Integrated Strategy Facilitates Understanding Of Pharmacophore Of Verucopeptin And Dissecting Its Cellular Targets

Natural products have long been in pivotal position in human health and drug development.However,drug discovery derived from natural products inevitably entered a period of decline in 1990s.High rate of re-discovery,scarce amount and poor reproducibility of natural products prevent this precious resource from being used as effective tools in various screening programs and in vivo study of drug efficacy.Besides,the elucidation of the molecular mechanism of action of natural products remains a formidable challenge in advancing natural product research owing to complex stereochemistry,difficult to modify to generate useable biochemical probe.New strategy was needed for advancing natural product research.In this thesis,we adopted an integrated strategy to study the structure basis and molecular mechanism of verucopeptin which is a famous anti-cancer agent.The core scaffold of verucopeptin,named piperazic acid containing pyranylated cyclodepsipeptide(PA-PCD),represents a powerful skeleton,we suppose the unique structure feature account for its splendid bioactivity.We profiled verucopeptin against 1094 cancer cell lines from the Cancer Cell Line Encyclopedia(CCLE at Massachusetts General Hospital Cancer Center,Harvard Medical School).Verucopeptin showed a broad anti-proliferation activity with half-maximum inhibitory concentration(IC50)values less than 100 nM against 66%of the cell lines tested.Furthermore,when applied to a multidrug resistant(MDR)gastric cancer cell line SGC7901/VCR,verucopeptin exhibited the most potent activity than the other 17 chemical reagents including well-known chemo-therapeutics and tool compounds。All of the above inspired us for deep exploration of pharmacophoric elements of verucopeptin as well as its cellular targets.We first sequenced the genome of Actinomadura sp.XM-4-3,the BGC(biosynthetic gene cluster)of verucopeptin was identified through a homology search program(anti-smash).By means of in silico analysis and genetic manipulation,we presented a hypothesis for the overall assembly of verucopeptin.Besides,the output of verucopeptin was significantly improved sustaining the consequent research via constitutively expression of putative luxR-like positive regulator verA.To gain deep insight into the structure determinants of activity in this specific niche VE family,we applied an approach combined with synthetic biology and organic diversification to generate a focus set of 18 VE oriented analogs.With a set of focused VE analogs in hand,we systematically evaluated their antiproliferative activities against cancer cells of A375,MDA-MB-231 and HL60.This systematic SAR data revealed that the functional elements piperazic acid,six-membered pyrane and adjacent alkene along with three-dimension cyclic-architecture constitute the key structure features of VE for its exquisite activities.VE-2 obtained via synthetic biology approach was emerged as the most active compound in this work,which also indicates hydroxyamino group can be modified to make clickable chemical probe to fish the molecular target of VE.We designed a probe derived from VE with a photo labile terminated alkyne at the hydroxyamino position,named VE-P.The in situ click chemistry and organelle orientation phenotype analysis revealed verucopeptin acts as a lysosomotropic agent which mediates the production of lysosomal ROS to trigger cancer cell apoptosis.Besides,via chemical proteomics employing a click chemistry strategy,we found that V-ATPase(vacuolar-type H+ ATPase)subunits exactly ATP6V1B2 and ATP6V1D has specific interaction with VE-P both in HL60 and transfected 293T cells,simultaneously,VE substantially inhibited lysosome acidification with higher potency than that of Baf A1 at the same concentration of 10 nM,which indicates V-ATPase act as the direct molecular target of verucopeptin.Moreover,we found that VE suppress mTORCl signaling via targeting V-ATPase.Here we delineated the molecular mechanism and pharmacophoric elements of verucopeptin with the combination of synthetic biology,organic synthesis diversification,and chemical proteomics coupled with phenotype analysis.Verucopeptin will serve as versatile tool to pharmacologically interrogate V-ATPase biology and study its roles in cancer treatment.The integrated strategy we presented holds the great promise to advance natural product sciences and hasten drug discovery and development.