Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit

Abstract

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α-and β-subunits that form active α n β m (n, m ¼ 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di-and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K i ¼ 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (K Ã i ¼ 17 nM; t 1∕2 ¼ 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E • ClFDP Ã by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, AEATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.allosteric regulation | oligomerization | slow-binding inhibitor

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule

“…Improved potency has been attained by several nucleotide analogs that are used clinically as drugs to target hRRM1 (10,13,22). Although the role of hRR inhibition is considered to be secondary to DNA chain termination as the primary mechanism of cytotoxicity by nucleoside drugs where the triphosphate form of the drug is incorporated (22), work reported by the Stubbe laboratory highlighted the importance of hRR inhibition by nucleotide analogs (10,11). The three analogs that are best characterized biochemically are gemcitabine (10), clofarabine (11,13), and cladribine (15).…”

“…1A). In the presence of effectors, α exists as a dimer which in the presence of the allosteric effectors dATP and ATP form α 6 β 2 and α 6 βn (n = 2, 4, 6) multimers, respectively (8)(9)(10)(11)(12). Current interest in RR research is increasing our understanding of higher-order oligomerization, enzyme turnover, and the mechanism of allosteric regulation that governs substrate specificity, catalysis, and inhibition of this critical enzyme (8,9,(11)(12)(13).…”

“…The nucleotide analogs of chemotherapeutics such as fludarabine, clofarabine, and cladribine target all three sites of hRRM1 for inhibition (11,14,15) (Fig. 1A).…”

“…In contrast, hydroxyurea (HU) blocks catalysis by targeting the diiron cluster of RR2 (16,17). Gemcitabine diphosphate, clofarabine, and cladribine nucleotides inhibit hRRM1 by stabilizing a form of the α 6 complex (10,11,13,15). Such nucleotide-analog inhibitors of hRR, especially gemcitabine, are effective anticancer agents and can sensitize cancer cells to ionizing radiation and to DNA-damaging drugs (18).…”

Diversity in Overall Activity Regulation of Ribonucleotide Reductase

“…Some clinically used drugs that target the allosteric and catalytic sites of RNR, such as clofarabine and gemcitabine, have direct effects on oligomerization as well as the affinity between ␣ and ␤ subunits in different RNRs (12,36,37). The allosteric a-site of the P. aeruginosa enzyme seems to have a broader specificity than most other members of class I RNRs.…”