Evaluation and improvement of the structural mimicry of gp120 mimics

QD Chemistry

Mickowska, Anna ; Liskamp, Robert

University:University of Glasgow

Department:School of Chemistry

关键词: Peptide cyclisation, protein mimicry, gp120 mimics, molecular scaffold.;   

Others  :  http://theses.gla.ac.uk/41164/1/2018mickowskaPhD.pdf

来源: University of Glasgow

【 摘 要 】

Despite 30 years of research dedicated to the development of an HIV 1 vaccine, a successful candidate capable of elicitation of neutralizing antibodies against the virus has not yet emerged. The Env gp120 mutation rate is very rapid, however in order to maintain its binding site towards CD4 receptor, certain crucial residues must be conserved within the viral strains, making it an interesting therapeutic target.This thesis describes the synthesis of discontinuous gp120 epitope mimics with a novel cyclisation linker, which was applied to improve the solubility and purification of the constructs. For the mimicry of gp120, cyclic peptides mimicking the discontinuous epitopes were assembled on a molecular scaffold. The synthesized linear and cyclic peptides, and final constructs with cyclic peptides assembled on a molecular scaffold were analysed by 1H NMR and CD spectroscopy. These spectroscopic methods allowed to gain a deeper understanding of the secondary structure of the synthesized compounds. It was found that cyclisation constrained the peptides, when compared to their linear counterparts. The NMR spectra of the final gp120 mimics were superimposed on each other and it was found, that two out of three compounds were almost identical. CD spectrometry confirmed the results obtained by NMR.Since binding studies of the synthesised gp120 mimics required access to CD4 and gp120 proteins, therefore approaches towards expression, purification and characterisation of these proteins were made. However, the challenging refolding of CD4D12 resulted in obtaining a heterogenous mixture of properly folded and misfolded protein. The expression of gp120 in mammalian cells was low-yielding and this protein could not be produced in an amount needed for binding studies.A reliable and reproducible technique was needed to evaluate binding of gp120 mimics to CD4 receptor. SPR was chosen to study the kinetics of the constructs. Careful optimisation of the experimental conditions yielded in a highly reproducible method for the evaluation of gp120 discontinuous mimics.