Madeline Moradi, Pacific University

Abstract

A Structural and Kinetic Basis for Cytarabine and Gemcitabine Inhibition

Moradi, Madeline A1,2, Chang, Caleb1, Zhou, Grace1, Eleraky, Sarah1, Lee Luo, Christie1, Gao, Yang1
1. Department of BioSciences, Rice University, Houston, TX
2. Pacific University, Forest Grove, OR

Gemcitabine (GemC) and Cytarabine (AraC) are common chemotherapy drugs that act as chain terminators to inhibit DNA synthesis. However, in the presence of repair polymerase, Pol η, synthesis proceeds. To combat this, GemC/AraC are administered in high doses, inducing toxic side effects. Previous studies have provided only a static kinetic analysis of GemC/AraC binding, leaving little information regarding their mechanism for inhibition of DNA elongation. We propose the application of both single-base replacement kinetic assays and time-resolved X-ray crystallography to capture the structural and mechanistic characteristics of GemC/AraC. Employing Pol η to bypass GemC/AraC inhibited elongation during an in crystallo analysis bears implications for the development of more potent and efficient drugs for cancer patients. Incorporation results characterized both AraC and GemC with lower binding affinity compared to the native nucleoside, dCTP. Structurally, however, crystallization of Pol η with AraC/GemC at the insertion site revealed minimal change from dCTP. Further, we will crystallize Pol η with AraC/GemC at the primer terminus to investigate their inhibition mechanisms.

Acknowledgement – Thank you to the National Science Foundation’s Biology with X-ray Free Electron Lasers (BioXFEL) Internship Program (NSF STC Award – 1231306) and Rice University for making this research possible. This work has also been supported by a fellowship from the Houston Area Molecular Biophysics Program (NIH Grant No. T32 GM008280), Program Director Dr. Theodore Wensel.