Identification of small molecule inhibitors of anthrax lethal factor

Panchal, Rekha G.; Hermone, Ann; Nguyen, Tam Luong; Wong, T.Y.; Schwarzenbacher, Robert; Schmidt, James J.; Lane, Douglas; McGrath, Connor F.; Turk, Benjamin E.; Burnett, James C.; Aman, M. Javad; Little, Stephen F.; Sausville, Edward A.; Zaharevitz, Daniel; Cantley, Lewis C.; Liddington, Robert; Gussio, Rick; Bavari, Sina

doi:10.1038/nsmb711

Cited by 135 publications

(170 citation statements)

References 24 publications

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“…We succeeded in detecting LF inhibition by 19 F-NMR using the fluorinated peptide Ac-A-R-R-K-K-V-Y-P-NH-Ph-CF 3 as an enzymatic substrate (24,25). Cleavage of the peptide occurring at the Pro-Xxx position deeply affects chemical environment of 19 F nuclei because of the conversion of the amide functionality into an amine with release of pCF 3 -aniline.…”

Section: Resultsmentioning

confidence: 99%

Efficient synthetic inhibitors of anthrax lethal factor

Forino

Johnson

Wong

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

124

144

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Inhalation anthrax is a deadly disease for which there is currently no effective treatment. Bacillus anthracis lethal factor (LF) metalloproteinase is an integral component of the tripartite anthrax lethal toxin that is essential for the onset and progression of anthrax. We report here on a fragment-based approach that allowed us to develop inhibitors of LF. The small-molecule inhibitors we have designed, synthesized, and tested are highly potent and selective against LF in both in vitro tests and cell-based assays. These inhibitors do not affect the prototype human metalloproteinases that are structurally similar to LF. Initial in vivo evaluation of postexposure efficacy of our inhibitors combined with antibiotic ciprofloxican against B. anthracis resulted in significant protection. Our data strongly indicate that the scaffold of inhibitors we have identified is the foundation for the development of novel, safe, and effective emergency therapy of postexposure inhalation anthrax.

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Section: Resultsmentioning

confidence: 99%

Efficient synthetic inhibitors of anthrax lethal factor

Forino

Johnson

Wong

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

124

144

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“…EF is an adenylate cyclase, and LF is a protease that cleaves mitogen-activated protein kinase kinases. The enzymatic activities of these proteins contribute to disease progression in several ways and at different stages of infection (16).Several anthrax-toxin inhibitors have been described that interfere with different steps in this intoxication pathway (8,11,12,16,(20)(21)(22)(23)(24)(25)(26), but none has targeted the host receptors. Here, we describe the development of a polyvalent receptor-directed anthrax-toxin inhibitor that binds both receptors and protects animals from toxin challenge.…”

mentioning

confidence: 99%

Polyvalent inhibitors of anthrax toxin that target host receptors

Basha

Rai

Poon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthraxtoxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.antimicrobial resistance ͉ phage display ͉ therapeutics P athogens can develop resistance to drugs directed against microbial targets by modifying the drug, by lowering the concentration of drug that reaches the target, or by mutating the target (1, 2). There is also an increasing concern that therapeutics developed for bioterrorism agents may be rendered ineffective if the microbial target is altered intentionally. This problem could be overcome, however, by designing inhibitors that block host proteins used by the pathogen or its toxins to cause disease.Microbial pathogens and their products interact with host structures to facilitate colonization or to promote cellular uptake. Many of these interactions are polyvalent, meaning that they involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another (3). The design of synthetic polyvalent (4-8) or oligovalent (9, 10) molecules also represents a promising approach to enhance the potency of inhibitors of microbial pathogens and toxins. Current examples of this approach have involved the design of molecules that bind directly to the pathogen or toxin. Inhibitors that bind host proteins would represent an effective way to attenuate virulence that may be less susceptible to resistance mechanisms, and the use of polyvalency could provide a significant enhancement in the potency of these inhibitors.ANTXR1 and ANTXR2 are host receptors that bind and internalize anthrax toxin (11,12). These proteins are likely important for anthrax pathogenesis because the toxin impairs the immune response and is responsible for the major symptoms and death associated with anthrax. Thus, blocking these receptors could represent a promising approach to anthrax therapy.ANTXR1 and ANTXR2 are widely expressed type I membrane proteins that bind components of the extracellular matrix (13). They both contain an extracellular I domain, which binds the protective antigen (PA) component of anthrax toxin. The two proteins are 40% identical overall and share 60% identity within ...

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“…FRET studies using small peptides demonstrated 100-fold faster cleavage of a consensus cleavage sequence (derived from MAPKK cleavage sites) when compared with natural sites (8). Using FRET peptides, small molecule inhibitors with K i 's ranging from 0.5 lM to 7 nM have been isolated (13,14). Absorbance methods using p-nitroanilide coupled peptides in HTS screens were used to select the peptide inhibitor IN-2-LF (9).…”

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confidence: 99%

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Saunders¹,

Kim²,

Woods³

et al. 2006

Cytometry Pt A

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Background: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. Methods: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCytÒ flow cytometry system. Results: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition

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Identification of small molecule inhibitors of anthrax lethal factor

Cited by 135 publications

References 24 publications

Efficient synthetic inhibitors of anthrax lethal factor

Efficient synthetic inhibitors of anthrax lethal factor

Polyvalent inhibitors of anthrax toxin that target host receptors

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

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