Dock-able linear and homodetic di,tri,tetra and pentapeptide library from canonical amino acids:SARS-CoV-2 Mpro as a case study / 药物分析学报

Peptide-based therapeutics are increasingly pushing to the forefront of biomedicine with their promise of high specificity and low toxicity.Although noncanonical residues can always be used,employing only the natural 20 residues restricts the chemical space to a finite dimension allowing for comprehensive in silico screening.Towards this goal,the dataset comprising all possible di-,tri-,and tetra-peptide com-binations of the canonical residues has been previously reported.However,with increasing computa-tional power,the comprehensive set of pentapeptides is now also feasible for screening as the comprehensive set of cyclic peptides comprising four or five residues.Here,we provide both the com-plete and prefiltered libraries of all di-,tri-,tetra-,and penta-peptide sequences from 20 canonical amino acids and their homodetic(N-to-C-terminal)cyclic homologues.The FASTA,simplified molecular-input line-entry system(SMILES),and structure-data file(SDF)-three dimension(3D)libraries can be readily used for screening against protein targets.We also provide a simple method and tool for conducting identity-based filtering.Access to this dataset will accelerate small peptide screening workflows and encourage their use in drug discovery campaigns.As a case study,the developed library was screened against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)main protease to identify po-tential small peptide inhibitors.

Mutant P21 Peptides Could Act As An Improved Cyclin A Inhibitors For Cancer Therapy: An In Silico Validation

Objective: The present study delineates the generation of mutant peptide library from a known anticancer peptide, p21 and in silico evaluation for their affinity towards cyclin. A substrate binding groove. Methods: Mutant peptide library was created based on their AntiCP score and was docked with cyclin A using ClusPro2.0 web server. The docked structures were further simulated into an aqueous environment using Gromacs 4.5.6. Visualization was performed using PyMol software and interaction analysis was done using Discovery Studio Visualizer 4.1 Client and LigPlot plus tool. Results: A total of 57 mutant peptides were generated; out of which only 3 namely, K3C (Lys3Cys), K3F (Lys3Phe), and K3W (Lys3Trp) had a greater affinity for cyclin A than WILD p21 peptide (HSKRRLIFS). Molecular dynamic simulation studies showed that the peptides remained docked into the substrate binding groove throughout the run. Among all the peptides, K3C showed a significantly higher negative binding energy with cyclin A as compared to WILD. Conclusion: The overall results suggested that K3C mutant peptide had ~30 % higher affinity towards cyclin A and thus, could further be explored for its anticancer potential. The study also provides an insight into the crucial interactions governing the recognition of substrate binding groove of cyclin A for the development of novel peptide-based anticancer therapeutics.

Phage display technology and tumor targeted therapy / 肿瘤研究与临床

Phage display technology is a kind of laboratory technique by means of genetic engineering to combine phage with exogenous genes, based on the presentation of a heterogeneous peptide or protein libraries on the surface of phage along with phage coat protein expressions. The displayed proteins or peptides could maintain their own relative spatial structure and biological activity, allowing them to direct phage binding to a target of interest. Therefore, the technology can be widely used as a simple and effective screening tool to screen successfully tumor-targeted peptides, deliver tumor-targeted drugs and prepare tumor antibody, which plays an important role in tumor-targeted therapy.

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of monoclonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and isolated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping.

