| Background: Cancer is one of the leading causes of death worldwide.Chemotherapy is one of the most important strategies in tumor treatment,while multidrug resistance(MDR)is usually induced during chemotherapy and causes treatment failure and disease progression.The overexpression of ATP-binding cassette(ABC)transporters such as P-glycoprotein(P-gp),multidrug resistance associated protein 1 and breast cancer resistance protein,is considered to be one of the main factors inducing MDR.However,most existing ABC modulators failed to provide ideal clinical outcomes,which is mainly due to that MDR is caused by multiple factors,thus modulating ABC transporters is insufficient to reverse MDR.Among these factors,the up-regulation of autophagy level is also widely recognized as an important cause of MDR.Studies have suggested that dual inhibition of ABC transporters and autophagy could be an effective strategy to reverse MDR.Natural isoquinoline alkaloids have shown good potential in inhibiting ABC transporters as well as regulating autophagy,thus are promising lead compounds to reverse MDR.However,hampered by their limited source,complex structures,difficult synthesis,comparatively high toxicity and poor water solubility etc.,the application of natural isoquinoline alkaloids are greatly limited.Therefore,simplifying and modifying the structures of isoquinoline alkaloids,optimizing the activity and exploring the mechanisms are important for further drug development,which is expected to provide a safer and more effective new strategy for the treatment of drug-resistant tumors.Objective: To synthesize simplified isoquinoline alkaloid derivatives and to obatain the simplified compounds with best reverse activity.To evaluate the druggability,pharmacokinetic properties and MDR reversal activity,as well as clarify the molecular mechanism of the best simplified compounds.Finally,to obtain candidate lead compounds with high activity and better accessibility to overcome the drawbacks of natural products.Methods: Molecular docking and dynamics simulation are used to analyze the binding patterns of small molecules to target proteins and guide the design of simplified derivatives.The compounds were synthesized via N-methylation,amide formation and oxidation/reduction reactions,etc.,purified by column chromatography with appropriate eluting system.The compounds were characterized by nuclear magnetic resonance and high resolution mass spectrometry,and their purity was measured by high performance liquid chromatography.CCK8 assay was used to determine the cytotoxicity of the compounds on drug-resistant cells,as well as the reverse activity of anticancer drugs in combination and the synergistic effects.The synergistic anticancer effect of the compounds was observed by plate cloning formation experiment.Flow cytometry was used to determine the accumulation of fluorescent dyes and the apoptosis of cells treated with the compounds.The in vivo pharmacodynamic evaluation of the best simplified compound was conducted by allograft tumor model.Laser Scanning Confocal Microscope(LSCM)was used to observe the cellular accumulation and localization of fluorescent dyes and transfected fluorescence-labeled proteins.Western blotting was used to detect the expression of drug-resistant related proteins and their changes after compounds treatment.The target was identified and verified by thermal shift assay and Activity-based Proteomic Profiling technique.Contents and Results:(1)The structure of isoquinoline alkaloid tetrandrine was simplified reasonably by molecular dynamics simulation and fragment growth,and 40 simplified derivatives were synthesized.Their structure-activity evaluation was conducted in vincristine(VCR)resistant cell line Eca109/VCR,and a novel simplified derivative OY-101,which is easy to prepare and has high MDR reversal activity and low cytotoxicity,was obtained.(2)The druggability evaluation,pharmacodynamic evaluation and molecular mechanism study of OY-101 were performed.Its pharmacokinetic and pharmacodynamic properties in vivo were also studied.Results indicated that the drug resistance of Eca109/VCR cells is related to the high expression of P-gp,while OY-101 can reverse the MDR of tumors in vivo and in vitro by inhibiting the function of P-gp.(3)Experiments results have figured out that OY-101 was similar to natural isoquinoline alkaloid,and could regulate P-gp function and autophagy at the same time.Therefore,further modification and multiple efficacy evaluation were carried out on the basis of OY-101,and a dual regulator of P-gp and autophagy-OY-102 with higher revere activity was obtained.(4)The pharmacokinetic and pharmacodynamic properties of simplified derivative OY-102 were studied in vitro and in vivo.The molecular mechanism of OY-102 regulating autophagy was also investigated.An active probe was synthesized for target fishing.These results indicated that the high drug resistance of SGC7901/VCR and MCF/ADR was associated with high autophagy flux and high expression of ABC transporters,and OY-102 effectively reversed MDR of the tumors in vivo and in vitro through dual inhibition of P-gp and autophagy.Conclusions and significance: Simplified derivative OY-101 is a novel P-gp inhibitor with high reverse activity of MDR in vivo and in vitro.Simplified compound OY-102 is a novel dual inhibitor of P-gp and autophagy,which has stronger synergistic effect on chemo-therapeutic drugs in vivo and in vitro.In conclusion,our study may provide a novel scaffold type as well as candidate molecules for the development of novel chemosensitizers,as well as providing a new promising solution to reverse MDR in tumors with the strategy of P-gp and autophagy dual inhibition. |
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