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

Walter Reed Army Institute of Research, Silver Spring, Maryland, United States
Bioorganic & Medicinal Chemistry (Impact Factor: 2.79). 02/2006; 14(2):395-408. DOI: 10.1016/j.bmc.2005.08.018
Source: PubMed


Botulinum neurotoxin serotype A (BoNTA) is one of the most toxic substances known. Currently, there is no antidote to BoNTA. Small molecules identified from high-throughput screening reportedly inhibit the endopeptidase--the zinc-bound, catalytic domain of BoNTA--at a drug concentration of 20 microM. However, optimization of these inhibitors is hampered by challenges including the computational evaluation of the ability of a zinc ligand to compete for coordination with nearby residues in the active site of BoNTA. No improved inhibitor of the endopeptidase has been reported. This article reports the development of a serotype-selective, small-molecule inhibitor of BoNTA with a K(i) of 12 microM. This inhibitor was designed to coordinate the zinc ion embedded in the active site of the enzyme for affinity and to interact with a species-specific residue in the active site for selectivity. It is the most potent small-molecule inhibitor of BoNTA reported to date. The results suggest that multiple molecular dynamics simulations using the cationic dummy atom approach are useful to structure-based design of zinc protease inhibitors.

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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]. But the information shows that small-molecules can significantly protect mammals against BoNT/A is scanty [31], [36]. "
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    ABSTRACT: 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 IC(50) 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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    • "In addition, adding more functional groups can hamper cell permeability. For these reasons, sophisticated computer simulations such as MMDSs employing the cationic dummy atom approach to simulating zinc proteins [19], [43]–[45] are needed to identify new functional groups. These groups must have high affinities for the BoNTAe active site and low molecular weights to maintain cell permeability. "
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    ABSTRACT: 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.
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