Studies On The Development And Evaluation Of Efficient Screening Methods For Breast Cancer Resistance Protein Inhibitors

Multidrug resistance(MDR)is a serious phenomenon of drug resistance in tumor cells and an important cause of chemotherapy failure.One common mechanism for MDR in tumors is the high expression of Breast cancer resistance protein(BCRP)in tumor stem cells and its wide substrate spectrum.Using BCRP inhibitors as chemosensitizers is an effective strategy to reverse MDR during chemotherapy.Despite the discovery of various BCRP inhibitors,none have been approved as drugs for clinical use due to severe side effects or lack of significant therapeutic effects.Traditional Chinese medicine has been widely used in tumor therapy in recent decades because of its advantages of multiple targets,pathways,and low toxicity.Therefore,screening for active and low-toxicity BCRP inhibitors from traditional Chinese medicine has become a hot spot in drug research and development for reversing tumor MDR.Currently,in vitro methods for screening BCRP inhibitors include bidirectional transport assays and ATPase activity assays.Bidirectional transport assay is the standard method for identifying and screening BCRP substrates and inhibitors,but it is time-consuming and has low detection throughput.The ATPase activity assay has a high false-positive rate and cannot provide parameters such as transport rate.Therefore,existing screening methods are unable to meet the demand for rapid and efficient screening of specific BCRP inhibitors.This paper focuses on the screening of BCRP inhibitors and addresses the challenges involved.Using BCRP as the target protein,the study employs a novel strategy involving styrene maleic acid(SMA)polymer-stabilized lipid particles(SMALPs)to obtain highly active BCRP-SMALPs.The study establishes BCRP-SMALPs-SPR and BCRP-SMALPs-BLI screening methods using surface plasmon resonance(SPR)and biolayer interferometry(BLI)technologies respectively,to screen potential BCRP protein inhibitors in traditional Chinese medicine,with the aim of providing more candidate compounds for reversing multidrug resistance(MDR).The research comprises three main parts.s:1.Construction of BCRP-SMALPs Based on Polymer Stabilization StrategyThe SMA polymer membrane protein stabilization strategy was applied on BCRP to construct BCRP-SMALPs,which avoid BCRP degradation or inactivation when leaving the membrane environment.The physical parameters and protein activities of BCRP-SMALPs were characterized.First,the porcine tubular epithelial cells LLC-PK1 and BCRP stable transfer cells LLC-PK1/BCRP were used as the cell model,the cell membrane were obtained by differential centrifugation,and they were co-incubated with SMA polymer to construct the corresponding protein liposomes(SMALPs),in which PK1-SMALPs were used as negative control and BCRP-SMALPs were used as positive liposomes.The preparation method for SMA extraction of BCRP was optimized by orthogonal experiments,and the results showed that the ultrasonic time was 9 min,the cell membrane concentration was 20mg/m L,the SMA concentration was 2%,and the incubation time was the optimal overnight.At the same time,the morphology and dispersion of the constructed SMALPs were characterized by transmission electron microscope(TEM)after the excess SMA separated by Size exclusion chromatograghy(SEC).TEM results showed that the liposomal particles of the constructed SMALPs were evenly distributed and their sizes were uniform.Finally,the activity of BCRP in BCRP-SMALPs liposomes was measured,and the results showed that the ATPase activity of BCRP in BCRP-SMALPs could be significantly inhibited by its inhibitor Ko143,which suggest BCRP maintains certain transport activity.This study constructs BCRP-SMALPs with uniform size and good activity of BCRP,which provides a research basis and technical support for the development of small molecule inhibitors for BCRP.2.Establishment and evaluation of BCRP inhibitor screening methods based on surface plasmon resonance technologyThis study combines the SMA polymer membrane protein stabilization strategy with SPR technology to establish a rapid screening method for potential inhibitors of breast cancer drug resistance proteins based on BCRP-SMALPs-SPR,aiming to