNT/A) is the most toxic protein known to man, and also a bioterrorism agent. As defined by our previous research targeting the etiological agent responsible for BoNT/A intoxication, a protease, we now report on the asymmetric synthesis of four new BoNT/ A inhibitors; the most potent of this series is roughly twofold more active than the best small molecule inhibitor currently known.The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) are proteins produced by bacteria of the genus Clostridium. 1 BoNTs are synthesized as ~150-kDa proteins consisting of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC) linked by disulfide bonds. 2 The HC is responsible for translocation and binding, while the LC is a zinc-dependent endopeptidase that cleaves soluble N-ethylmaleimide-sensitive fusion proteins (SNARE) located at nerve endings. paralysis and subsequent death due to inhibition of the release of acetylcholine from synaptic terminals.4Current treatment for BoNT intoxication employs an antitoxin to sequester the toxin followed by removal of the complex from the body. However, this treatment option is dependent upon prompt diagnosis of intoxication, and must be administered rapidly before the toxin can enter the cell. 5 The antitoxin also suffers from the risk of allergic reaction, 6 long term effects of unknown origin 7 and insufficient available quantities for treatment of a large scale attack. 8BoNT serotype A (BoNT/A) is considered to be the most deadly BoNT serotype based on the following: first, and most importantly, BoNT/A has an extremely long duration of action, i.e. weeks to months. Second, there is no antidote for BoNT/A intoxication, with severe intoxication cases requiring mechanical ventilation due to respiratory paralysis. 9 Third, BoNT/ A, together with serotype B, is responsible for ~1000 cases of human BoNT poisoning per year.1 Finally, BoNT/A has a high potential for bioterrorism use due to its extreme toxicity and ease of dissemination through the food or water supply. Thus, the inhibition of BoNT/A LC is an attractive target for non-peptidic small molecules that can target the enzyme within an intoxicated cell.Previous research from one of our laboratories screened a diverse library of small molecules and discovered several inhibitors of BoNT/A, with trans-cinnamic hydroxamate 1 being our initial lead ( Figure 1). Hydroxamate 2 stemmed from structure activity studies, and is one of the most potent inhibitors known, having a K i value of 300 ± 12 nM.10 From these studies several lessons were learned including: (1) all structures required the hydroxamate functionality, a monoanionic bidentate chelator of zinc that has been previously used for the inhibition of metalloproteinases, 11 (2) the need for a para-halogenated phenyl ring 12 and (3) an ortho, para-dihalogenated ring was superior to para-substition alone 10 ( Figure 1). Indeed, x-ray crystallographic analysis of the bound inhibitors revealed hydroxyl coordination of the hydroxamate to the Zn ...

Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A

“…While, in a vast majority of cases, one enantiomer of a racemic drug or drug candidate has significantly greater pharmacological activity than the other, as has recently been demonstrated for one BoNT/A inhibitor, 27 we found essentially equivalent BoNT/A-inhibitory activity in (+)-( R )- 1 and (−)-( S )- 1 . However, this unexpected finding can be explained by the proposed docking motifs for the two enantiomers – different ensembles, but nearly equivalent in energy.…”

Synthesis/biological evaluation of hydroxamic acids and their prodrugs as inhibitors for Botulinum neurotoxin A light chain

“…17 Thus, dichlorobenzaldehyde was condensed with dimethyl malonate in the presence of piperidine to provide the α, β-unsaturated diester 5 . Dimethyl malonate underwent a Michael addition with 5 under basic conditions, affording tetraester 6 , which was then subjected to decarboxylation to furnish diacid 7 .…”

“…Previously our laboratory disclosed cinnamic hydroxamic acid 1 as a potent hit from hydroxamic acid library screening, 15,16 and a congener of this molecule, hydroxyethyl hydroxamate 2 as an inhibitor with an additional element of chemical functionality. 17 To further diversify this line of research, we devised two other scaffolds: amides ( 3 ) and ethers ( 4 ) as a means to further increase hydrophobic interactions within the active site of enzyme. Herein, we communicate the synthesis/molecular modeling of amides ( 3 ), ethers ( 4 ), and β-(2,4-dichlorophenyl)-hydroxamic acid O -carbamates along with their inhibitory activity as determined through enzymatic as well as cellular assays.…”

3-Hydroxy-1-alkyl-2-methylpyridine-4(1H)-thiones: Inhibition of the Pseudomonas aeruginosa Virulence Factor LasB

“…Our laboratories have invested much effort in exploring the modulation of metalloproteases for human health, such as identifying small molecule inhibitors of the botulinum neurotoxins, 12–18 anthrax lethal factor 19–22 and matrix metalloproteinases. 21, 22 To do so, we have designed and developed non-peptidic small molecules containing a variety of metal-chelating warheads as active site inhibitors.…”