Inhibition of PRKAA/AMPK (Ser485/491) phosphorylation by crizotinib induces cardiotoxicity via perturbing autophagosome-lysosome fusion.

Crizotinib, a small-molecule tyrosine kinase inhibitor targeting ALK, MET and ROS1, is the first-line drug for ALK-positive metastatic non-small cell lung cancer and is associated with severe, sometimes fatal, cases of cardiac failure, which increases the risk of mortality. However, the underlying mechanism remains unclear, which causes the lack of therapeutic strategy. We established in vitro and in vivo models for crizotinib-induced cardiotoxicity and found that crizotinib caused left ventricular dysfunction, myocardial injury and pathological remodeling in mice and induced cardiomyocyte apoptosis and mitochondrial injury. In addition, we found that crizotinib prevented the degradation of MET protein by interrupting autophagosome-lysosome fusion and silence of MET or re-activating macroautophagy/autophagy flux rescued the cardiomyocytes death and mitochondrial injury caused by crizotinib, suggesting that impaired autophagy activity is the key reason for crizotinib-induced cardiotoxicity. We further confirmed that recovering the phosphorylation of PRKAA/AMPK (Ser485/491) by metformin re-activated autophagy flux in cardiomyocytes and metformin rescued crizotinib-induced cardiomyocyte injury and cardiac complications. In summary, we revealed a novel mechanism for crizotinib-induced cardiotoxicity, wherein the crizotinib-impaired autophagy process causes cardiomyocyte death and cardiac injury by inhibiting the degradation of MET protein, demonstrated a new function of impeded autophagosome-lysosome fusion in drugs-induced cardiotoxicity, pointed out the essential role of the phosphorylation of PRKAA (Ser485/491) in autophagosome-lysosome fusion and confirmed metformin as a potential therapeutic strategy for crizotinib-induced cardiotoxicity.Abbreviations and Acronyms: AAV: adeno-associated virus; ACAC/ACC: acetyl-Co A carboxylase; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; CHX: cycloheximide; CKMB: creatine kinase myocardial band; CQ: chloroquine; c-PARP: cleaved poly (ADP-ribose) polymerase; DAPI: 4'6-diamidino-2-phenylindole; EF: ejection fraction; FOXO: forkhead box O; FS: fractional shortening; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HF: heart failure; HW: TL: ratio of heart weight to tibia length; IR: ischemia-reperfusion; KEGG: Kyoto encyclopedia of genes and genomes; LAMP2: lysosomal-associated membrane protein 2; LDH: lactate dehydrogenase; MCMs: mouse cardiomyocytes; MMP: mitochondrial membrane potential; mtDNA: mitochondrial DNA; MYH6: myosin, heavy peptide 6, cardiac muscle, alpha; MYH7: myosin, heavy peptide 7, cardiac muscle, beta; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; PI: propidium iodide; PI3K: phosphoinositide 3-kinase; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; qPCR: quantitative real-time PCR; SD: standard deviation; SRB: sulforhodamine B; TKI: tyrosine kinase inhibitor; WGA: wheat germ agglutinin.

Mutation-mediated influences on binding of anaplastic lymphoma kinase to crizotinib decoded by multiple replica Gaussian accelerated molecular dynamics.

Anaplastic lymphoma kinase (ALK) has been thought to be a prospective target of anti-drug resistance design in treatment of tumors and specific neuron diseases. It is highly useful for the seeking of possible strategy alleviating drug resistance to probe the mutation-mediated effect on binding of inhibitors to ALK. In the current work, multiple replica Gaussian accelerated molecular dynamics (MR-GaMD) simulations, molecular mechanics generalized Born surface area (MM-GBSA) and free energy landscapes were coupled to explore influences of mutations L1198F, L1198F/C1156Y, and C1156Y on the binding of the first ALK inhibitor crizotinib to ALK. The results suggest that three mutations obviously affect structural flexibility, motion modes and conformational changes of ALKs. L1198F and L1198F/C1156Y strengthen the binding of crizotinib to the mutated ALKs but C1156Y induces evident drug resistance toward crizotinib. Analyses of free energy landscapes show that stability in the orientation and positions of crizotinib relative to ALK plays a vital role in alleviating drug resistance of mutations toward crizotinib. Residue-based free energy decomposition method was utilized to evaluate the contributions of separate residues to the binding of crizotinib. The results not only indicate that the tuning of point mutation L1198F on interaction networks of crizotinib with ALK can be regarded as a possible strategy to relieve drug resistance of the mutated ALK but also further verify that residues L1122, V1130, L1196, L1198, M1199, and L1256 can be used as efficient targets of anti-drug resistance design induced by mutations.

Drug-drug interaction between crizotinib and entecavir via renal secretory transporter OCT2.