obtain active ingredients that reverse tumor MDR mediated by BCRP from traditional Chinese medicine resources.Firstly,BCRP-SMALPs were coupled to L1 chip to construct BCRP-SMALPs-L1 chip,and protein activity,stability and SMA interference were investigated.The results showed that the negative drug dexamethasone had no binding signal on the chip,and the positive drug doxorubicin and mitoxantrone had a good response signal on the chip,and their K_D was 5.29μM and 4.68μM,respectively,which suggested the good activity of the chip.The K_D of doxorubicin was 5.29μM and 3.95μM after SMALPs coupled on the chip at 0 h and 48 h,respectively,indicating that the protein chip had good stability within 48 h.Interference experiments showed that SMA did not interfere with the screening method.3 active compounds were screened out from 26 traditional Chinese medicine monomers by BCRP-SMALPs-SPR screening method and the affinity were verified.The K_D of 3 active compounds(Zanthoxytoxin,bergamot and naringenin)were 5.14μM,4.57μM and 3.72μM,respectively.The in vitro activity verification showed that Zanthoxytoxin,bergamot and naringenin could improve the sensitivity of LLC-PK1/BCRP cells to mitoxantrone and reverse BCRP-mediated MDR activity.The BCRP-SMALPs-SPR screening method constructed in this study has the advantages of rapidity,efficiency and high specificity,which can significantly improve the evaluation or screening speed of BCRP modulators,and provide more scientific basis for clinical reversal of MDR and improvement of drug disposal.3.Establishment and evaluation of BCRP inhibitor screening methods based on biolayer interferometry technologyThis study is based on the characteristics of BLI immersion reading,which shifts the analysis from structurally clear and simple traditional Chinese medicine monomers to more complex traditional Chinese medicine extracts.Combining with the stable strategy of SMA membrane protein,we constructed the SSA-Biotin-BCRP-SMALPs protein probe and successfully established a new screening method for BCRP potential inhibitors based on BCRP-SMALPs-BLI.Firstly,BCRP-SMALPs were successfully immobilized on the surface of SSA probe by biotin-avidin reaction to construct the SSA-Biotin-BCRP-SMALPs protein probe.The activity of the probe was verified:the positive drug doxorubicin has a good response signal on the probe and its K_D is 129μM while there is no binding signal of negative drug dexamethasone,which proved the good activity of the protein probe.The BCRP-SMALPs-BLI screening method was used to screen BCRP small molecule ligands from Ginkgo biloba extract,and 3 target compounds(quercetin,kaempferol,and luteolin)were screened out.The biological activity verification experiments showed that kaempferol and luteolin could significantly increase the sensitivity of LLC-PK1/BCRP cells to mitoxantrone,and the activity of kaempferol was higher than that of the positive inhibitor Ko143.The BCRP-SMALPs-BLI screening method constructed in this study realizes the rapid and efficient screening of BCRP inhibitors from TCM.Compared with the traditional cell plate experiment(usually takes a few days),this method only takes tens of minutes to screen and enrich active compounds in Chinese medicine extracts based on the advantage of BLI immersion and reading,which greatly improves the speed and throughput of drug screening.This study provides a new strategy for the inhibitor development of BCRP.In summary,this study integrated SMA membrane protein stabilization technology,SPR technology and BLI technology to establish two new screening systems for inhibitors of BCRP(BCRP-SMALPs-SPR and BCRP-SMALPs-BLI).3 active compounds,naringenin,bergamot and xanthotoxin were screened from the TCM monomer library by using the BCRP-SMALPs-SPR method;3 active compounds,quercetin luteolin,kaempferol and luteolin were screened from ginkgo biloba extract by using the BCRP-SMALPs-BLI method.Biological verification results showed that naringenin,bergamotide,xanthoxylin,kaempferol,and luteolin could increase the sensitivity of LLC-PK1/BCRP cells to anticancer drugs.This study provides a new platform and new ideas for screening and evaluation of novel inhibitor of BCRP,and also provides technical reference for the drugs screening target membrane protein.