Inhibitors of anthrax lethal factor

doi:10.1016/j.bmcl.2007.05.089

Bioorganic & Medicinal Chemistry Letters

Volume 17, Issue 16, 15 August 2007, Pages 4575-4578

https://doi.org/10.1016/j.bmcl.2007.05.089 Get rights and content

Abstract

An inhibitor of anthrax lethal toxin mediated cell death (1) was identified by a medium throughput cell-based screen. This compound was determined to specifically inhibit anthrax lethal factor (LF), and subsequent SAR studies produced an even more potent inhibitor (4). Mechanistic studies identified these agents as uncompetitive inhibitors of LF with K_i values of 3.0 and 1.7 μM, respectively, with good cell potency and low cytotoxicity.

Graphical abstract

An inhibitor of anthrax lethal factor (1) was discovered. A subsequent SAR and mechanistic study was performed to identify uncompetitive inhibitors of anthrax lethal factor with good cell potency and low cytotoxicity.

Section snippets

Acknowledgments

This work was supported by a grant from the Showalter Foundation. The authors are grateful to Mark Lipton for helpful discussions, Andrew Mesecar for PA, and Jenna Rickus for use of the Flex Station II fluorometer.

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Cited by (23)

Highly predictive support vector machine (SVM) models for anthrax toxin lethal factor (LF) inhibitors
2016, Journal of Molecular Graphics and Modelling
Citation Excerpt :
Antibiotics such as fluoroquinolones are capable of eradicating the bacilli, however, host death from residual toxemia can occur even after B. anthracis is cleared from the system, and there is currently no extant therapeutic modality to directly combat LF-mediated cytotoxicity [6,7]. As B. anthracis continues to pose a significant threat as a biological weapon, various experimental and computational efforts have been focused on identifying small-molecule LF inhibitors as potential drugs as adjunct therapeutics with antibiotics [4,8–33]. Previous computational modeling efforts have been primarily directed toward structure-based virtual screening, pharmacophore mapping, and 3D-QSAR model development [28–33].
Anthrax is a highly lethal, acute infectious disease caused by the rod-shaped, Gram-positive bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), a zinc metalloprotease secreted by the bacilli, plays a key role in anthrax pathogenesis and is chiefly responsible for anthrax-related toxemia and host death, partly via inactivation of mitogen-activated protein kinase kinase (MAPKK) enzymes and consequent disruption of key cellular signaling pathways. Antibiotics such as fluoroquinolones are capable of clearing the bacilli but have no effect on LF-mediated toxemia; LF itself therefore remains the preferred target for toxin inactivation. However, currently no LF inhibitor is available on the market as a therapeutic, partly due to the insufficiency of existing LF inhibitor scaffolds in terms of efficacy, selectivity, and toxicity. In the current work, we present novel support vector machine (SVM) models with high prediction accuracy that are designed to rapidly identify potential novel, structurally diverse LF inhibitor chemical matter from compound libraries. These SVM models were trained and validated using 508 compounds with published LF biological activity data and 847 inactive compounds deposited in the Pub Chem BioAssay database. One model, M1, demonstrated particularly favorable selectivity toward highly active compounds by correctly predicting 39 (95.12%) out of 41 nanomolar-level LF inhibitors, 46 (93.88%) out of 49 inactives, and 844 (99.65%) out of 847 Pub Chem inactives in external, unbiased test sets. These models are expected to facilitate the prediction of LF inhibitory activity for existing molecules, as well as identification of novel potential LF inhibitors from large datasets.
D-Proline-based peptidomimetic inhibitors of anthrax lethal factor
2012, European Journal of Medicinal Chemistry
Citation Excerpt :
Indeed, a possible therapeutic approach envisages the combination of antibiotics with LF inhibitors to clear the active infection while blocking the toxin already present in the cells. There are a number of successful LF inhibitors reported to date [16–21], and important structure–activity studies for understanding binding mode features of chemically diverse inhibitors have been published during the last decade [22,23]. Also, recent reports on potent hydroxamic acid-based LF inhibitors [24,25], and new SAR studies have been reported [26].
In this work we reported the generation of d-proline-derived hydroxamic acids as inhibitors of anthrax lethal factor (LF), taking advantage of a pyrrolidine ring as the central scaffold and a hydroxamate group as the Zn²⁺ chelating agent. The introduction of two hydrophobic groups addressing the S1′ subsite and a long substrate-binding groove was conceived by overlapping the bioactive conformations of two reported LF inhibitors. Micromolar affinity of compound 38 suggested cis-3-substituted-1-sulfonamido-d-proline hydroxamic acids as a promising class of peptidomimetic inhibitors for developing novel LF inhibitors.
Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors
2011, Biochemical and Biophysical Research Communications
Citation Excerpt :
In summary, this study describes a novel LF inhibitor screening system in a high-throughput format. Despite these concerns like differences between yeast and mammalian cells, the screening system reported here has an advantage over existing animal cell-based screens [26,27] in that the hits are specific for LF activity inhibition rather than blocking entry or altering host response to toxin action. Therefore, target identification appears to be straight forward to obtain LF inhibitors.
Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.
Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F
2008, Protein Expression and Purification
Lethal factor (LF) is a 90 kDa zinc metalloprotease that plays an important role in the virulence of anthrax. Recombinant LF (rLF) is an effective tool to study anthrax pathogenesis and treatment. In this study, the LF gene was cloned into the Escherichia coli expression vector pGEX-6P-1 and expressed as a GST fusion protein (GST-rLF) in E. coli BL21-codonPlus (DE3)-RIL cells with 0.2 mM IPTG induction at 28 °C. The GST-rLF protein was purified and the GST-tag was then cleaved in a single step by combining both GST-affinity column and treatment with 3C protease. This procedure yielded 5 mg of rLF protein per liter of culture. The purified rLF was functional as confirmed by cytotoxicity assay in RAW264.7 cells and Western blot assay. Furthermore, the rLF could induce strong immune response in BALB/c mice and the presence of a specific antiserum could neutralize the cytotoxicity of rLF in vitro. In addition, a novel inactive mutant (rLFm-Y236F) was obtained. Compared to the wild-type rLF, an increase by 3700 folds of the purified rLFm-Y236F was needed to achieve a similar level of cytotoxicity of the wild-type rLF. This mutant might be of significance in the study of anthrax pathogenesis and treatment.
Inhibitors of anthrax lethal factor based upon N-oleoyldopamine
2008, Bioorganic and Medicinal Chemistry Letters
The structural features of an anthrax lethal factor inhibitor, N-oleoyldopamine (OLDA, 1) have been probed. The oleic acid moiety is critical, but, more interestingly, the presence of the double bond and its geometry were found to play an essential role. One compound, 5, was found to be an uncompetitive inhibitor of lethal factor (LF) with a K_i value of 2.2 μM and a cell-based IC₅₀ value of 4.3 μM.
Targeting metalloenzymes for therapeutic intervention
2019, Chemical Reviews

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Inhibitors of anthrax lethal factor

Abstract

Graphical abstract

Section snippets

Acknowledgments

Biochem. Biophys. Res. Comm.

Trends Biochem. Sci.

Cell. Signalling

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

J. Biol. Chem.

Science

Biochem. J.

Biochem. J.

Biochem. J.

Infect. Immun.

Nature

EMBO Reports

ChemBioChem

Nat. Struct. Biol.