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TOWARDS DEVELOPING SPECIFIC INHIBITORS OF THE ATP-DEPENDENT LON PROTEASE

Frase, Hilary
http://rave.ohiolink.edu/etdc/view?acc_num=case1175637588

Abstract Details

2007, Doctor of Philosophy, Case Western Reserve University, Chemistry.
The ATP-dependent serine protease Lon is responsible for degrading damaged and certain regulatory proteins in vivo. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics however no potent or specific inhibitors had been reported. This study focused on identifying a lead compound(s) for the development of potent inhibitors of the proteolytic activity of Lon. Steady-state kinetic characterization of the ATP and peptide hydrolysis activities of human and Salmonella enterica serovar Typhimurium (S. Typhimurium) Lon revealed no kinetic differences in ATP hydrolysis, but marked differences in substrate specificity. This suggests a peptide-based inhibitor may be developed which exploits these differences to target an inhibitor to a single homolog, minimizing cross-reactivity. Screening of commercially available peptide-based inhibitors highlight the utility of transition state analogs in inhibiting peptide hydrolysis. The peptidyl boronate, MG262, was the most potent inhibitor tested and was effective against both human and S. Typhimurium homologs (IC50 = 160 ± 10 nM and 122 ± 9 nM, respectively). Peptidyl boronates inhibit peptide hydrolysis through a two-step time-dependent mechanism with an overall Ki of ~ 20 nM. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. A second slow step occurs in which the protease undergoes a conformational change or isomerization to enhance the interaction of the inhibitor with the proteolytic active site. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease which requires the binding of ATP to observe inhibition. Finally, the purification of the human homolog of the steroidogenic acute regulatory protein (StAR) is described. It is shown to be a substrate of human Lon and provides a starting point for the development of a physiologically relevant peptide substrate(s) for the human enzyme. This peptide(s) will be useful in studying the kinetic mechanism of human Lon and for understanding the effect of mutations on the turnover of StAR in patients suffering from congenital lipoid adrenal hyperplasia.
Irene Lee (Advisor)
284 p.
Chemistry, Biochemistry
Lon protease; serine protease; steady-state kinetics; enzyme kinetics; time-dependent inhibition; enzyme inhibition

Recommended Citations

Citations

  • Frase, H. (2007). TOWARDS DEVELOPING SPECIFIC INHIBITORS OF THE ATP-DEPENDENT LON PROTEASE [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1175637588

    APA Style (7th edition)

  • Frase, Hilary. TOWARDS DEVELOPING SPECIFIC INHIBITORS OF THE ATP-DEPENDENT LON PROTEASE. 2007. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1175637588.

    MLA Style (8th edition)

  • Frase, Hilary. "TOWARDS DEVELOPING SPECIFIC INHIBITORS OF THE ATP-DEPENDENT LON PROTEASE." Doctoral dissertation, Case Western Reserve University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1175637588

    Chicago Manual of Style (17th edition)

case1175637588
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© 2007, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.