Tapping into Regulation of Human Ribonucleotide Reductase: Inhibitor-Promoted Assembly of Persistent Hexamers & Insights into Metallocofactor Integrity

Ribonucleotide reductase (RNR), a linchpin enzyme in DNA synthesis, is a proven target of cancer drugs. Two distinct subunits, α and β, complete the active unit required for the catalysis of NDP (N=A,C,G,U) reduction to dNDPs. β harbors a diferric-tyrosyl radical cofactor that initiates intersubunit electron transfer between itself and α, the NDP reduction site. My studies aim to unravel inhibition mechanisms of Clolar® (ClF) and Triapine® (3AP) that respectively target α and β. Allosteric regulation on α directly governs dNTP pools homeostasis and in vitro this leads to specific changes in quaternary structure; yet, whether such oligomeric equilibria are relevant in vivo has remained unanswered. Studies with ClF show (i) both di- and triphosphates (ClFDP and â€"TP) are reversible hRNR inhibitors; (ii) ClFDP, which was not considered to be an active form of the drug, is a slow-release inhibitor; and (iii) α-targeted inhibition occurs via assembly into hexameric states that remarkably persist beyond inhibitor departure. We progressed to demonstrate that persistent hexamerization is a hallmark of hRNR down-regulation in vivo; thus, ClF induces in-cell assembly of kinetically stable α hexamers. Our data unveil a new avenue to target a key regulatory enzyme, identify a tractable platform to readout hRNR down-regulation, and shed light on the mechanisms of small-molecule-induced enzyme inactivation by persistent oligomerization. My parallel studies with 3AP suggest that β inhibition principally arises from radical quenching as opposed to ironchelation. We are progressing to examine these in vitro observations in whole cells to garner insights into 3AP inhibition in vivo.

For more information contact Prof. Wendy Kelly (404-385-1154).