Background/Objectives: Bcr-Abl inhibitors such as imatinib have been used to treat chronic myeloid leukemia (CML). However, the efficacy of these drugs has diminished due to mutations in the kinase domain, notably the T315I mutation. Therefore, in this study, new purine derivatives were designed as Bcr-Abl inhibitors based on 3D-QSAR studies. Methods: A database of 58 purines that inhibit Bcr-Abl was used to construct 3D-QSAR models. Using chemical information from these models, a small group of new purines was designed, synthesized, and evaluated in Bcr-Abl. Viability assays were conducted on imatinib-sensitive CML cells (K562 and KCL22) and imatinib-resistant cells (KCL22-B8). In silico analyses were performed to confirm the results. Results: Seven purines were easily synthesized (7a-g). Compounds 7a and 7c demonstrated the highest inhibition activity on Bcr-Abl (IC50 = 0.13 and 0.19 μM), surpassing the potency of imatinib (IC50 = 0.33 μM). 7c exhibited the highest potency, with GI50 = 0.30 μM on K562 cells and 1.54 μM on KCL22 cells. The GI50 values obtained for non-neoplastic HEK293T cells indicated that 7c was less toxic than imatinib. Interestingly, KCL22-B8 cells (expressing Bcr-AblT315I) showed greater sensitivity to 7e and 7f than to imatinib (GI50 = 13.80 and 15.43 vs. >20 μM, respectively). In silico analyses, including docking and molecular dynamics studies of Bcr-AblT315I, were conducted to elucidate the enhanced potency of 7e and 7f. Thus, this study provides in silico models to identify novel inhibitors that target a kinase of significance in CML.

• Keywords
• 3D-QSAR, Bcr-Abl inhibitors, chronic myeloid leukaemia, docking studies, molecular dynamics, purine derivatives,
• Publication type
• Journal Article MeSH

OBJECTIVE: A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. METHODS: HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. RESULTS: This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. CONCLUSIONS: This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

• Keywords
• CML, Carnitine, Imatinib, OCTN2, TKI therapy side effects,
• MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * drug therapy   metabolism   MeSH
• Energy Metabolism drug effects   MeSH
• Imatinib Mesylate * pharmacology   adverse effects   MeSH
• Protein Kinase Inhibitors pharmacology   adverse effects   MeSH
• Carnitine * metabolism   pharmacology   MeSH
• Humans MeSH
• Mitochondria metabolism   drug effects   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Antineoplastic Agents adverse effects   pharmacology   MeSH
• Solute Carrier Family 22 Member 5 * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Imatinib Mesylate * MeSH
• Protein Kinase Inhibitors MeSH
• Carnitine * MeSH
• Antineoplastic Agents MeSH
• Solute Carrier Family 22 Member 5 * MeSH
• SLC22A5 protein, human MeSH Browser

• MeSH
• Fusion Proteins, bcr-abl genetics   MeSH
• Blast Crisis * genetics   drug therapy   pathology   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * drug therapy   genetics   pathology   MeSH
• Imidazoles * therapeutic use   administration & dosage   MeSH
• Protein Kinase Inhibitors therapeutic use   pharmacology   MeSH
• Humans MeSH
• Mutation * MeSH
• Mice MeSH
• Niacinamide analogs & derivatives   MeSH
• Antineoplastic Combined Chemotherapy Protocols therapeutic use   MeSH
• Pyrazoles MeSH
• Pyridazines * therapeutic use   administration & dosage   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Letter MeSH
• Names of Substances
• asciminib MeSH Browser
• Fusion Proteins, bcr-abl MeSH
• Imidazoles * MeSH
• Protein Kinase Inhibitors MeSH
• Niacinamide MeSH
• ponatinib MeSH Browser
• Pyrazoles MeSH
• Pyridazines * MeSH

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 μM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 μM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 μM) compared to 11b-f (GI50 = 6.4-11.5 μM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

• Keywords
• Bcr-Abl inhibitors, TKI-resistant cells, chronic myeloid leukemia, in silico studies, purine derivatives,
• Publication type
• Journal Article MeSH