Sequence analysis and antigenic identification of mimic epitopes of peptidoglycan / 中国免疫学杂志

Objective:To analysis the Mimotopes of the peptide mimics to PGN using online softwares.Methods: Mimotopes of PGN were screened from 12-mers linear phage display peptide library by using anti-PGN McAb and the antigenicity of selected clones was identified by ELISA.The B cell epitopes,T cell epitopes of ′GRWxHxVxWAGL′ were estimated by DNAstar and online softwares.Results: 16 phage clones that bound with anti-PGN McAb were screened from 12-mers linear phage display peptide library.Among these positive clones,phage clones No.39 shared the conserved sequence:WxHx……AGL found in previous clone No.31(ATWxHxLxSAGL),which provoked an effective protective immunity against infection with S.aureus.To enhance the stability of the conformation as well as adding biotin on the N-ter-minal as a tag,the sequences ′39′ were redesigned and synthesized by adding S(serine)A(alanine) and GG(glycine)on the C-terminal of origin sequence(named SP39).Next,we estimated or predicted antigenic epitopes,T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares(http://bio.dfci.harvard.edu/Tools/antigenic.pl,www.syfpeithi.de,http://www.darrenflower.info/mhcpred),indicating that SP39 contains sites bound both mice and human MHC.The sequences ′WxHxVxW-′ may be antigenic epitope as SP39,which contains a T cell epitope.Our results showed that both SP39 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus.Moreover PGN could inhibit the binding of SP39 to the anti-PGN McAb.These data indicated that SP39 mimic to epitopes on PGN.Conclusion: SP39(GRWxHxVxWAGLAGGS) probably display the mimotopes of PGN.

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of mono-clonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and iso-lated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping.

Screening and identification of peptide mimics to lipoteichoic acid by phage displayed random peptide library / 中国免疫学杂志

Objective:To screen epitope mimics to lipoteichoic acid from a random 12-mer phage display peptide library and i-dentify the specificity of the mimotopes of LTA.Methods:The monoclonal antibody against LTA was used as a target to screen the 12-mer phage display peptide library and the specificity of phage clones were identified by sandwich ELISA.The amino acid sequences of positive phage clones were deduced from DNA sequencing.The specificity of synthetic peptide were identified by sandwich ELISA.Results:4 clones were obtained after 3 rounds of screening.Amino acid sequence analysis revealed four different types of mimotope sequence.A linear peptide (GHxDFRQxxQPS),named L2,which derived from positive sequence was synthesized.ELISA result indicates that L2 can bind to anti-LTA mAb specifically in a dose-dependent manner.Conclusion:The mimotopes of LTA were obtained by using the phage display technology.

Screening and identification of the polypeptides specifically binding to the adhesion protein of Myco-plasma genitalium / 中华微生物学和免疫学杂志

Objective To screen and identify the polypeptides specifically binding to the adhesion protein of Mycoplasma genitalium(MgPa) by using the Ph. D.-12TM phage display peptide library for further understanding the biological function and the possible pathogenic mechanism of the MgPa. Methods The Ph. D.-12TM phage display peptide library was used for 3 rounds of biopanning with the purified recombinant MgPa ( rMgPa) as the given target. The phages were collected for amplification after biopanning. The single strand DNA of phage clones were extracted and purified by using the sodium iodide method for further se-quencing. ELISA, competitive binding assay and dot immunobinding assay were performed to analyze the specific binding of positive phages to rMgPa. Results A significant enrichment of phages was achieved after 3 rounds of biopanning. Eleven different phage exogenous sequences (P1-P11) were detected among the 38 phages randomly selected from the agar. Two core sequences were deduced according to the repeating times of amino acids among the 11 polypeptide sequences, which were V-H-W-D-F-R-Q-W-W-Q-P-S and D-W-S-S-W-V-H/Y-R-D-P-Q-T/S. Ten out of the 11 representative phages ( P1-P10 ) specifically combined with the rMgPa. Conclusion Two polypeptides specifically binding to rMgPa were successfully screened out, which provided the tool for further investigation on the biological function of MgPa and the pathogenic mecha-nism of Mycoplasma genitalium.

