From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia

Abstract

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Fig. 1. The two first masitinib derivatives show increased affinity to dCK and enzymatic inhibition.

a Masitinib, dCKi1 and dCKi2 structures showing rings and linkers. b dCK thermal stabilization by masitinib, dCKi1 and dCKi2 measured by TSA (n = 3). c dCK binding to masitinib, dCKi1 and dCKi2 determined by ITC (n = 3). d Superimposition of crystal structures of dCK (gray ribbons) in complex with UDP (yellow) and dCKi1 (orange) (PDB 7ZI1) or dCKi2 (green) (PDB 7ZI2). e Representative experiment showing the effect of masitinib, dCKi1 and dCKi2 on substrate phosphorylation by dCK in the presence of UTP (n = 3). f Representative experiment showing the effect of masitinib, dCKi1 and dCKi2 on cell proliferation of the IL3 dependent Ba/F3 cell line (expressing WT human c-KIT) in the absence of IL3 and presence of the Cell Stem Factor (SCF) (proliferation depending on c-KIT) (48 h) (n = 3). g Effect of masitinib, dCKi1 and dCKi2 (10 µM) on cell proliferation of the leukemia cell line CCRF-CEM (T-ALL) in the absence or presence of dT (111 µM) and dC (1 µM) (72 h). NS represents no statistical significance between masitinib and masitinib+dT + dC. Statistical significance is **P = 0.0011 and ***P = 0.0001 for dCKi1 and dCKi2, respectively (dCKi + dT + dC compared to dCKi alone, two-tailed unpaired t-test). All data are presented as the mean ± SD (n = 3).

Fig. 2. Optimization of dCK inhibitors with improved affinity, enzymatic inhibition and cellular activity.

a Effect on dCK thermal stabilization (determined by TSA) and dCK cell proliferation assay (CCRF-CEM cell line) produced by the 74 synthetized compounds. Data are presented as the mean. b dCK thermal stabilization by selected compounds measured by TSA. Data are presented as the mean ± SD (n = 3). c Representative curves of one of two independent experiments showing the effect of selected compounds on substrate phosphorylation by dCK in the presence of ATP. Data are presented as the mean of three technical replicates ± SD. d Representative experiment showing the effect of selected compounds on cell proliferation of the CCRF-CEM cell line in the presence of dT (200 µM) and dC (1 µM) Data are presented as the mean ± SD (n = 3). e Chemical structure of OR0642. f Structure of dCK (gray ribbon) in complex with OR0642 (magenta) and UDP (yellow) (PDB 7ZI3) showing the hydrogen bond network to dCK (black dashed lines). VdW interactions between OR0642 CF₃ group and dCK ILE-200 and PRO-201 are shown as green dashed lines in the zoom insert. g The meshes around the compound shows the |2Fo|−|Fc| electron density map contoured at 2.0σ around the ligand (blue) and the |Fo|−|Fc| electron density map (orange) contoured at 1.0σ generated with the model compound omitted before refinement (omit map).

Fig. 3. Hit-to-lead decision tree.

Decision tree of the hit-to-lead optimization campaign showing the 9 key milestone compounds that drove the SAR optimization from masitinib (blue), dCKi1 (orange) and dCKi2 (green) to ORO0642 (magenta). For each compound the TSA (ΔT_m, °C) and dCK cellular (IC₅₀, nM) measured values are indicated. At each step, the modified portion of the molecule is highlighted by a colored disk and the overall modifications with regard to masitinib are highlighted in red.

Fig. 4. OR0642 in combination with dT has strong antileukemic effect in vivo.

a Schedule of the vehicle and drugs administration mice groups (vehicle (n = 8), dT 1.5 g/kg (n = 7), OR0642 40 mg/kg (n = 9) and dT 1.5 g/kg + OR0642 40 mg/kg (n = 9)) (once/day (QD) and twice/day (BID)) against CCRF-CEM leukemia bearing mice. b Effect of treatments on the quantification of whole body radiance of CCRF-CEM leukemia bearing mice (bioluminescence). The yellow arrow highlights the end of the treatment. Data are presented as the mean ± SEM and compared using two-way ANOVA test and Turkey’s multiple comparison test (vehicle (n = 8), dT (n = 7), OR0642 (n = 9) and dT + OR0642 (n = 9)). NS represents no statistical significance, *P < 0.05, and **P = 0.001. c Representative bioluminescence images of mice at day 21 (NI = non injected mouse; without graft) (n = 3). d Effect of treatments on the quantification of whole body radiance at day 21. (NI = non injected mouse; without graft). Data are presented as the mean ± SEM. Comparison of bioluminescence data at day 21 was performed using one-way ANOVA Kruskal–Wallis test and Dunn’s multiple comparison test (vehicle (n = 8), dT (n = 7), OR0642 (n = 9) and dT + OR0642 (n = 9)). NS represents no statistical significance and ***P = 0.0002. e Effect of treatments on hCD45⁺ populations determined by flux cytometry at day 21. Data are presented as the mean ± SEM. Comparison of hCD45⁺ populations was performed using one-way ANOVA Kruskal–Wallis test and Dunn’s multiple comparison test (vehicle (n = 8), dT (n = 7), OR0642 (n = 9) and dT + OR0642 (n = 9)). NS represents no statistical significance and ***P = 0.0005. f Survival analysis of CCRF-CEM leukemia bearing mice treated with vehicle and drugs. The yellow arrow highlights the end of the treatment. Median survival times were compared using Log-Rank (Mantel–Cox) Test. NS represents no statistical significance and ****P < 0.0001.

Fig. 5. Effect of the combined therapy on PDX models.

a Effect of a dT concentration range (10 to 3000 µM) on PDX samples in the absence or presence of dC (1 µM) and/or OR0642 (0.25 µM). Data are presented as the mean ± SD (n = 3). b Correlation between the response at dT 100 µM and the dividing fraction. Two-tailed Pearson r correlation was computed after verification of the normal distribution of the dataset (P = 0.0012). c Effect of the combined therapy on the PDX sample UPNT730 in the absence and presence of proliferative conditions (OP9-DL1 monolayer) dC (1 µM) and/or OR0642 (0.25 and 0.5 µM). Data are presented as the mean ± SD (n = 3). d Treatments on the PDX mouse model UPNT525. Schedule of the vehicle and drugs administration mice groups (vehicle (n = 6), dT 1.5 g/kg (n = 6), and dT 1.5 g/kg + OR0642 40 mg/kg (n = 7)) (twice/day (BID)) against UPNT525 bearing mice. e Effect of treatments on hCD45⁺ populations determined by flux cytometry at day 19. Data are presented as the mean ± SEM. Comparison of hCD45⁺ populations was performed using one-way ANOVA Kruskal–Wallis test and Dunn’s multiple comparison test (vehicle (n = 6), dT (n = 6), and dT + OR0642 (n = 7)). NS represents no statistical significance and ***P = 0.0002. f Survival analysis of UPNT525 bearing mice treated with vehicle and drugs. The yellow arrow highlights the end of the treatment. Median survival times were compared using Log-Rank (Mantel–Cox) Test. NS represents no statistical significance, *P = 0.02, and ***P < 0.0003.