Three‐Dimensional Database Mining Identifies a Unique Chemotype that Unites Structurally Diverse Botulinum Neurotoxin Serotype A Inhibitors in a Three‐Zone Pharmacophore

Hermone, Ann; Burnett, James C.; Nuss, Jonathan E.; Tressler, Lyal E.; Nguyen, Tam Luong; Šolaja, Bogdan A.; Vennerstrom, Jonathan L.; Schmidt, James J.; Wipf, Peter; Bavari, Sina; Gussio, Rick

doi:10.1002/cmdc.200800241

Cited by 37 publications

(47 citation statements)

References 47 publications

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“…Though the above approaches have resulted in the identification of a number of small-molecules as BoNT/A inhibitors, no compound has yet advanced to pre-clinical development [24], [29], [30], [31]. The majority of such molecules reportedly demonstrated to be effective in enzymatic assays [21], [23], [27], [28], [32], [33] and a few small-molecules have been tested in cell-based assays [34], [35], [36], [37].…”

Section: Introductionmentioning

confidence: 99%

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Singh

Chaudhary

et al. 2012

PLoS ONE

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Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC50 values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).

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Section: Introductionmentioning

confidence: 99%

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Singh

Chaudhary

et al. 2012

PLoS ONE

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“…Previous research [15] led to the identification of NSC 104999, a terephthalamide-based SMNPI of the BoNT/A LC metalloprotease (Figure 1). As part of the current study, various analogs of this SMNPI chemotype were obtained and examined for in vitro potency employing an HPLC-based assay.…”

Section: Resultsmentioning

confidence: 99%

Post-Intoxication Inhibition of Botulinum Neurotoxin Serotype A within Neurons by Small-Molecule, Non-Peptidic Inhibitors

Ruthel

Burnett

Nuss

et al. 2011

Toxins

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Botulinum neurotoxins (BoNTs) comprise seven distinct serotypes that inhibit the release of neurotransmitter across neuromuscular junctions, resulting in potentially fatal flaccid paralysis. BoNT serotype A (BoNT/A), which targets synaptosomal-associated protein of 25kDa (SNAP-25), is particularly long-lived within neurons and requires a longer time for recovery of neuromuscular function. There are currently no treatments available to counteract BoNT/A after it has entered the neuronal cytosol. In this study, we examined the ability of small molecule non-peptidic inhibitors (SMNPIs) to prevent SNAP-25 cleavage post-intoxication of neurons. The progressive cleavage of SNAP-25 observed over 5 h following 1 h BoNT/A intoxication was prevented by addition of SMNPIs. In contrast, anti-BoNT/A neutralizing antibodies that strongly inhibited SNAP-25 cleavage when added during intoxication were completely ineffective when added post-intoxication. Although Bafilomycin A1, which blocks entry of BoNT/A into the cytosol by preventing endosomal acidification, inhibited SNAP-25 cleavage post-intoxication, the degree of inhibition was significantly reduced versus addition both during and after intoxication. Post-intoxication application of SMNPIs, on the other hand, was nearly as effective as application both during and after intoxication. Taken together, the results indicate that competitive SMNPIs of BoNT/A light chain can be effective within neurons post-intoxication.

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“…BoNT antagonist development has been largely limited to studies of small sets of compounds or peptides chosen through rational design, computer-assisted, or other methods [54][55][56][57][58][59]. Although these studies have provided a considerable amount of information about BoNT catalytic mechanism and substrate requirements, few of the small molecule inhibitors that have emerged from these studies have sufficient potency to be effective BoNT therapeutics.…”

Section: Discussionmentioning

confidence: 99%

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Ruge

Dunning

Piazza

et al. 2011

Analytical Biochemistry

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a b s t r a c tMethods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra-and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.Ó 2011 Elsevier Inc. All rights reserved.Botulinum neurotoxins (BoNTs), 1 produced by the bacteria of the genus Clostridium, are the most lethal substances known. The zinc-dependent endopeptidases act by entering neurons and cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and, thus, compromise the protein machinery responsible for neurotransmitter release [1][2][3][4][5][6][7][8]. Failure to promptly treat a victim of BoNT poisoning can result in flaccid paralysis, respiratory failure, or death [9]. The combination of extreme potency and a lack of medical treatments other than antitoxin administration and intensive care has made BoNT a biodefense priority requiring the discovery and development of assays to quantify toxins and to identify antagonists to counteract intoxication [10][11][12][13]. Despite their lethality, BoNTs are widely used for cosmetic and pharmaceutical applications due in part to their exquisite specificity for the neuromuscular junction. BoNTs provide relief of muscle tension and pain by inhibiting neurons that cause excessive muscle contractions. Therapeutic preparations of BoNT/A and B serotypes are Food and Drug Administration (FDA) approved for treating glabellar lines, strabismus, cervical dystonia, blepharospasm, cranial nerve VII disorders, and primary axillary hyperhidrosis. Dozens of ''off-label'' BoNT clinical applications have also been documented [14][15][16][17].The mouse bioassay, or lethality test, has been the standard for testing BoNT-containing samples for the past 30 years [18][19][20]. Government agencies use this method for testing food and serum samples for the presence of BoNT, whereas the pharmaceutical industry uses it for quality control and to quantify ''for human use'' BoNT preparations. The test is carried out by injecting mice intraperitoneally with approximately 0.5 ml of sample per mouse and recording the number of deaths over a 1-to 7-day period. The assay is very sensitive, with a detection limit of 5-10 pg for BoNT/A [21,22]. Results for the mouse bioassay are reported in...

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Three‐Dimensional Database Mining Identifies a Unique Chemotype that Unites Structurally Diverse Botulinum Neurotoxin Serotype A Inhibitors in a Three‐Zone Pharmacophore

Cited by 37 publications

References 47 publications

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Post-Intoxication Inhibition of Botulinum Neurotoxin Serotype A within Neurons by Small-Molecule, Non-Peptidic Inhibitors

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

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