Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.     

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.     

Molecular level studies on binding modes of labeling molecules with polyalanine peptides

In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(II) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO(3)Na)(4))) on pentaalanine, which is a model peptide segment of amyloid peptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalanine peptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO(3)Na)(4) display different adsorption affinity with the pentaalanine peptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides.     

In Silico Design of MDM2-Targeting Peptides from a Naturally Occurring Constrained Peptide

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (K_D=6.6±2.6 nm ) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved “FWL” epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.     

HLA-A3 supermotif defined by quantitative structure-activity relationship analysis

Activation of a cytotoxic T cell requires specific binding ofantigenic peptides to major histocompatibility complex (MHC)molecules. This paper reports a study of peptides binding tomembers of the HLA-A3 superfamily using a recently developed2D-QSAR method, called the additive method. Four alleles withhigh phenotype frequency were included in the study: A*0301,A*1101, A*3101 and A*6801. The influence of each of the 20 aminoacids at each position of the peptide on binding was studied.A refined A3 supertype motif was defined in the study.     

Multipose Binding in Molecular Docking

Molecular docking has been extensively applied in virtual screening of small molecule libraries for lead identification and optimization. A necessary prerequisite for successful differentiation between active and non-active ligands is the accurate prediction of their binding affinities in the complex by use of docking scoring functions. However, many studies have shown rather poor correlations between docking scores and experimental binding affinities. Our work aimed to improve this correlation by implementing a multipose binding concept in the docking scoring scheme. Multipose binding, i.e., the property of certain protein-ligand complexes to exhibit different ligand binding modes, has been shown to occur in nature for a variety of molecules. We conducted a high-throughput docking study and implemented multipose binding in the scoring procedure by considering multiple docking solutions in binding affinity prediction. In general, improvement of the agreement between docking scores and experimental data was observed, and this was most pronounced in complexes with large and flexible ligands and high binding affinities. Further developments of the selection criteria for docking solutions for each individual complex are still necessary for a general utilization of the multipose binding concept for accurate binding affinity prediction by molecular docking.     

Molecular modeling of the interaction of polyproline-based peptides with the Abl-SH3 domain: rational modification of the interaction

A molecular model of the interaction of polyproline-rich peptideswith the Abl-SH3 domain is proposed, based on docking calculationswith the DOCK program coupled with molecular dynamics simulations.Two distinct binding modes of the peptide to the same aromatic-richregion (TyrlO, Phel2, Trp39, Trp50, Tyr55) of the domain wereobtained. It is proposed that these two models could representdifferent binding modes of proline-rich peptides to Src homologyregion 3 domains. Several peptide mutants were designed to determinewhether the two orientations were possible. Analysis of theKd values and fluorescence emission of these peptides indicatethat one of the orientations is more plausible and that residuesat position 4 of the peptide interact with the RT loop, beingimportant in modulating the peptide affinity for the Abl-SH3domain     

Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity

Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure.     

What Is the Role of HLA-I on Cancer Derived Extracellular Vesicles? Defining the Challenges in Characterisation and Potential Uses of This Ligandome

The Human Leukocyte Antigen class I (HLA-I) system is an essential part of the immune system that is fundamental to the successful activation of cytotoxic lymphocytes, and an effective subsequent immune attack against both pathogen-infected and cancer cells. The importance of cytotoxic T cell activity and ability to detect foreign cancer-related antigenic peptides has recently been highlighted by the successful application of monoclonal antibody-based checkpoint inhibitors as novel immune therapies. Thus, there is an increased interest in fully characterising the repertoire of peptides that are being presented to cytotoxic CD8+ T cells by cancer cells. However, HLA-I is also known to be present on the surface of extracellular vesicles, which are released by most if not all cancer cells. Whilst the peptide ligandome presented by cell surface HLA class I molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles remains relatively poorly defined. Here, we will describe the current understanding of the HLA-I peptide ligandome and its role on cancer-derived extracellular vesicles, and evaluate the aspects of the system that have the potential to advance immune-based therapeutic approaches for the effective treatment of cancer.     

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling

The molecular interaction of the Fab fragment of the human monoclonalantibody 3D6, directed against the transmembrane protein gp41of human immunodeficiency virus (HTV) 1, with its peptide epitopeis characterized by a panel of overlapping peptides, a peptideepitope library and molecular modeling techniques. The sequenceCSGKLICTTAVPW, corresponding to amino acids 605–617 ofgp41, was identified as the best binding peptide (K_D = 1x10^-8mol/1). This peptide served as a starting point to prepare acellulose-bound peptide epitope library in which each residueof the epitope is substituted by all L- and D-amino acids, resultingin 494 epitope peptide variants which were subsequently analyzedfor binding 3D6. The library was synthesized to identify residuescritical for binding and to obtain information about the molecularenvironment of the epitope peptide bound to 3D6. Both cysteineresidues, as well as isoleucine 6, threonine 8 and proline 12,of the epitope were highly sensitive to substitution. Usingthe data obtained from the epitope characterization, as wellas a low-resolution electron density map of a 3D6 Fab-peptidecomplex, a 3-D model of the Fab-peptide complex was generatedby molecular modeling. The modeling experiments predict bindingof the peptide, which is cyclized via the two cysteine residues,to a pocket formed dominantly by the hypervariable loops complementaritydetermining regions CDR3L, CDR2H and CDR3H.     

