The Anti-Renal Carcinoma Mechanism And ADMET Research Of Marine Drug Candidates PA/GPA

The high level of reactive oxygen species(ROS)in renal cell carcinoma(RCC)makes it insensitive to conventional radiotherapy and chemotherapy drugs.Therefore,there is an urgent need to find drug candidates with new target or new structure for the treatment of renal cell carcinoma.Marine microbial natural products are an important source of novel drug molecules.In this paper,anti-renal cell carcinoma drug candidates were screened form marine natural products.At the same time,in accordance with the principle of "Fail early,Fail cheap" for new drug development,ADMET research is produced into the drug development in order to conduct early draggability evaluation,forming a complete research system from the screening of new anti-renal cell carcinoma marine drug candidates with new targets and mechanisms to draggability evaluation.Screening of novel anti-renal carcinoma targets:We identified 33 Piericidins from two Marine actinomycetes,including 21 novel structural compounds,of which a new piericidin triglycoside was reported for the first time.PA and its monoglycoside GPA,as the most yielding piericidin compounds,had an IC50 for 0.4μM and 0.2 μM towards ACHN cell,respectively,indicating that the anti-renal cell carcinoma activity of PA/GPA was significantly better than that of Sorafenib(P<0.001).Therefore,the exploration of new mechanism of anti-renal cell carcinoma is initial for the development of piericidins candidates.In this study,we first found that peroxide oxidoreductase protein 1(PRDX1)directly interacts with PA in ACHN cells using the protein fishing technique.PRDX1 mainly plays the function of scavenging reactive oxygen species to maintain homeostasis in vivo.When the intracellular reactive oxygen species level is abnormally elevated or the protein concentration of PRDX1 is increased,PRDX1 forms dimer or decamer which acts as a functional regulatory protein to regulate the activity of NF-κB and other proteins,or plays the role of molecular chaperone,while losing the activity of peroxidase reductase instead.According to literature reports,high expression of PRDX1 in different tumor tissues promotes the occurrence and development of tumors,whereas renal cell carcinoma tissues exhibit low PRDX1 expression.Therefore,to clarify the relationship between PRDX1 and ROS is of great importance to explore the new mechanism of PA/GPA against renal cell carcinoma.Research on new anti-renal carcinoma mechanism:Using clinical human renal cell carcinoma(RCC)samples combined with transcriptome data from 2 cohorts of RCC and paracancerous tissues,low expression of PRDX1 was confirmed in RCC tissues(P<0.0001).Transcriptome analysis showed that PA up-regulated the expression of PRDX1 in ACHN cell.Therefore,we designed and constructed PRDX1 knockdown stably transfected ACHN cell line to investigate the effect of up-regulated PRDXl on ROS level.The results showed that PA/GPA could significantly down-regulate ROS levels in ACHN cells.When the expression of PRDX1 protein was inhibited,the inhibitory effect of ROS by PA/GPA was disappeared.Thus,we confirm that PRDX1 is the target of PA/GPA.As a class of multifunctional proteins,the enzyme activity and protein regulation function of PRDX1 play a key role in maintaining homeostasis.We detected the phosphorylation of Tyr194 of PRDX1 protein and found that PA/GPA could inhibit the phosphorylation of PRDX1 and increase its total protein level.To further investigate whether the increase concentration of PRDX1 protein affects its peroxide-reductase activity,we detected the enzyme activity and found that after 500 nM PA/GPA treatment,the activity of PRDX1 were increased to 1.36-and 1.60-fold,respectively(P<0.01).Besides,the surface plasmonic resonance(SPR)analysis have found that PA/GPA could directly bind to PRDX1 protein,which may be the reason why the PRDX1 inhibited the phosphorylation of Tyr194 of PRDX1 and maintained the enzyme activity.In addition,cell co-localization techniques showed that PA/GPA could force PRDX1 to enter the nucleus inhibiting the expression of NF-kB served as functional regulatory protein.In a word,we demonstrate that the novel mechanism of PA/GPA against renal cell carcinoma is related to the enhanced ROS scavenging ability of PRDX1,which provides a new research idea for the development of natural anti-renal cancer products.The ADMET evaluation:To further confirm the anti-renal cell carcinoma effect of PA/GPA,we constructed tumor-bearing nude mice with ACHN cells.Tumor volume decreased significantly after 3 weeks of PA/GPA administration to 50%(P<0.01)and 41%(P<0.001),respectively.However,the occurrence of hepatotoxicity prompted an exploration of the toxic targets and relationship between toxicity and efficacy of PAs compounds through ADME/T studies.Combined with quantitative proteomic and transcriptome analysis,it was found that PA had a significant effect on bile secretion pathway.SPR studies have shown that PA directly binds to liver nuclear receptor LXRa to regulate CYP7A1 and LDLR,therefore increase cholesterol absorption and inhibit its metabolism,causing aggravated cholesterol accumulation as well as mitochondrial toxicity in hepatocytes,which lead to cell apoptosis.The pharmacokinetic results of PA/GPA showed that PA could concentrate in the liver as liver drug concentration was 29-fold higher than that of plasma causing hepatotoxicity,while GPA was converted into PA in the liver with conversion rate up to 29.6%.We performed SPR and molecular docking experiments with 5 PA glycosides and 12 glycoside PA analogues with LXRα,and found that the presence of glycoside prevented the binding between compounds and LXRα protein.This may be a reason why GPA has no LXRα-binding activity but induces liver injury.In addition,we used the human liver microsomes and recombinant human CYP/UGT isoforms to confirm that PA mainly underwent phase Ⅱ metabolism,and UGT1A7,1A8,1A9 and 1A10 were the main metabolic enzymes with metabolic rates were all higher than 40%.In conclusion,this study clarified the new mechanism and the relationship of toxicity and efficacy of the candidate compounds of piericidins against renal cell carcinoma,confirmed that GPA has better draggability than PA,which laid a foundation for searching drug candidates against renal cancer with better medicinal properties.However,the action site of PRDX1 decamer in this study has not been fully elucidated,and how to reduce hepatotoxicity based on the toxicity-activity relationship is still to be solved.In addition,structural optimization of PAs compounds based on the propertiy of ADME/T can improve the bioavailability and renal tumor targeting,as well as the problem of poor druggability caused by respiratory chain inhibition activity.On the basis of this study,we must continue to search for novel anti-renal carcinoma active compounds with better druggability,so as to further promote the research of original anti-renal carcinoma targeted drugs with independent intellectual property rights in China.