Combinatorial Peptide Libraries Research Articles

Pharmacokinetic Positron Emission Tomography Imaging of an Optimized CD38-Targeted 68Ga-Labeled Peptide in Multiple Myeloma: A Pilot Study.

Multiple myeloma (MM) is an incurable disease characterized by its clinical and prognostic heterogeneity. Despite conventional chemotherapy and autologous hematopoietic stem cell transplantation, the management of relapsed and refractory MM disease poses significant challenges, both medically and socioeconomically. CD38, highly expressed on the surface of MM cells, serves as a distinct tumor biological target in MM. Peptides offer advantages over antibodies, enabling precise tumor imaging and facilitating early tumor diagnosis and dynamic immunotherapy monitoring. In this study, we developed PF381, a CD38-targeted peptide, and investigated its role in diagnosis, biodistribution, and dosimetry through 68Ga-labeling for preclinical evaluation in tumor-bearing models. We screened a microchip-based combinatorial chemistry peptide library to obtain the amino acid sequence of PF381. Affinity for human CD38 was evaluated by SPRi. PF381 was conjugated with DOTA for radiolabeling with 68Ga, and the complex was characterized by HPLC. PET imaging was performed in murine tumor models after the administration of [68Ga]Ga-DOTA-PF381. Biodistribution analysis compared CD38-positive H929 and CD38-negative U266 tumors, and human radiation dosimetry was estimated. Tumor sections were stained for CD38 expression. SPRi showed that PF381 had a high affinity for CD38 with a KD of 2.49 × 10-8 M. HPLC measured a radiolabeling efficiency of 78.45 ± 7.91% for [68Ga]Ga-DOTA-PF381, with >98% radiochemical purity. PET imaging revealed rapid and persistent accumulation of radioactivity in CD38-positive H929 tumors, contrasting with negligible uptake in CD38-negative U266 tumors. Biodistribution confirmed higher uptake in H929 tumors (0.75 ± 0.03%ID/g) vs U266 (0.26 ± 0.08%ID/g, P < 0.001). The kidney received the highest radiation dose (3.57 × 10-02 mSv/MBq), with an effective dose of 1.41 × 10-02 mSv/MBq. Immunofluorescence imaging supported PET and biodistribution findings. We developed a novel peptide targeting CD38 and proved that 68Ga-labeled PF381 had rapid targeting and good tumor penetration capabilities. Therefore, 68Ga-labeled PF381 could achieve high sensitivity in vivo imaging for CD38-positive hematological malignancies.

Structural insights into molecular-targeting helix–loop–helix peptide against vascular endothelial growth factor-A

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries

Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed having triple-helical peptides with a “host-guest” design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.

HLA A*24:02-restricted T cell receptors cross-recognize bacterial and preproinsulin peptides in type 1 diabetes.

CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I-restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02-peptide-TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.

In Silico Analysis of Conformational Dynamics and Energetic Landscapes of Putative Insulinase PF11_0189 From the Plasmodium falciparum Genome With Insulin‐Derived Peptides: Approach to Rationale Design of Insulin Peptide‐Based Inhibitors

ABSTRACTGlucose abnormality is one of the atypical symptoms mostly overlooked during severe malaria. Screening for proteases involved in glucose abnormality suggests a potential new drug target. PF11_0189 is a putative insulin degrading enzyme found in the genome of Plasmodium falciparum. In silico approach revealed that PF11_0189 is similar to the metalloproteases exhibiting a zinc binding motif. The substrate binding region of PF11_0189 catalytic domain is lined by residues of a mixed nature, enabling the accommodation of both hydrophobic and hydrophilic residues from substrates. Analysis of each amino acid position from combinatorial peptide library suggests Thr and Ser to be the most crucial residues as upon its mutation significant improvement is observed in the binding. The peptides P‐21 (VPICSLY), P‐70 (TMICVLY), and P‐121 (AVICSLY) demonstrate significantly better interaction within the active site than a template peptide (TSICSLY). Molecular dynamic simulations confirm the complex's integrity, with all structures within the qualitative limit of compactness and stability during the simulation time. Principal component analysis (PCA) reveals major conformational motions and different energy landscapes, while dynamic cross‐correlation matrix (DCCM) highlights residue interactions of the complexes. The insights obtained through computational methods provide clues about substrate selectivity in PF11_0189, which can be utilized for peptide‐based therapeutics against severe malaria.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 μM and 7.5 μM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 μM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation

AbstractMacrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol‐containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol‐containing elements and used them for the combinatorial synthesis of a 2,688‐member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X‐ray structure analysis of macrocycle‐target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry.

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3' specificities of PPEP-1 and PPEP-2, while identifying the endogenous substrate of PPEP-2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.

Analytical Tools for Dynamic Combinatorial Libraries of Cyclic Peptides.

Target-directed dynamic combinatorial chemistry is a very attractive strategy for the discovery of bioactive peptides. However, its application has not yet been demonstrated, presumably due to analytical challenges that arise from the diversity of a peptide library with combinatorial side-chains. We previously reported an efficient method to generate, under biocompatible conditions, large dynamic libraries of cyclic peptides grafted with amino acid's side-chains, by thiol-to-thioester exchanges. In this work, we present analytical tools to easily characterize such libraries by HPLC and mass spectrometry, and in particular to simplify the isomers' distinction requiring sequencing by MS/MS fragmentations. After structural optimization, the cyclic scaffold exhibits a UV-tag, absorbing at 415 nm, and an ornithine residue which favors the regioselective ring-opening and simultaneous MS/MS fragmentation, in the gas-phase.

Computational Design, Combinatorial Screening and Experimental Analysis of Lung Cancer EGFRVIII-Binding Peptides

Human epidermal growth factor receptor mutation variant III ([Formula: see text] is a cancer-specific cell surface oncogenic marker and has been observed to be widely involved in the formation, progression and metastasis of lung cancer and some other tumors. Previously, a massive quantity of [Formula: see text]-binding peptides were enriched via random phage display (RPD) targeted against the protein. In this study, we reported rational discovery of 12-mer peptides with high affinity to [Formula: see text] and strong selectivity for [Formula: see text] over wild-type EGFR ([Formula: see text]. A combinatorial peptide library was designed to target [Formula: see text] based on over ten thousands of known [Formula: see text] binders enriched from RPD analysis, and a virtual high-throughput screening protocol was then systematically performed against the library to derive those potential candidates, which were further examined rigorously at structural and energetic levels to identify few promising hits. Anisotropy binding assays were carried out to substantiate the computational findings. Consequently, eight 12-mer peptides were designed as effective binders that can target the [Formula: see text] extracellular subdomain 3 (SD3). In particular, two potent peptides (T1: FLHRYEIVTSYF and T3: FLQKYEWNTSYW) were found to have a high affinity to [Formula: see text] and a good selectivity for [Formula: see text] over [Formula: see text]. Structural analysis revealed that the peptide-binding site can be divided into hydrophobic, polar and mixed regions, which correspond to the nonpolar [Formula: see text]-terminal section, polar/charged middle section and hybrid C-terminal section of the peptide. The peptide selectivity originated from the middle section, which can form a different hydrogen bond network between the two proteins upon the mutating perturbation, whereas the N- and C-terminal sections are primarily responsible for the peptide stability but not specificity.

Multi-tiered approach to detect autoimmune cross-reactivity of therapeutic T cell receptors.