Modeling of the intestinal peptide transporter hPepT1 and analysis of its transport capacities by docking and pharmacophore mapping

An early pharmacokinetic screen for peptidomimetic drugs should have the ability to predict molecules with high affinity for intestinal transporters, as peptide-like derivatives are seldom absorbed passively. Hence, the first objective of this study was to generate a reliable model for the structure of the hPepT1 protein, which is the main intestinal transporter involved in the absorption of both dietary peptides and peptidomimetics. The modeling was based on the resolved structure of the homologous bacterial lactose permease LacY using a fragmental strategy. The interaction capacities of the hPepT1 model were explored by docking a set of 50 known ligands. Despite the known predilection of hPepT1 for hydrophobic ligands, docking results unveiled the key role of the polar interactions stabilized by charged termini, especially concerning the ammonium head group. The docking results were further verified by developing a pharmacophore model that confirmed the key features required for optimal hPepT1 affinity. The consistency of the docking results and the agreement with the pharmacophore model afford an encouraging validation for the proposed model and suggest that it can be exploited to design peptide-like molecules with an improved affinity for such a transporter.     

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.     

Construction and Validation of a New Naïve Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides

Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation.     

A Facile Cyclization Method Improves Peptide Serum Stability and Confers Intrinsic Fluorescence

Peptides are a growing class of macromolecules used in pharmaceutics. The path toward the clinical use of candidate peptides involves sequence optimization and cyclization for stability and affinity. For internalized peptides, tagging is also often required for intracellular trafficking studies, although fluorophore conjugation has an impact on peptide binding, permeability, and localization. Herein, a strategy based on cysteine arylation with tetrafluoroterephthalonitrile (4F‐2CN), which simultaneously cyclizes peptides and imparts fluorescence, is reported. The 4F‐2CN cyclization of an M2 macrophage‐targeting peptide yields, in a single step, a peptide with improved serum stability, intrinsic fluorescence, and increased binding affinity. In a murine breast cancer model, it is demonstrated that the intrinsic fluorescence from the cyclized peptide is sufficient for monitoring biodistribution by whole‐organ fluorescence imaging and cell internalization by flow cytometry.     

Advances and challenges in protein-ligand docking

Molecular docking is a widely-used computational tool for the study of molecular recognition, which aims to predict the binding mode and binding affinity of a complex formed by two or more constituent molecules with known structures. An important type of molecular docking is protein-ligand docking because of its therapeutic applications in modern structure-based drug design. Here, we review the recent advances of protein flexibility, ligand sampling, and scoring functions-the three important aspects in protein-ligand docking. Challenges and possible future directions are discussed in the Conclusion.     

Rapid microsphere-assisted peptide screening (MAPS) of promiscuous MHCII-binding peptides in Zika virus envelope protein

Despite promising developments in computational tools, peptide-class II MHC (MHCII) binding predictors continue to lag behind their peptide-class I MHC counterparts. Consequently, peptide–MHCII binding is often evaluated experimentally using competitive binding assays, which tend to sacrifice throughput for quantitative binding detail. Here, we developed a high-throughput semiquantitative peptide–MHCII screening strategy termed microsphere-assisted peptide screening (MAPS) that aims to balance the accuracy of competitive binding assays with the throughput of computational tools. Using MAPS, we screened a peptide library from Zika virus envelope (E) protein for binding to four common MHCII alleles (DR1, DR4, DR7, DR15). Interestingly, MAPS revealed a significant overlap between peptides that promiscuously bind multiple MHCII alleles and antibody neutralization sites. This overlap was also observed for rotavirus outer capsid glycoprotein VP7, suggesting a deeper relationship between B cell and CD4⁺ T cell specificity which can facilitate the design of broadly protective vaccines to Zika and other viruses.     

Conversion of Low‐Affinity Peptides to High‐Affinity Peptide Binders by Using a β‐Hairpin Scaffold‐Assisted Approach

Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.     

Diamondoid Amino Acid-Based Peptide Kinase A Inhibitor Analogues

The incorporation of diamondoid amino acids (DAAs) into peptide-like drugs is a general strategy to improve lipophilicity, membrane permeability, and metabolic stability of peptidomimetic pharmaceuticals. We designed and synthesized five novel peptidic DAA-containing kinase inhibitors of protein kinase A using a sophisticated molecular dynamics protocol and solid-phase peptide synthesis. By means of a thermophoresis binding assay, NMR, and crystal structure analysis, we determined the influence of the DAAs on the secondary structure and binding affinity in comparison to the native protein kinase inhibitor, which is purely composed of proteinogenic amino acids. Affinity and binding pose are largely conserved. One variant showed 6.5-fold potency improvement, most likely related to its increased side chain lipophilicity. A second variant exhibited slightly decreased affinity presumably due to loss of hydrogen-bond contacts to surrounding water molecules of the first solvation shell.