Poster abstracts

Poster number 67 submitted by Andrew Knappenberger

Enzymatic investigation of ribonucleotide reductase as a chemotherapy target

Andrew Knappenberger (Biochemistry, Case Western Reserve University), Md. Faiz Ahmad (Pharmacology, Case Western Reserve University), Chris Dealwis (Pharmacology, Case Western Reserve University), Michael Harris (Biochemistry, Case Western Reserve University)

Abstract:
Ribonucleotide reductase (RR) converts RNA nucleotides to DNA nucleotides, and is an attractive and well-established chemotherapy target, but present drugs are largely nucleotide inhibitors which target the enzyme’s active site (1). The nature of these compounds predisposes them to causing deleterious off-target effects which have no therapeutic value (2, 3). A demand therefore exists for RR inhibitors which are not nucleotide analogs. Our group seeks to identify such compounds by a combination of in vivo and in vitro drug screens for compounds which can disrupt pools of dNTPs in vivo, and which alter the relative second-order rate constants for RR’s four substrates (A, G, C, UDP) in vitro. This disruption of dNTP pools will interfere with DNA replication and repair without disrupting other essential processes, slowing tumor growth and potentiating radiation therapy (4, 5). The normal balanced pool of dNDPs is generated via an elegant crosstalk system between the active site of the large subunit and an allosteric site called the specificity site, where dNTP effectors (ATP/dATP, dGTP, dTTP) bind and modulate RR’s preferences among its substrates (6). Disrupting this allosteric transfer of information is vital for the desired perturbation of dNTP pools. We will observe relative rates of formation for each of the four dNDP products in vitro using borate chromatography and HPLC. Because each substrate acts as a competitive inhibitor for the other three, determining the relative rates of formation at known substrate concentrations is sufficient for the determination of relative second-order rate constants (7). We will use this assay to interrogate the effects of newly screened compounds known to inhibit RR in human cancer cell lines. These data will provide insight into the mechanisms of action for the screened compounds, which will lead to iterative rational design of progressively more effective chemotherapeutic agents.

References:
1. Wijerathna, S.R.; Ahmad, M.F.; Xu, H.; Fairman, J.W.; Zhang, A.; Kaushal, P.S.; Wan, Q.; Kiser, J.; Dealwis, C.G. Pharmaceuticals 2011, 4, 1328-1354.
2. Burstein, Harold J. Journal of Clinical Oncology 18.3 (2000): 693-693.
3. Galmarini, Carlos M., John R. Mackey, and Charles Dumontet. The lancet oncology 3.7 (2002): 415-424.
4. Shewach, Donna S., and Theodore S. Lawrence. Investigational new drugs 14.3 (1996): 257-263.
5. Oliver, F. J., M. K. L. Collins, and A. Lopez-Rivas. Experientia 52.10-11 (1996): 995-1000.
6. Xu, Hai, et al. Proceedings of the National Academy of Sciences of the United States of America 103.11 (2006): 4022-4027.
7. Cornish-Bowden, Athel. Journal of theoretical biology 108.3 (1984): 451-457.

Keywords: Enzymology, Drug Design