Structural Studies Of Chemokine Receptor CCR5 In Complex With New Antagonists And Staphylococcus Aureus Leucotoxin

G protein-coupled receptors(GPCRs)comprise the largest protein superfamily in human genome.There are more than 800 GPCRs,which sense diverse ligands and induce multiple downstream signaling pathways to modulate physiological activities.Chemokine receptor CCR5 belongs to class A GPCR sub-family.It is expressed on the surfaces of T cells,macrophages,dendritic cells,eosinophils,and microglia,and is involved in modulating inflammation,cell migration,and angiogenesis in cancer cells.CCR5 also plays a key role in the process of human immunodeficiency virus type 1(HIV-1)infection.It acts as a co-receptor of HIV-1 to mediate the membrane fusion between the virus and human cell by interacting with the viral envelope glyco-protein gp120.In addition,as an important receptor on the immune cell surface,CCR5 has also been identified as the specific receptor for leucocidin E(LukE)secreted by staphylococcus aureus.As the HIV-1 co-receptor,CCR5 has been considered as an important drug target for the treatment of HIV-1 infection.Many CCR5 antagonists have shown therapeutic potential in clinical trials.However,only maraviroc(pfizer)was approved by FDA in 2007,while the other drug candidates ended in phase ? or phase ? due to toxic or side effects.Even maraviroc has exposed some side effects as well as drug resistance.CCR5 structure-based drug design is one of the strategies to solve the above issues,and will enable the development of novel anti-HIV drugs.Staphylococcus aureus is a versatile and dangerous pathogen which is second to E.coli in morbidity and mortality world-widely.But its detailed mechanism of infecting immune cells is still unknown.Latest studies have reported that Staphylococcus aureus infects immune cells by secreting leukotoxin E(LukE)which will specifically interact with chemokine receptor CCR5,forms pores on cell membrane and eventually leads to the destruction of immune cells.Traditional antibiotic treatment has caused serious antibiotic resistance of Staphylococcus aureus and even multi-antibiotic resistance.Developing drugs targeting CCR5 as immune system evasion factor inhibitor will provide a new perspective in treating infection of S.aureus.Such strategy has great advantages in reducing the screening pressure of pathogenic bacteria,weakening the lethal effect of bacteria to immune cell and enhancing immune defensive function of human body.In the first part of this study,we have solved the crystal structures of CCR5 in complex with three drug candidates(PF-232798,A18 and A22).PF-232798,a second generation of anti-HIV drug,shows increased binding affinity and improved oral absorption as well as alternative drug resistance profile compared to maraviroc,a marketed anti-HIV drug.The structure of CCR5 bound to PF-232798 reveals the molecular mechanism of PF-232798 in anti-drug-resistance when compared to the previous determined structure of CCR5-maraviroc.Meanwhile,we have collected with our collaborators to develop new anti-HIV-1 drug based on the CCR5-maraviroc structure.By solving the structure of CCR5 in complex with two best drug candidates A18 and A22,we provided evidences to guide the drug optimization.These two complex structures indicate that A18 and A22 occupy the same binding pocket as maravitoc,and their optimized substitute heteroaromatic ring enhanced the interaction with CCR5 in hydrophobic pocket.Recently,these drug candidates have been applied for clinical approval.In the second part,we studied the interaction mechanism between CCR5 and LukE of S.aureus with structural and biochemical methods.After extensive optimization of complex expression and purification,we obtained protein complex with high quality.After rounds of crystallization optimization,we successfully obtained the complex crystals that diffracted to 7 ?.Single particle cryo-electron microscopy(cryo-EM)was also applied to solve the complex structure.We have gotten homogeneous cryo specimen with good dispersity.The optimization of crystallization and cryo-EM is in progress.This thesis focused on chemokine receptor CCR5 to study the molecular mechanisms of its interactions with anti-HIV drug candidates and pathogen protein.The findings provide structural basis for carrying out drug discovery for the treatment of pathogen infection.