Analysis by mutagenesis of the ATP binding site of the .gamma. subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

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Identification of the ATP binding site in tyrocidine synthetase 1 by selective modification with fluorescein 5'-isothiocyanate.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36560-2 ◽

1994 ◽

Vol 269 (21) ◽

pp. 14962-14966

Author(s):

M. Pavela-Vrancic ◽

E. Pfeifer ◽

W. Schröder ◽

H. von Döhren ◽

H. Kleinkauf

Keyword(s):

Binding Site ◽

Atp Binding ◽

Atp Binding Site

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Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

Annals of Hematology ◽

10.1007/s00277-020-04357-z ◽

2021 ◽

Author(s):

Afsar Ali Mian ◽

Isabella Haberbosch ◽

Hazem Khamaisie ◽

Abed Agbarya ◽

Larissa Pietsch ◽

...

Keyword(s):

Binding Site ◽

Kinase Inhibitors ◽

Therapeutic Potential ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Binding Pocket ◽

Atp Binding ◽

Atp Binding Site ◽

Lung Cancer Patients

AbstractResistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

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