Chemical Modification Of The Pharmaceutical Peptide Hepcidin And Semi-Synthesis Of Ubiquitin Reagent

Polypeptides for medical use have received increasing attention in drug development because of their specificity and low immunogenicity.In recent years,with the development of research on pharmaceutical polypeptides and disease-related peptide reagents,the problems of discovery and treatment of a series of diseases have been solved.Pharmaceutical polypeptides,especially those rich in disulfide bonds,are important for studying the functional effects of their disulfide bond folding or allosteric modification.Therefore,it is of great significance and application value to develop a new method to study the disulfide bond function and modification of medicinal peptides rich in disulfide bonds.And,the development of peptide reagents for detection will provide a powerful tool for disease detection and drug screening.The main content and conclusions of this paper are divided into the following two parts:Hepcidin(Hep)is a signal peptide containing four pairs of disulfide bonds that bind to the receptor iron transporter(FPN)and allow endocytosis and degradation of FPN.Inhibition of FPN to over-transport intracellular iron ions to the blood can avoid the occurrence of symptoms such as hemochromatosis and Alzheimer’s disease.Clinically,Hep has been partially successful as a treatment for related diseases.However,it is controversial whether the binding mode of Hep and FPN depends on the disulfide bond modification in Hep,which hinders the further modification and optimization of Hep.In order to slove this hinder,a strategy was proposed at the first time:replacement with non-reducing thioether bonds to detect disulfide exchange between small disulfide-rich proteins and their receptors.This strategy has the advantage of less perturbation of the protein structure and stability under reducing conditions.The diaminodiacid synthon is separately inserted into the position of the four pairs of disulfide bonds in the Hep protein by SPPS technique to form a Hep derivative containing a thioether bond.We used Hep derivatives to verify the binding mode of Hep to FPN,and got a clearer conclusion: there is no classical disulfide bond mode between Hep and FPN,especially between Hep Cys7 and FPN Cys326.This provides a new idea for the modification of medicinal Hep: Cys7 is not a decisive factor in the activity of Hep production.After modification,we finally obtained a highly active Mini-Hep.Ubiquitination,a novel protein post-translational modification discovered in recent years,plays a key role in physiological health regulation.In the ubiquitination pathway,deubiquitinating enzymes act as a class of ubiquitin-modified negative regulatory proteins,and its dysfunction is closely related to the occurrence of cancer.At present,the development of small molecule inhibitor drugs using deubiquitinating enzymes as inhibitory targets has received increasing attention.In the pre-screening strategy,Ub-PA is a novel active probe.Compared with Ub-AMC,there is no false positive result caused by the overlapping of absorption or the excitation bands of small molecule inhibitors and AMC fluorescent molecules.Thus,it’s becoming the preferred reagent for deubiquitination enzyme screening.However,Ub-PA has a problem of low productivity in the chemical synthesis process.In order to better meet the needs of the tools in the screening process,a new semi-synthetic strategy was developed: using biological expression to obtain a large number of modules required for synthesis and combining chemical methods to prepare the key intermediate ubiquitin hydrazide,and then through the acylation chemical reaction to assemble the alkyne molecules.Using this strategy,it’s easily to acquire hundreds of milligrams of active probes in a short period of time.The probe was used to screen the USP14 deubiquitinating enzyme inhibitor IU1,demonstrating that the probe tool obtained using the new synthetic strategy has good biological activity.