Studies On Bifunctional Molecule Degraders Targeting Carbonic Anhydrase â…¨(CAâ…¨)

Objective:Hypoxia and acidic microenvironment can promote tumor cell proliferation and reduce the anticancer effect of chemotherapy drugs.It has been reported that inhibition of carbonic anhydrase 9（CAⅨ）can not only effectively improve the characteristics of hypoxic and acidic microenvironment unique to tumor tissue,but also interfere with tumor metabolism.Therefore,in this study,CAâ…¨ was selected as a target to design a bifunctional small molecule degrader that could efficiently degrade CAâ…¨ in an integrin-and lysosome-dependent manner.By gradually optimizing and screening to obtain the optimum small-molecule drug targeting CAâ…¨ degradation,and the integrin receptor-mediated internalization and degradation pathways of membrane protein CAâ…¨ are elucidated,so as to clarify the antitumor activity and mechanism of CAâ…¨ small molecule degraders.This study is expected to develop novel degraders targeting CAâ…¨ that will provide new strategies for the treatment of solid tumors based on hypoxia and acidic microenvironment characteristics.Methods:1.In this work,we selected cyclicRGDy K（cRGD）with high stability and hydrophilicity as the integrin-recognition ligand,the cRGD-target proteins of interest（POI）ligand-conjugated small molecule drugs for integrin-mediated extracellular proteins internalization were designed and synthesized to verify the feasibility of the integrin-mediated extracellular proteins degradation via the endosomal-lysosomal pathway.Neutr Avidin Protein Dylight 650（NAP-650）,engineered Halo Tag-m Cherry fusion protein,or Alexa Flour 488 labeled apolipoprotein E4（ApoE4-AF488）and programmed death-ligand 1（PD-L1-AF488）proteins were used as extracellular targets,and the proteins were traced based on their labeled fluorescence signals,and the efficiency of small molecule drugs in mediating extracellular membrane proteins into cells by binding to integrin receptors was evaluated by the fluorescence experiment.Moreover,the effect of lysosome inhibitor CQ on extracellular protein degradation and the colocalization of target protein with lysosomal marker（Lyso Tracker）or early endosome marker（Rab5）were also studied to verify the activity and mechanism of small molecule drugs promoting extracellular protein entry into lysosome degradation through integrin receptors.2.A series of bifunctional small molecule compounds targeting CAâ…¨ were designed and synthesized,and the optimum small molecule drugs targeting the degradation of CAâ…¨ protein activity were screened out according to western blot experiments.The degradation pathway of CAâ…¨ was investigated by the effects of lysosome inhibitor bafilomycin A1 or proteasome inhibitor MG132 on the degradation of CAâ…¨.In addition,the effects of CAâ…¨ degrader on integrinβ₃,integrinβ₅ and integrinβ₁ protein levels were analyzed by western blot to elucidate the mechanism of integrin-mediated internalization of target protein CAâ…¨,and the effect of CAâ…¨ degrader on tumor cell viability was studied by CCK8 assay.Results:1.The binding of cRGD-POI ligand conjugate to integrin receptor can mediate the cellular uptake of the extracellular POI,effectively verifying the feasibility of integrin-mediated protein endocytosis;The accumulation of the POI in cells reached the highest level due to the inhibition of protease hydrolytic activity by CQ;The fluorescence signals of target proteins are highly colocalized with lysosome marker and early endosome marker.2.Three compounds with different linker lengths,Sul-L1-RGD,Sul-L2-RGD and Sul-L3-RGD,could induce the degradation of CAâ…¨,among which Sul-L1-RGD had the highest degradation efficiency for CAâ…¨,with a maximal percent degradation(D_max)of roughly59%,and the optimal drug concentration and degradation time were 5 n M and 8 h,respectively;Degradation of CAâ…¨ can be mitigated by lysosome inhibitors but not proteasome inhibitors;Sul-L1-RGD can significantly degrade the protein level of integrinβ₃,with a D_max of 55%,but the degradation efficiency of integrinβ₅ is relatively low,with a D_max of 49%,and it has almost no degradation effect on Integrinβ₁,with a D_max of 6%;CCK8 results showed that Sul-6-RGD,a degrader of CAâ…¨,could significantly inhibit the viability of tumor cells under hypoxia condition.Conclusion:The molecular degrader targeting CAâ…¨ designed in this study is a bifunctional compound containing a target protein recruiting ligand and an integrin-recognition ligand,which can induce the internalization and subsequent degradation of CAâ…¨ by bridging them with integrins on the cell surface.Compared with other technologies for degrading POI through the lysosomal pathway,the bifunctional small molecule degrader formed by coupling monocyclic cRGD and small molecule inhibitor of POI in this study has the advantages of easy synthesis,small size,and controllable pharmacological and pharmacokinetic properties.Furthermore,the availability of small molecule inhibitors of many disease-associated proteins also facilitates the design of bifunctional molecular degraders,which will provide new opportunities for targeted degradation of secreted proteins and membrane proteins,thus having great potential applications in chemical biology and drug discovery.