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Research Highlight - Inhibitors of Multidrug Resistance

Bacterial infections are arguably some of the most serious threats to humankind to date. The options for treating infections have dwindled substantially and the reports of antibiotic resistant pathogens are increasing at an alarming rate. Once potent antibiotics such as penicillin nowadays are hardly effective by themselves including against certain strains of Bacillus anthracis, the causative agent of anthrax.

This antibiotic resistance is due in large part to bacterial beta-lactamases capable of degrading penicillin-like drugs. A powerful avenue for treating infections was the administration of a beta-lactamase inhibiting co-drug in addition to prescribing penicillin-like antibiotics. There are currently three beta-lactamase inhibitors on the market, tazobactam, sulbactam, and clavulanic acid each with an annual sales of close to or over a billion dollar.

The bacterial response to this co-drug combination was not too surprising and beta-lactamase variants were soon found to confer. Detailed structural knowledge on how these inhibitors function and how mutations in beta-lactamases confer resistance to these inhibitors is desperately needed.

Protein crystallography has been a tremendous tool to study beta-lactamases with and without substrates or inhibitors yet the complexity of the enzymatic degradation pathway of such compounds has often precluded obtaining clear crystallographic snapshots of reaction intermediates.

A novel solution to this underlying problem of not knowing what intermediates are formed at what rate in the crystal once soaking is commenced has been developed by our collaborative team including Drs. Paul Carey, Marion Helfand, and Robert Bonomo. This innovative technique, termed Raman crystallography, has led to the identification of the trans-enamine intermediate peaking at 20-30 minutes inside the deacylation deficient E166A mutant of SHV-1 beta-lactamase. Our lab subsequently determined the 1.63 Ang crystal structure of this intermediate complex for tazobactam yielding a wealth of detailed information of how this drug inhibits this enzyme resulting in ideas on how to rationally improve this drug (tazobactam has an annual sales of close to a billion dollars in the US). This work was published in Biochemistry.

Based on this tazobactam complex, we have designed a novel inhibitor SA2-13 with an intend to stabilize the trans-enamine intermediate. Our designed SA2-13 compound yielded a 10-fold improvement of the longevity of the trans-enamine intermediate and, unlike the starting tazobactam compound, SA2-13 could now readily be trapped in wt SHV-1 crystals. This work was published in JACS and in collaboration with Dr. John Buynak.

Selected References

  • Padayatti, P.S., Helfand, M.S., Totir, M.A., Carey, M.P., Hujer, H.M., Carey,P.R., Bonomo, R.A., & van den Akker, F.
    “Tazobactam forms a stoichiometric trans-enamine intermediate in the E166A variant of SHV-1 β-lactamase: 1.63 Å crystal structure ”
    Biochemistry 43, 843-848 (2004)
  • Padayatti, P.S., Sheri, A., Totir, M.A., Helfand, M.S. Carey, M.P., Anderson, V.E., Carey,P.R., Bethel, C.R., Bonomo, R.A., Buynak, J.D. & van den Akker, F.
    “Rational design of a beta-lactamase inhibitor achieved via stabilization of the trans-enamine intermediate: 1.28 Å crystal structure of wt SHV-1 complex with a penam sulfone ”
    J. Am. Chem. Soc. 128, 13235-13242 (2006)