Ribonucleotide reductases have been designated some of the most interesting enzymes in the world due to their unusual free radical mechanism that reduces ribonucleotides to deoxyribonucleotides, a process essential for DNA replication and repair. 3-AP is a potent inhibitor of RNR. Upon forming a bis-iron complex in the cell, it directly quenches a tyrosyl radical on the RNR beta subunit, thereby depleting de novo dNTP and selectively starving fast-replicating cancer cells of nucleotides. However, the structure-function relationship of this drug remains poorly understood. Using the distinct absorbances of the iron (II) and (III) complexes of 3-AP, a series of spectroscopic assays were developed to determine the relative binding affinities of the analogs for iron and the stability of the active reduced complex. We found that compounds that bound iron irreversibly were inactive in the cell. Additionally, our studies showed that complexes with very high redox potentials were unable to get oxidized by RNR, showing no S-phase arrest in vitro, while complexes within the reduction potential of the cellular environment were potent RNR inhibitors. Certain modifications of the structure made the drug more potent in vitro, and our experiments found that this is due to slower oxidation of the active iron (II) complex. Making structural modifications, we enhanced the activity of this drug and shed light on its mechanism of action.

Development of iron binding assays to explore structure activity relationships of 3-AP analogs

255th National Meeting (2018)

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