| Cathelicidins is one of the host defense peptide(HDP)families widely distributed in vertebrates.They usually possess a broad spectrum antimicrobial activity against bacteria,fungi including clinical isolated drug-resistant strains,enveloped viruses,parasites,mycoplasmas and and even chlamydiae.In addition,cathelicidins also possess various biological functions including immunoregulation,wound repair,etc.However,the low bioavailability,susceptibility to enzymatic degradation and short half-life usually restrict their therapeutic use.The substitution of D-amino acids to L-amino acids,chemical modification and cyclizing were usually employed to enhance the stability of peptidic drugs.However,these means on the other hand enhance the toxicity but compromise the pharmacological activity.Two sections were conjugated in this work to endow HDP enzyme-resistant ability while maintain bactericidal activity,compared with other means conjugation preparation technology was simpler with lower cost.Firstly,the optimization and screening of pharmacological section were carry out.Here,using our previously characterized Hc-CATH as template,we designed a series of shortened variants by truncating from N terminal or C terminal,respectively,named Hc1-15.Determination of MICs indicated that variants showed decreased antimicrobial activities compared with Hc-CATH,among them Hc1-3 and Hc8 performed relatively better on antimicrobial potency than other analogs,while Hc3 possessed the best bioactivity.It is also found that once the peptide(analogs of Hc-CATH)reaches a certain minimal length of 17 residues,its antimicrobial activity seemingly achieves the optimum,and then drastically decreases as the sequence length gets shorter(Hc10-15).In addition,analogs with one Phe longer presenting much lower MIC values in contrast to their corresponding analogs indicated the importance of Phe towards antibacterial activity.Secondly,trypsin inhibitor(TI)section was chosen.Basically,there were 3 types of peptide-based trypsin inhibitor: Kazal,Kunitz and Bowmane-Birk.Kazal and Kunitz were not suitable for this subject for their large molecular weights,complicated structures and possessing of 3 disulfide bonds.Bowmane-Birk had relative small molecular weights with one disulfide bond and presented cyclic structure.ORB,which is our previously characterized peptide with both antibacterial and trypsin inhibitor activity,was similar to Bowmane-Birk and its core,named ORB-C,was composed of 11 amino acids.Hence,ORB-C was chose as trypsin inhibitor section.Finaly,conjugation(hybridization)modes were designed.Hc3 was engineered with ORB-C to finally obtain four conjugates: H3 TI,TIH3,H3 TIF and TIH3 F.All of the four conjugates except for TIH3 were found inheriting antimicrobial and proteases inhibitory characteristics from pharmacological section and TI section without increasing cytotoxicity which was also supported by hemolysis activity.Among them,TIH3 F exhibited the most potent antimicrobial and anti-inflammatory activities,the latter were proved by q PCR and ELISA.Investigation of structures and action mechanism indicated that conjugates inherent structures and bioactivities of conjugated sections.Besides,changes of TIH3F's killing rate exhibited its good tolerance to salt,p H,and temperature.Most importantly,in the case of resistance against serum and proteases hydrolysis,current hybrid peptides demonstrated a remarkable stability than any other pharmaceutical proteins.In conclusion,the optimization design in this research endowed Hc-CATH stability against proteinase while maintained its bioactivity.Guidance may be provided by this method for the design of clinical applied pharmaceutical proteins. |
PDF Full Text Request