Effect of chemotherapy on detection of colorectal cancer autoantibodies by phage display peptide method / 第二军医大学学报

Objective: To evaluate the effect of chemotherapy on detection of colorectal cancer autoantibodies by phage display peptide method. Methods: The five-phage peptide clones (No. 95, No. 149, No. 174, No. 396, and No. 1009) with high discriminatory ability of colorectal cancer patients were selected as the study subjects. We compared the reactivity of autoantibodies against each of the five-phage peptide clones among 20 colorectal cancer patients receiving chemotherapy, 40 colorectal cancer patients receiving no chemotherapy, and 40 healthy controls by enzyme linked immunosorbent assay (ELISA). Results: The seroreactivity of No. 95 clone was significantly lower in the patients with chemotherapy than those without chemotherapy (P<0.05), and was similar between normal control group and patients with chemotherapy (P = 0.074). For the seroreactivity of the other four clones (No. 149, No. 174, No. 396, No. 1009), there were significant differences between patients with chemotherapy and normal controls (all P<0.01). Conclusion: The detection ability of No. 95 clones for colorectal cancer autoantibodies is greatly influenced by chemotherapy, while chemotherapy shows no notable influence on the reactivity of other four-phage peptide clones.

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.

Identification of novel peptides that stimulate human neutrophils

Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we identified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified peptides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-dependent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leukocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimulated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide receptor (FPR)1 while the other peptide (MMHWAM) acted on FPR2. Since the three novel peptides were specific agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling.

Screening of HIV-1 neutralizing antibody mimotopes in slow progressor from phage display peptide library / 中华检验医学杂志

ObjectiveTo screen mimetic HIV-1 neutralizing epitopes from plasma with high level neutralizing antibody,and to provide useful information for further study of the interaction between antigen and antibody.MethodsIn order to gain neutralizing antibody recognized mimotopes, we detected neutralizing antibodieslevelsof 11HIV-1infectedslowprogressorsbyPBMC-basedneutralization assays.High-titer HIV-neutralizing antibodies from plasma of SPs was used as the ligand for biopanning by phage-displayed random peptide library.Positive phage clones was evaluated by ELISA,sequenced,and analyzed for homology to HIV-1 env by local BLAST to deduce the neutralizing peptide.ResultsTwenty-two clones were obtained consistent with requirement through three rounds biopanning.After comparison analysis,twelve clones include C8 were obtained as mimotopes of neutralizing antibody,C40 located in gp41Ⅱ cluster with the highest titer by inhibition ratio may be as neutralizing epitope.Conclusion By the use of IgG antibodies from SPs to screen the phage random polypeptide library,one can acquire multiple phage mimetic peptides of HIV related antigen epitope.(Chin J Lab Med,2012,35:838-842 )

Screening and identification of the mimic peptide of Mycobacterium tuberculosis antigen / 中华微生物学和免疫学杂志

ObjectiveTo immunoscreen the mimic peptides of Mycobacterium tuberculosis antigen from phage displayed 12-mer peptide library.MethodsSpecific IgG was purified from sera of patients with TB and used as the target to immunoscreen a phage random peptide library of 12 amino acids.Positive clones which were obtained after three rounds of biopanning were detected by ELISA and sequenced.The diagnostic value of the high frequent positive clones were observed by ELISA.Results After 3 rounds of immunoscreening,the eluted phages were enriched effectively.Six kinds of animo acid sequence were obtained from twelve positive phage clones.Sensitivity of the two high frequent positive clones were 71.4％ (A2)and 55.4％ (A7) respectively.ConclusionThe antigen-mimic peptide was successfully screened from 12 random phage peptide library and the peptides can be recognized by tuberculosis patients' polyclonal antibodies.

Screening and identification of antigenic mimics of the adhesion protein of Mycoplasma genitalium by phage display peptide library / 中华微生物学和免疫学杂志

ObjectiveTo screen a 12-mer phage display peptide library by the polyclonal antibody (pAb) against the recombinant adhesion protein of Mycoplasma genitalium (rMgPa) in order to obtain the antigenic mimic epitopes of MgPa.MethodsThe purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library.Seventy-four recombinant phage clones were randomly selected,and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX.The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA,competitive ELISA and Western blot analysis.Results After four rounds of biopanning,a significant enrichment of phages was achieved,the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences.Amongst 45 peptides,36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa.Immunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M.genitalium,P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I.Results of bioinformatics analysis by MIMOX demonstrated that S,A,F for cluster 1,A,K,I,T and L for cluster 2,K,S,L,R,D and I for cluster 3,may be the key consensus amino acid residues in the aligned mimotopes,respectively.ConclusionAntigenic mimics on MgPa were successfully identified and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent the immunodominant mimic epitopes of MgPa.And S,A,F K,I,T,L,R and D may be the key amino acid residues for the epitopes of MgPa.

The screening of liver cancer specific binding peptide from serum / 中华肝胆外科杂志

Objective To find the liver cancer specific peptide for serological screen of liver cancer patients via screening phage-display peptide library. Methods Fifteen sera from liver cancer patients and physical examinates were collected for the four-round screening with Ph. D. 12TM phage display peptide kit. Highly specific phage monoclones were selected based on the ELISA results of the serological assay. The peptide labeled with FITC was synthesized according to the DNA sequencing of the optimal monoclone and tested with serum via fluorescent imagery. Results Nine highly specific monoclones were found among 50 selected ones after 4 rounds of screenings. The positive rate of the optimal monoclone,ZH-3, reached 46.7 %. The peptide sequence of ZH-3 was concluded by DNA sequencing as SAHGTSTGVPWP. Desirable specificity and affinity were also shown in the serum of liver cancer patients. Conclusion The peptide ZH-3 can be used as a diagnostic reagent for liver cancer.

Screening, identification and significance of the phage-display random 7 amino acid peptide specific to the sera of patients with systemic lupus erythematosus / 中华微生物学和免疫学杂志

Objective To screen and identify the phage-display random 7 amino acid peptide specific to the systemic lupus erythematosus(SLE) and analyze its practical significance. Methods Using the phage random 7 peptide library screening, the SLE specific phage clones are obtained after binding with the mixture of sera from 30 SLE patients and 30 normal controls as ligand respectively. Then the Dot-ELISA is used to identify the SLE specific phage clones reactive to sera of the SLE patients and normal controls individually. Finally the identified phage-display random 7 amino acid peptides are sequenced and it's homology with the antigenic epitope of human being and other are also analyzed. Results Total 12 of the phage-display random 7 amino acid peptide are obtained by phage peptide library screening and the Dot-ELISA identification. Sequence analysis shows that the identified phage-display random 7 amino acid peptide epitope have homology with E. coli, Salmonella and human immunodeficiency virus, but not with that of human being. Conclusion SLE-specific peptides screened by phage random peptide library maybe used to diagnosis the SLE. Meanwhile, the antibodies in SLE patients which are combined with the Pathogen epitope, suggest that SLE maybe relate to pathogen infection.

Biopanning of HCV antigen epitopes from a random 12-peptide library by anti-hepatitis C virus polyclonal antibody / 中南大学学报(医学版)

Objective To analyze the epitopes of anti-hepatitis C virus(HCV)antibodies by peptide library biopanning. Methods Phage random peptide library of 12 amino acids was immunoscreened with purified IgG from the sera of hepatitis C patients. Positive clones which were obtained after 3 rounds of biopanning were detected by ELISA and 4 of them were sequenced. Results After 3 rounds of screening, the radio of output to input increased from (4.6×10~(-4))% to (5.3×10~(-2))%, meaning the enrichment was effective. At the third round of screening, all the selected clones proved to specifically react with the sera for immunoscreening. Four positive phage clones were sequenced, which shared a very conservative sequence and was named as C1. Its inserting sequence in the coat protein III was deduced to be GSMSPYVRWYTP, and the positive rate of C1 reacted with 20 cases of HCV patients was 85%.Conclusion The antigen-mimic peptide C1 is successfully screened from 12 random phage peptide library and it has some diagnostic value.

Screening of binding proteins of HMGB1 promoter by phage display technique / 中国病理生理杂志

AIM: To screen the binding proteins to HMGB1 promoter by phage display technique. METHODS: HMGB1 promoter was incubated with phage display library. Unbound phages were eluted and phages bound to HMGB1 promoter were amplified. Twenty individual clones were randomly selected and identified by enzyme-linked immunosorbent assay (ELISA). Positive clones were characterized by DNA sequencing and the sequences were subjected for computer analysis. RESULTS: Positive phages binding to HMGB1 promoter were enriched after 4 rounds of biopanning. Twenty phage clones were selected and eleven clones of which were identified to bind specifically to HMGB1 promoter. The sequences in full length were obtained and searched for homologous sequences from GenBank. Altogether eight coding sequences were obtained, six of which were known proteins including activator protein-1(AP-1) and two of which were uncharacterized ones. CONCLUSION: Several proteins were obtained that bind specifically with HMGB1 promoter. The results will be useful for further studying the expression and regulation mechanism of HMGB1.

Screening of molecular markers for early diagnosis of colorectal cancer by phage-peptide library / 第二军医大学学报

Objective: To establish a colorectal cancer phage-peptide library and to screen for biomarkers for early detection of colorectal cancer. Methods: A T7 phage display peptide library was constructed using 30 surgical colorectal cancer specimens from Changhai Hospital, Second Military Medical University. Protein-A/G was used to enrich IgG from control sera as well as colorectal cancer sera. Five biopanning protocols were carried out for enrichment of colorectal cancer-specific phage clones, and 2 000 phage clones were randomly selected. ELISA was used for further screening of clones of different reactivities between the cancer serum and control serum; and the selected clones were subjected to DNA sequencing and the cloned protein function was forecasted by Chilibot for validation. Results: (1) The titer of the colorectal cancer phage display peptide library was 3.0×106pfu, with a recombination rate of 60% as showed by PCR identification and a storage capacity of 1.8×106 pfu. (2) Of the 18 phage clones selected by ELISA, 12 were cancer-related genes. Conclusion: ELISA for screening the recombinant tumor antigen phage display peptide library can be used to discover new differentially expressed antigens ; and the selected phage clones expressing antigen might be used for early detection of colorectal cancer.

Construction of phage-displayed random combinatorial library of IgA affibodies and its directed in vitro evolution / 第二军医大学学报

Objective: To construct a phage-displayed random combinatorial library of IgA affibodies and to analyze its directed in vitro evolution, so as to study the relationship of IgA affibody structure with its function. Methods: The coding sequences of two affibodies, ZA1 and ZA2, were generated by overlapping PCR. The affibodies with a random linking peptide coding sequence in the 3′ terminal were randomly ligated and cloned into the K pn I site of the phagemid pCANTAB5S to construct a combinatorial phage library. Totally four rounds of in vitro human IgA directed evolution were conducted, and selected phage clones were prepared individually to test the IgA binding activity by ELISA technology. Results: The combinatorial phage library was successfully constructed; it contained about 3.4 × 107 clones with a titer of 1.6 × 1012 TU/L, and the positive clones accounted for more than 79%. Sequence analysis showed that the two single affibodies were randomly linked by random linking peptides. The composition of the phage clones displaying three or four affibodies in tandem increased remarkably along with the rounds of selection, which indicated the successful IgA directed evolution. Three new arrangements of two, three or four affibodies in tandem were obtained. ELISA results demonstrated a significantly enhanced IgA binding activity of three or four affibodies in tandem. Conclusion: A series of new IgA binding recombinants have been obtained by directed in vitro evolution of combinatorial phage library displaying randomly linked IgA affibodies. The three or four affibodies in tandem have much higher IgA binding activity than others, indicating that the in vitro molecular evolution is an effective way to produce IgA binding proteins with high activity.