Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A - PubMed (original) (raw)

Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A

Virginia Roxas-Duncan et al. Antimicrob Agents Chemother. 2009 Aug.

Abstract

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-nitro-anilino)(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 microM. 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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Figures

FIG. 1.

FIG. 1.

Integrated experimental flow chart for identification of BoNT/A small-molecule inhibitors.

FIG. 2.

FIG. 2.

Structures of selected hits and percent inhibition against recombinant full-length BoNT/A LC (rALC). Structures were obtained from the NCI Developmental Therapeutics Program website (

http://dtp.nci.nih.gov/index.html/

). Compounds were tested in an HPLC-based assay using recombinant full-length BoNT/A LC (140 nM) in the presence of 0.8 mM 17-mer SNAP-25 peptide substrate (see Materials and Methods for details). Percentages of inhibition at 200 and 20 μM are indicated in parenthesis and were determined by the amounts of peptide substrate cleaved in the presence or absence of inhibitors under the same conditions.

FIG. 3.

FIG. 3.

Structural formula and IC50 values of selected analogs against recombinant full-length BoNT/A LC (rALC) and truncated BoNT/A LC (tALC; residues 1 to 425). Structures of NSC compounds were obtained from the NCI Developmental Therapeutics Program (

http://dtp.nci.nih.gov/index.html/

), and those of CB compounds were from Chembridge screening compounds and building blocks (

http://www.hit2lead.com/

). Each compound structure was redrawn by using ChemDraw Ultra 11 (

http://www.cambridgesoft.com/

). All compounds tested were racemates. The structure and purity of these analogs were confirmed by liquid chromatography-mass spectrometry and nuclear magnetic resonance. IC50 values for rALC and tALC are indicated in parentheses and were determined from nine concentrations of each inhibitor by using GraphPad Prism 4 (GraphPad Software, La Jolla, CA). rALC (140 nM final concentration) or tALC (620 nM) was incubated with 0.8 mM 17-mer SNAP-25 peptide substrate and various concentrations of inhibitor (dissolved in DMSO at 10× the final concentration) at 37°C for 5 min in 50 mM HEPES, pH 7.3. Reactions were stopped by adding 0.7% trifluoroacetic acid and analyzed by reverse-phase HPLC (see Materials and Methods for details).

FIG. 4.

FIG. 4.

Efficacy of small-molecules in inhibiting SNAP-25 cleavage. (A) Gel cleavage assay using recombinant BoNT/E LC (rELC). Inhibitors (240 μM) and rELC (6.0 μM) were added to recombinant SNAP-25 (S25; 12.9 μM) and incubated at 37°C for 30 min. The reaction products were analyzed on SDS-PAGE gels. Lane 1, S25 alone; lanes 2 to 7, S25 with rELC (lane 2), NSC 84096 (lane 3), CB 7967495 (lane 4), CB 7969312 (lane 5), NSC 84094 (lane 6), and CB 79698218 (lane 7). Panels B and C. Inhibition of BoNT/A-mediated SNAP-25 cleavage by small-molecules in cell-based assay. N2a cells were grown in EMEM with supplements at 37°C in an atmosphere of 5% CO2 and 95% O2. Upon 70 to 80% confluence, the cells were used to seed a six-well plate and incubated for 48 h. The medium was removed and replaced with serum-free medium. Cells were grown for an additional 24 h with either 10 nM BoNT/A-DMSO or inhibitor with 10 nM BoNT/A. The cells were harvested and the samples were analyzed by Western blotting (see Materials and Methods for details). Efficacy of five small-molecule inhibitors at 15 μM (B) and at 10 μM (C). Lane 1, with BoNT/A; lane 2, without BoNT/A; lanes 3 to 7, BoNT/A with inhibitor NSC 84096 (lane 3), CB 7967495 (lane 4), CB 7969312 (lane 5), NSC 84094 (lane 6), and CB 7968218 (lane 7). These blots represent three independent experiments.

FIG. 5.

FIG. 5.

Effects of BoNT/A small-molecule inhibitors in MPNHDA. Mouse hemidiaphragms were attached to isometric force transducers, and the phrenic nerves were secured to stimulating electrodes. Inhibitors (dissolved in DMSO at 0.3% final assay concentration) were mixed with BoNT/A toxin (30 pM final concentration) and incubated at 37°C for 15 min prior to addition to the bath after baseline stabilization. Twitch tension data collection continued for 5 h or until muscle twitch tension ceased (for details, see Materials and Methods). In all assays, BoNT/A (+) and No Toxin (▵) controls were run to demonstrate the difference in twitch tension between BoNT-intoxicated and normal tissues (n = 18 for both groups). The twitch tension time courses are the averages of results from triplicate assays. The time courses include only the effective concentration of inhibitor required for maximum protection, except for CRATKML, which was not effective at the tested concentration: CB 7969312 (500 nM) (▪), CB 7967495 (5 μM) (▴), NSC 84094 (10 μM) (○), CB 7968218 (10 μM) (♦), NSC 84096 (20 μM) (x), and CRATKML (20 μM) (•).

FIG. 6.

FIG. 6.

Binding mode of NSC 84094 into BoNT/ALC substrate binding cleft showing the quinolinol group (light green) interacting with the Zn atom (light blue), while the pyridyl substituent can form hydrogen bond with Arg363. Atom colors: nitrogen (blue), oxygen (red), hydrogen (white), enzyme C atoms (dark green), and inhibitor C atoms (light green).

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