Both entecavir and crizotinib are substrates of organic cation transporter 2 (OCT2). The aim of present study was to investigate the mechanisms of drug interactions between these two drugs. Kinetic analysis of entecavir on crizotinib uptake was conduct. Plasma concentration of crizotinib in rats and lung cancer patients, uptake of crizotinib in kidney slices and OCT2 transfected cells, were determined by LC-MS/MS. The clinical pharmacokinetic interactions and impact on adverse reaction of crizotinib in lung cancer patients were investigated. Steady-state through concentration of crizotinib was measured. The crizotinib-related adverse reactions were recorded in lung cancer patients with and without entecavir. Entecavir and 1-methyl-4-phenylpyridinium iodide significantly inhibited the uptake of crizotinib in kidney slices. Kinetic constants for crizotinib uptake by OCT2 were Km 1.16 ± 0.26 µM, Vmax 12.05 ± 0.53 µmol/min mg-1 protein and Ki 9.711 nM. Entecavir can inhibit crizotinib transport by OCT2 in kidney. Co-administration of entecavir significantly reduced the elimination of crizotinib in rats. In lung cancer patients, the steady-state AUCss of crizotinib increased approximately 1.2 fold (p < 0.05) but clearance was decreased by approximately 15% in the presence of entecavir. Steady-state through concentration of crizotinib significantly increased 1.3-fold when co-administrated with entecavir (p>0.001). Co-medication of entecavir significantly (p < 0.05) increased the risks of vision disorders, diarrhea and vomiting 1.6-, 2.3- and 1.8-fold. Entecavir could increase the exposure and reduce the elimination of crizotinib in lung cancer patients. Moreover, the presence of entecavir could significantly increase the incidences of adverse reaction of crizotinib.

Identification and biological evaluation of glycol diaryl ethers as novel anti-cancer agents through structure-based optimization of crizotinib.

Crizotinib, a drug for anaplastic lymphoma kinase (ALK) positive and c-ros oncogene 1 receptor tyrosine kinase (ROS1) positive non-small cell lung cancer (NSCLC), was structurally optimized via a strategy of structure-based fragment replacing. Computational study showed it was beneficial for interaction of crizotinib and ALK to increase the distance between pyridyl ring and phenyl ring in crizotinib, and thus, a series of novel glycol diaryl ethers were synthesized. The in vitro anti-tumor activity of synthesized compounds was studied in NSCLC cell line H2228 and neurobalstoma cell line SH-SY5Y. Among the synthesized compounds, 9e exhibits stronger anti-cancer activity than crizotinib toward H2228 cell line with an IC50 value of 0.22 µM. Molecular docking indicated that a longer chain between pyridyl ring and phenyl ring enabled molecule to have new interaction with a neighboring small hydrophobic pocket.

Evolution strategy of ROS1 kinase inhibitors for use in cancer therapy.

The abnormal expression of c-ros oncogene1 receptor tyrosine kinase (ROS1) has been identified as clinically actionable oncogenic driver in non-small-cell lung cancer. Since crizotinib was approved by the US FDA for the treatment of advanced ROS1-positive non-small-cell lung cancer, ROS1 kinase has become a promising therapeutic target. Under the guidance of some advanced computer-assisted technologies, such as structure-based drug design, homology modeling and lipophilic efficiency parameters, several potent and selective inhibitors against wild-type and mutant ROS1 were designed and synthesized. In this article, we will review a series of scaffolds targeting ROS1 kinase from the hit-to-drug evolution strategies of their representative compounds and it is hoped that these design strategies would facilitate medicinal chemists to optimize the process of drug design.

Insight into the key features for ligand binding in Y1230 mutated c-Met kinase domain by molecular dynamics simulations.

c-Met kinase has been considered as an attractive target for developing antitumor agents. The strong interactions between Tyr1230 and the inhibitors emphasized its importance for ligand binding. The clinically related Tyr1230 mutations have made negative impacts on current c-Met kinase inhibitors, especially the exquisitely selective ones, like PF-04217903, while the multi-targeted inhibitors, like Crizotinib, were not affected so much. In this study, the protein-ligand interactions between c-Met kinase domain (wild, Y1230C and Y1230H) and these inhibitors were compared. The binding site was expanded and the post-mutated regions became solvent accessible. The heavy dependency of PF-04217903 on the interactions with Tyr1230 resulted in the steep decrease of its potency against the Y1230 mutants. It was found that the ligand entrance region contributed consistently to the binding of Crizotinib, but not PF-04217903. Additional groups substituted in the ligand entrance region with stable interactions should be beneficial for improving the inhibitory activity of PF-04217903 against the Y1230 mutants. These findings will facilitate the discovery of potent inhibitors against Y1230 mutated c-Met kinase.

Highly Sensitive, Engineered Magnetic Nanosensors to Investigate the Ambiguous Activity of Zika Virus and Binding Receptors.

The aim of this research is twofold: 1) to shed light on zika's binding and entry mechanism while 2) demonstrating the effectiveness of our magnetic relaxation platform to achieve this goal. Magnetic relaxation-sensitive nanoparticles (MRNPs) are used in a novel fashion to analyze binding interactions between the zika envelope protein (ZENV) and proposed host cell receptors: AXL, HSP70, and TIM-1. Computational analysis is also utilized to examine these binding interactions for the first time. In addition, the role of crizotinib as a potential binding inhibitor is demonstrated and the possibility of ligand-independent phosphatidylserine-mediated binding is explored. Our findings suggest that while the extracellular domain of AXL has the highest affinity for ZENV; HSP70, TIM-1, and phosphatidylserine might also play active roles in zika tropism, which offers a potential explanation for the variety of zika-associated symptoms. This is, to our knowledge, the first time that MRNPs have been used to examine and quantify host-zika interactions. Our magnetic relaxation platform allows for timely and sensitive analysis of these intricate binding relationships, and it is easily customizable for further examination of additional host-pathogen interactions.