Abstract
The metalloprotease lethal factor (LF) from Bacillus anthracis plays a vital role in anthrax toxin action, and thus becomes a target for anti-anthrax therapy. Following the guidelines based on existing metalloprotease inhibitors, we designed a ‘first-generation’ LF inhibitor R9LF-1. This inhibitor was shown to be very stable by itself in a wide range of pH and temperature and able to inhibit LF activity in vitro. However, as we reported previously in the presence of LF, this inhibitor was degraded to a small molecular weight species, resulting in a significantly decreased ability to protect MAPKK from cleavage by LF as well as to protect murine macrophages from lethal toxin. In order to elucidate this unusual phenomenon to build solid basis for high-efficiency LF inhibitor development, we performed extensive research to study the effect of LF on its peptide-based inhibitor. Effects of temperature and incubation period of time on generation of the smaller peptide (short version R9LF-1) by LF as well as its catalytic domain were analyzed. We found that LF degraded R9LF-1 with maximum efficiency in the pH range of 7.0–8.5, which correlates well with the range of LF enzymatic activity with its native substrate. The degradation showed a deviation from normal hyperbolic kinetics but a similarity to the kinetics profile of an enzyme-catalyzed reaction with positive cooperativity. The short version R9LF-1 had decreased inhibitory activity toward LF; surprisingly, BIAcore results suggested a better affinity for its binding to LF. In addition, R9LF-1 was not hydrolyzed by other common proteases, such as chymotrypsin and pepsin, suggesting hydrolysis of the bond between amino acid and hydroxamate groups is unique to LF. This study calls for caution when designing peptide-based LF inhibitors and when interpreting effects of these types of inhibitors.
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Abbreviations
- LF:
-
Lethal factor
- LT:
-
Lethal toxin
- LF-N:
-
N-terminal domain of wild type LF
- MAPKK:
-
Mitogen-activated protein kinase kinase
- MALDI-TOF:
-
Matrix-assisted laser desorption/ionization time of flight
- MW:
-
Molecular weight
- PA:
-
Protective antigen
- SPR:
-
Surface plasmon resonance
- WT:
-
Wild type
References
Ascenzi P, Visca P, Ippolito G, Spallarossa A, Bolognesi M, Montecucco C (2002) Anthrax toxin: a tripartite lethal combination. FEBS Lett 531:384–388
Mourez M (2004) Anthrax toxins. Rev Physiol Biochem Pharmacol 152:135–164
Christensen KA, Krantz BA, Collier RJ (2006) Assembly and disassembly kinetics of anthrax toxin complexes. Biochemistry 45:2380–2386
Turk BE (2007) Manipulation of host signalling pathways by anthrax toxins. Biochem J 402:405–417
Young JA, Collier RJ (2007) Anthrax toxin: receptor binding, internalization, pore formation, and translocation. Annu Rev Biochem 76:243–265
Gujraty K, Sadacharan S, Frost M, Poon V, Kane RS, Mogridge J (2005) Functional characterization of peptide-based anthrax toxin inhibitors. Mol Pharm 2:367–372
Lim NK, Kim JH, Oh MS, Lee S, Kim SY, Kim KS, Kang HJ, Hong HJ, Inn KS (2005) An anthrax lethal factor-neutralizing monoclonal antibody protects rats before and after challenge with anthrax toxin. Infect Immun 73:6547–6551
Mourez M, Kane RS, Mogridge J, Metallo S, Deschatelets P, Sellman BR, Whitesides GM, Collier RJ (2001) Designing a polyvalent inhibitor of anthrax toxin. Nat Biotechnol 19:958–961
Rainey GJ, Young JA (2004) Antitoxins: novel strategies to target agents of bioterrorism. Nat Rev Microbiol 2:721–726
Turk BE, Wong TY, Schwarzenbacher R, Jarrell ET, Leppla SH, Collier RJ, Liddington RC, Cantley LC (2004) The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor. Nat Struct Mol Biol 11:60–66
Forino M, Johnson S, Wong TY, Rozanov DV, Savinov AY et al (2005) Efficient synthetic inhibitors of anthrax lethal factor. Proc Natl Acad Sci USA 102:9499–9504
Min DH, Tang WJ, Mrksich M (2004) Chemical screening by mass spectrometry to identify inhibitors of anthrax lethal factor. Nat Biotechnol 22:717–723
Panchal RG, Hermone AR, Nguyen TL, Wong TY, Schmidt J et al (2004) Identification of small molecule inhibitors of anthrax lethal factor. Nat Struct Mol Biol 11:67–72
Li F, Chvyrkova I, Terzyan S, Wakeham N, Turner R, Ghosh AK, Zhang XC, Tang J (2012) Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group. Appl Microbiol Biotechnol 94:1041–1049
Tonello F, Ascenzi P, Montecucco C (2003) The metalloproteolytic activity of the anthrax lethal factor is substrate-inhibited. J Biol Chem 278:40075–40078
Tonello F, Seveso M, Marin O, Mock M, Montecucco C (2002) Pharmacology: screening inhibitors of anthrax lethal factor. Nature 418:386
Jacobsen FE, Lewis JA, Cohem SM (2007) The design of inhibitors for medicinally relevant metalloproteins. Chem Med Chem 2:152–171
Acknowledgments
This work was supported by the grants from Shandong NSFC of China (ZR2013CM031).
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Qingsheng Kong and Yuezhang Song have contributed equally to this work.
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Kong, Q., Song, Y., Mu, M. et al. Effects of metalloprotease anthrax lethal factor on its peptide-based inhibitor R9LF-1. Mol Cell Biochem 406, 293–299 (2015). https://doi.org/10.1007/s11010-015-2447-6
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DOI: https://doi.org/10.1007/s11010-015-2447-6