Dataset Information

Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A.

ABSTRACT: The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K(i) values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2' position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.

SUBMITTER: Zuniga JE

PROVIDER: S-EPMC2894858 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A.

Zuniga Jorge E JE Hammill Jared T JT Drory Omri O Nuss Jonathan E JE Burnett James C JC Gussio Rick R Wipf Peter P Bavari Sina S Brunger Axel T AT

PloS one 20100630 6

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K(i) values are in the nM-range. A co-c ...[more]

PMID: 20614028

Similar Datasets

Project description:Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K(i) (app) value of 3.8+/-0.8 microM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC(50) value of 100 microM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 A(2) for BoNTAe is about four times larger than the typical protein-protein interface area of 750-1,500 A(2). Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K(i) value of 760+/-170 nM using synthesis-based computer-aided molecular design (SBCAMD). This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight into structural modification of known small-molecule BoNTAe inhibitors. It also demonstrates that SBCAMD is capable of improving potency of an inhibitor lead by nearly one order of magnitude, even for BoNTAe as one of the most challenging protein targets. The results are insightful for developing effective small-molecule inhibitors of protein targets with large active sites.

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

Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A.

Publications

Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets