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Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.

ABSTRACT: Botulinum neurotoxins (BoNTs) are the deadliest of microbial toxins. The enzymes' Zinc(II) metalloprotease, referred to as the light chain (LC) component, inhibits acetylcholine release into neuromuscular junctions, resulting in the disease botulism. Currently, no therapies counter BoNT poisoning post-neuronal intoxication; however, it is hypothesized that small molecules may be used to inhibit BoNT LC activity in the neuronal cytosol. Herein, we describe the pharmacophore-based design and chemical synthesis of potent (non-Zinc(II) chelating) small molecule (non-peptidic) inhibitors (SMNPIs) of the BoNT serotype A LC (the most toxic of the BoNT serotype LCs). Specifically, the three-dimensional superimpositions of 2-[4-(4-amidinephenoxy)-phenyl]-indole-6-amidine-based SMNPI regioisomers (K(i) = 0.600 μM (± 0.100 μM)), with a novel lead bis-[3-amide-5-(imidazolino)-phenyl]-terephthalamide (BAIPT)-based SMNPI (K(i) = 8.52 μM (± 0.53 μM)), resulted in a refined 4-zone pharmacophore. The refined model guided the design of BAIPT-based SMNPIs possessing K(i) values = 0.572 μM (± 0.041 μM) and 0.900 μM (± 0.078 μM).

SUBMITTER: Nuss JE

PROVIDER: S-EPMC2992458 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.

Nuss Jonathan E JE Dong Yuxiang Y Wanner Laura M LM Ruthel Gordon G Wipf Peter P Gussio Rick R Vennerstrom Jonathan L JL Bavari Sina S Burnett James C JC

ACS medicinal chemistry letters 20101001 7

Botulinum neurotoxins (BoNTs) are the deadliest of microbial toxins. The enzymes' Zinc(II) metalloprotease, referred to as the light chain (LC) component, inhibits acetylcholine release into neuromuscular junctions, resulting in the disease botulism. Currently, no therapies counter BoNT poisoning post-neuronal intoxication; however, it is hypothesized that small molecules may be used to inhibit BoNT LC activity in the neuronal cytosol. Herein, we describe the pharmacophore-based design and chemi ...[more]

PMID: 21116458

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Project description:Background & objectivesBotulism, a potentially fatal paralytic illness, is caused by the botulinum neurotoxins (BoNTs) secreted by Clostridium botulinum. It is an obligate anaerobic, Gram-positive, spore-forming bacterium. BoNTs are classified into seven serotypes based on the serological properties. Among these seven serotypes, A, B, E and, rarely, F are responsible for human botulism. The present study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA)-based detection system for the detection of BoNT/E.MethodsThe synthetic gene coding the light chain of BoNT serotype E (BoNT/E LC) was constructed using the polymerase chain reaction primer overlapping method, cloned into pQE30UA vector and then transformed into Escherichia coli M15 host cells. Recombinant protein expression was optimized using different concentrations of isopropyl-β-D-1-thiogalactopyranoside (IPTG), different temperature and the rBoNT/E LC protein was purified in native conditions using affinity column chromatography. The purified recombinant protein was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and further confirmed by western blot and matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF). Polyclonal antibodies were generated against rBoNT/E LC using Freund's adjuvant in BALB/c mice and rabbit. Sandwich ELISA was optimized for the detection of rBoNT/E LC and native crude BoNT/E, and food matrix interference was tested. The developed antibodies were further evaluated for their specificity/cross-reactivity with BoNT serotypes and other bacterial toxins.ResultsBoNT/E LC was successfully cloned, and the maximum expression was achieved in 16 h of post-induction using 0.5 mM IPTG concentration at 25°C. Polyclonal antibodies were generated in BALB/c mice and rabbit and the antibody titre was raised up to 128,000 after the 2nd booster dose. The developed polyclonal antibodies were highly specific and sensitive with a detection limit about 50 ng/ml for rBoNT/E LC and 2.5×10[3] MLD50 of native crude BoNT/E at a dilution of 1:3000 of mouse (capturing) and rabbit (revealing) antibodies. Further, different liquid, semisolid and solid food matrices were tested, and rBoNT/E LC was detected in almost all food samples, but different levels of interference were detected in different food matrices.Interpretation & conclusionsThere is no immune detection system available commercially in India to detect botulism. The developed system might be useful for the detection of botulinum toxin in food and clinical samples. Further work is in progress.

Dataset Information

Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.

Publications

Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.

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