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 = 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) = 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(i)*; t(1/2) = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP* 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, ± ATP 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.

Fig. 1.

(A) Time-dependent inhibition by ClFDP. Inhibition mixtures (IMs) contained 1.2 μM [α] = [β] = [ClFDP], 0.1 mM dGTP, and 0 (♦) or 3 (•) mM ATP (in assay for α); 0 (▪) or 3 (▴) mM ATP (for β). (▾) was identical to (•) except it contained 6.0 μM ClFDP. (○) was identical to (▾) except IM contained D57N-α in place of WT-α. (B) Initial nearly complete inhibition by ClFTP and subsequent recovery of approximately 50% activity. IM contained 1.2 μM [α] = [β], 1.2 (•), 6.0 (▪) or 12 (♦) μM ClFTP (in assay for α), 1.2 (▾, ○) μM ClFTP (for β), and 0 (▪,♦,▾) or 3 (•, ○) mM ATP. D57N-α CDP reductase activity was unaffected (▴) under identical conditions to (•).

Fig. 2.

ClFDP is a C-site-directed, slow-binding reversible inhibitor of hRNR. (A) Progress curves for ADP reduction. [ClFDP] was varied from (★) 0, (•) 1.5, (▴) 3.0, (♦) 6.0, (▪) 9.0 to (▾) 12.0 μM. The solid curves are the best nonlinear fits of the data to Eq. 1. The dotted line is the uninhibited progress curve. (Inset) A replot of k_obs for the progress curves as a function of [ClFDP]. The hyperbolic curve is the best nonlinear fit of the data to Eq. 2. (B) Perturbations of [8-³H]-ClFDP residence t_1/2 in response to ternary complex formation. Release buffer contained: 0.1 mM dGTP (A); 0 or 5 μM ClFDP (B); and 5 μM [ClFTP] = [ClFDP] (C). (C) CDP protects α from ClFDP inhibition. The preincubation mixture contained hRNR (5 μM) and either CDP alone [0.5 (▴) or 3 (•) mM], or ClFDP alone [10 μM (▾)], or both [10 μM ClFDP and 0.5 (▪) or 3 (♦) mM CDP].

Scheme 1.

Two possible kinetic mechanisms that describe slow-binding inhibition.

Fig. 3.

ClFTP rapidly and reversibly inactivates human α, approaching maximum inhibition of approximately 50%. (A) Progress curves for CDP reduction. [ClFTP] was varied from (♦) 0, (•) 150, (▪) 300, (▴) 600, (▾) 900, (♦) 1,200 to (○) 1,500 nM. The solid lines are the best nonlinear fits of the data to straight lines through the origin. The fitted linear lines were omitted for [ClFTP]≥600 nM for clarity. (Inset) A plot of fractional inactivation as a function of [ClFTP]. Each data point was derived from the respective slope generated by nonlinear fitting of the data in A to straight lines. The dotted line represents the best nonlinear fit to Eq. 4. (B) Activity of WT-α (•) vs. D57N-α (♦) as a function of [ClFTP]. dCDP production rates with 0–6.0 μM [ClFTP], 0.3 μM α, 3.0 μM β, 2 mM [5-³H]-CDP, and 3 mM ATP measured over 20 min.