PROJECT SUMMARY/ABSTRACT The equilibrium of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA, is critical for maintaining human health. Known as the regulator of dNTP biosynthesis, ribonucleotide reductases (RNRs) are essential enzymes found in all organisms that catalyze the reduction of ribonucleotides to deoxyribonucleotides, an essential reaction for DNA replication and repair. Failure of cells to maintain appropriate dNTP concentrations can lead to increased mutagenesis and uncontrolled proliferation, characteristics that promote cancer development. RNR inhibition has been implicated in several types of cancers and is a target for drug design. Although current drugs in clinical use are effective, our understanding of the inhibition mechanism is incomplete. Specifically, nucleoside analogs are used as α-inhibitors and have been shown to cause a distinct conformational change upon addition to the RNR α-subunit. Upon addition of nucleoside analogs, α-hexamer rings are formed. α-hexamerization has been observed with three triphosphorylated nucleoside analogs, clofarabine, cladribine and fludarabine; however, there are no near- atomic resolution structures available. The work described in this proposal aims to obtain high resolution structures of each α-inhibitor with Human RNR and to design and evaluate new RNR α-inhibitors. Cryo- electron microscopy will be used to examine the structures of α-hexamers after addition of triphosphorylated cladribine, clofarabine, and fludarabine to determine α-inhibitor binding locations, possible conformational changes and noncovalent interactions that could explain α-hexamer stability, and how α-hexamerization prevents RNR activity. Furthermore, new nucleoside analogs will be designed with the goal of increasing binding affinity of the nucleoside analogs to study the effects of electronic properties on the stability of α- hexamers. Together, this work aims to deepen our understanding of the mechanism of RNR inhibition by understanding the formation of α-hexamers and utilize structure-based drug design to expand the library of nucleoside analogs that can induce α-hexamerization.