Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A

Park, Jewn Giew; Sill, Peter C.; Makiyi, Edward F.; García-Sosa, Alfonso T.; Millard, Charles B.; Schmidt, James J.; Pang, Yuan Ping

doi:10.1016/j.bmc.2005.08.018

Cited by 71 publications

(71 citation statements)

References 56 publications

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“…The HEXXH motif that is characteristic of the catalytic site of Zn-endopeptidase is conserved. For instance, the active sites of BoNT/A and BoNT/B endopeptidases differ only in two residues: F162 and F193 in BoNT/A correspond to N169 and S200 in BoNT/B (32). Thus, it is conceivable for some BoNT/A inhibitors to be also active against other serotypes.…”

Section: Discussionmentioning

confidence: 99%

“…Though the above-mentioned approaches have resulted in the identification of a number of small-molecule BoNT/A inhibitors, no compound has yet advanced to preclinical development. The majority of these leads have been demonstrated to be effective only in enzymatic assays (11,12,29,32,47). Only a few small molecules have been tested in cell-based assays (5, 9, 15) that involved mixing the compound with the toxin, and not by preloading the inhibitor.…”

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Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Roxas-Duncan

Enyedy

Montgomery

et al. 2009

Antimicrob Agents Chemother

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An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitroanilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 M. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Roxas-Duncan

Enyedy

Montgomery

et al. 2009

Antimicrob Agents Chemother

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“…Die Verbindung wirkt kompetitiv, was die geäußerte Hypothese widerlegt, dass mindestens zehn Kohlenstoffatome für eine effektive BoNT-Inhibierung notwendig seien. [118,119] Kristallstrukturen von LC/A im Komplex mit den Inhibitoren l-Arg-Hydroxamat, 4-Chlorzimtsäure und 2,4-Dichlorzimtsäure weisen auf eine gewisse Flexibilität des aktiven Zentrums hin und unterstreichen damit die Wahrscheinlichkeit, dass die Substratspezifität von Wechselwirkungen an sekundären Bindungsregionen herrührt. [120] Der Metallchelatbildner TPEN wurde an hemidiaphragmatischen Nervus-Phrenicus-Präparationen aus Mäusen sowie an einem vollständigen Mausmodell untersucht.…”

Section: +unclassified

“…Mit dem gleichen Ziel wurde in einer weiteren Studie ein computerbasiertes Screening von 2.5 Millionen Verbindungen ausgeführt. [118] Ausgangspunkt waren Moleküldynamik-simulationen (Cationic-Dummy-Ansatz; CaDA) des ZinkEndopeptidase-Inhibitorkomplexes. [130] Nach umfangreichen strukturgelenkten Modifizierungen wurde ein kompetitiver Inhibitor (34 a, Schema 8) mit einem K i -Wert von 12 mm erhalten.…”

Section: Niedermolekulare Inhibitoren Der Leichten Kette Vonunclassified

Der seltsame Fall des Botulinum‐Neurotoxins: chemische und biologische Modulierung des tödlichsten aller Gifte

et al. 2008

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In seiner klassischen Erzählung “Der Seltsame Fall des Dr. Jekyll und Mr. Hide” beschreibt Robert Louis Stevenson einen gespaltenen Menschen zwischen Gut und Böse. Botulinum‐Neurotoxin (BoNT), der wirksamste bekannte Giftstoff, ist von ähnlich zwiespältiger Natur: BoNT zeigt einerseits eine ausgeprägte Morbidität und Mortalität, kommt andererseits aber – wenn auch in viel geringeren Dosen – in einem breiten Spektrum klinischer Szenarien zum Einsatz. In jüngerer Zeit sind enorme Fortschritte beim Verständnis der Struktur und Funktion von BoNT erzielt worden, die eine intensive Erforschung von biomakromolekularen und niedermolekularen Modulatoren der BoNT‐Aktivität nach sich zogen. Ein Schwerpunkt dieser Projekte lag auf der Identifizierung von Inhibitoren, die einer BoNT‐Exposition, z. B. im Falle eines bioterroristischen Anschlags, entgegenwirken können. Dieser Aufsatz fasst die aktuellen Fortschritte bei der Entwicklung von Therapeutika zur Prävention und Behandlung von Botulismus zusammen, mit einem Schwerpunkt auf Impfstoffen sowie peptidischen und niedermolekularen Inhibitoren.

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“…[15][16][17] Investigators are limited, however, in their ability to assess the efficacy of these inhibitors in living cells and to evaluate additional critical parameters such as potential toxicity or ability to traverse cell membranes. Although testing in live intoxicated animals may ultimately be necessary, it is impractical to test large numbers of potential inhibitors initially identified as hit or lead compounds by high-throughput screening or other cell-free assays.…”

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confidence: 99%

Primary Cultures of Embryonic Chicken Neurons for Sensitive Cell-Based Assay of Botulinum Neurotoxin: Implications for Therapeutic Discovery

Stahl¹,

Ruthel²,

Torres-Melendez³

et al. 2007

SLAS Discovery

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Botulinum toxin is an exceedingly potent inhibitor of neurotransmission across the neuromuscular junction, causing flaccid paralysis and death. The potential for misuse of this deadly poison as a bioweapon has added a greater urgency to the search for effective therapeutics. The development of sensitive and efficient cell-based assays for the evaluation of toxin antagonists is crucial to the rapid and successful identification of therapeutic compounds. The authors evaluated the sensitivity of primary cultures from 4 distinct regions of the embryonic chick nervous system to botulinum neurotoxin A (BoNT/A) cleavage of synaptosomal-associated protein of 25 kD (SNAP-25). Although differences in sensitivity were apparent, SNAP-25 cleavage was detectable in neuronal cells from each of the 4 regions within 3 h at BoNT/A concentrations of 1 nM or lower. Co-incubation of chick neurons with BoNT/A and toxin-neutralizing antibodies inhibited SNAP-25 cleavage, demonstrating the utility of these cultures for the assay of BoNT/A antagonists. (Journal of Biomolecular Screening 2007:370-377)

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Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A

Cited by 71 publications

References 56 publications

Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Der seltsame Fall des Botulinum‐Neurotoxins: chemische und biologische Modulierung des tödlichsten aller Gifte

Primary Cultures of Embryonic Chicken Neurons for Sensitive Cell-Based Assay of Botulinum Neurotoxin: Implications for Therapeutic Discovery

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