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The Natural and Pharmacological Inhibition of Ribonucleotide Reductase

Misko, Tessianna, Misko
http://rave.ohiolink.edu/etdc/view?acc_num=case1537128885029723

Abstract Details

2019, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleotide 5`-diphosphate (NDP) to deoxyribonucleotide 5`-diphosphate (dNDP), maintaining balanced and adequate deoxyribonucleotide triphosphate (dNTP) pools for DNA replication and repair. As an essential enzyme in the cell, RR is subjected to many different forms of regulation, including transcriptional, allosteric, compartmentalization and small protein inhibition of Saccharomyces cerevisiae RR (ScRR) by Sml1. Sml1 is an intrinsically disordered protein inhibitor of the large subunit of ScRR (ScRR1). Sml1 has been extensively studied; however, it remains unknown where Sml1 targets ScRR1 for inhibition. Here we show that Sml1 inhibition is dependent on the effector/substrate pair bound to ScRR1. Kinetic studies indicate that Sml1 exhibits a mixed mode of inhibition where the affinity of binding is greater in the presence of effector/substrate when compared to free enzyme. dGTP/ADP bound ScRR1 elicits cooperative binding of Sml1, whereas ATP/CDP does not. Sml1 has a higher affinity of binding to the hexameric ScRR1 compared to the dimeric ScRR1. These differences in inhibition due to the binding of effector/substrate pairs and oligomerization states suggest that conformational changes as a result of these conditions influence the binding of Sml1. Previous literature had suggested Sml1 binds to the C-terminus of ScRR1, but examination of inhibition of ScRR1 lacking the CXXC motif crucial to the redox mechanism indicated Sml1 does not bind to this region. Future structural and mechanistic studies to gain insight into the mechanism of Sml1 inhibition of ScRR would pave the way for the design of new RR-targeted anti-cancer therapeutics. Current clinical RR inhibitors are fairly toxic to patients, presumably due to their lack of specificity for RR. Our laboratory’s discovery of a non-nucleoside inhibitor of, NSAH, is proposed to improve upon the pitfalls of the current RR inhibitors. Here we determined that NSAH sensitizes cancer cells to ionizing radiation (IR) and arrests cells in the S-phase. A library of NSAH derivatives was investigated for their improved interactions with hRR. Docking studies predict that all the derivatives have improved interactions with the phosphate-binding site of the C-site of hRRM1, with the tryptophan-quenching assay supporting this prediction, exhibiting an improved KD for all compounds with respect to NSAH. TP7 showed the best-predicted binding, interacting with the phosphate-binding site as well as near loop 2; however, cell inhibition studies indicated TP7 was not effective against human pancreatic cancer cells (Panc1). TP6 showed the greatest potency against Panc1 cells, with a 2-fold improvement compared to NSAH. Binding studies indicated that TP6 was one of the top binding compounds to hRRM1. Further studies of TP6 and TP7 will aid in the design of the next generation of RR inhibitors.
Derek Taylor (Committee Chair)
Chris Dealwis (Advisor)
Nancy L. Oleinick (Committee Member)
Michael E. Harris (Committee Member)
Marvin Neiman (Committee Member)
261 p.
Biomedical Research; Pharmacology
Ribonucleotide Reductase, Sml1, protein inhibitor, enzyme kinetics, radiation, small molecule derivative, drug inhibitors

Recommended Citations

Citations

  • Misko, Misko, T. (2019). The Natural and Pharmacological Inhibition of Ribonucleotide Reductase [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1537128885029723

    APA Style (7th edition)

  • Misko, Misko, Tessianna. The Natural and Pharmacological Inhibition of Ribonucleotide Reductase. 2019. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1537128885029723.

    MLA Style (8th edition)

  • Misko, Misko, Tessianna. "The Natural and Pharmacological Inhibition of Ribonucleotide Reductase." Doctoral dissertation, Case Western Reserve University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1537128885029723

    Chicago Manual of Style (17th edition)

case1537128885029723
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.