T cell receptor (TCR)-engineered T cell therapy using high-affinity TCRs is a promising treatment modality for cancer. Discovery of high-affinity TCRs especially against self-antigens can require approaches that circumvent central tolerance, which may increase the risk of cross-reactivity. Despite the potential for toxicity, no standardized approach to screen cross-reactivity has been established in the context of preclinical safety evaluation. Here, we describe a practical framework to prospectively detect clinically prohibitive cross-reactivity of therapeutic TCR candidates. Cross-reactivity screening consisted of multifaceted series of assays including assessment of p-MHC tetramer binding, cell line recognition, and reactivity against candidate peptide libraries. Peptide libraries were generated using conventional contact residue motif-guided search, amino acid substitution matrix-based search unguided by motif information, and combinatorial peptide library scan-guided search. We demonstrate the additive nature of a layered approach, which efficiently identifies unsafe cross-reactivity including one undetected by conventional motif-guided search. These findings have important implications for the safe development of TCR-based therapies.

Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy

The Tcells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific Tcell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual Tcells to attack cancer in several ways simultaneously. Such "multipronged" Tcells exhibited superior recognition of cancer cells compared with conventional Tcell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

Establishing Fluorine-Containing Amino Acids as an Orthogonal Tool in Coiled Coil Assembly.

The α-helical coiled coil (CC) is one of the best-characterized folding motifs in the protein world. In this context, fluorinated amino acids have been shown to be capable of tuning the properties of CC assemblies, and especially fluorinated derivatives of aliphatic amino acids can significantly increase the stability of this folding motif when placed in the hydrophobic a and d positions. However, it has not been shown yet whether fluorinated amino acids, by means of rational design, can be used as an orthogonal tool to control CC assembly processes. In the current work, we approached this question by creating a combinatorial peptide library based on a VPE/VPK heteromeric CC system previously established and characterized in our group. This CC model allowed us to screen fluorinated amino acids for interaction with different potential binding partners in position a of the VPE/VPK model with a particular emphasis on studying the impact of stereochemistry within the side chain of α-branched aliphatic fluorinated amino acids on CC properties such as oligomerization state, thermodynamic stability, and orientation. 28 combinations of library members were characterized regarding structure, oligomerization, and thermal stability utilizing circular dichroism, size exclusion chromatography, and Förster resonance energy transfer measurements. This detailed approach showed that the stability and oligomerization state of the motif were not only dependent on the steric demand and the fluorination of corresponding amino acids but also on the stereochemistry within the side chain. The results were applied for a rational design of the fluorine-driven orthogonal assembly, and we could show that CC dimer formation occurred based on specific interactions between fluorinated amino acids. These results demonstrate the potential of fluorinated amino acids as an orthogonal tool besides classical electrostatic and hydrophobic interactions for the fine-tuning and direction of peptide-peptide interactions. Furthermore, within the space of fluorinated amino acids, we could demonstrate the specificity of interactions between differently fluorinated side chains.

Low-Abundance Protein Enrichment for Medical Applications: The Involvement of Combinatorial Peptide Library Technique.

The discovery of low- and very low-abundance proteins in medical applications is considered a key success factor in various important domains. To reach this category of proteins, it is essential to adopt procedures consisting of the selective enrichment of species that are present at extremely low concentrations. In the past few years pathways towards this objective have been proposed. In this review, a general landscape of the enrichment technology situation is made first with the presentation and the use of combinatorial peptide libraries. Then, a description of this peculiar technology for the identification of early-stage biomarkers for well-known pathologies with concrete examples is given. In another field of medical applications, the determination of host cell protein traces potentially present in recombinant therapeutic proteins, such as antibodies, is discussed along with their potentially deleterious effects on the health of patients on the one hand, and on the stability of these biodrugs on the other hand. Various additional applications of medical interest are disclosed for biological fluids investigations where the target proteins are present at very low concentrations (e.g., protein allergens).

New Neutralizing Epitope Exposed on the Domain II of Tick-Borne Encephalitis Virus Envelope Glycoprotein E.

Orthoflavivirus encephalitidis, formerly tick-borne encephalitis virus (TBEV), belongs to the Orthoflavivirus genus. TBEV is transmitted by tick bites and infection with TBEV can lead to serious disorders of the central nervous system. In this study, a new protective monoclonal mouse antibody (mAb) FVN-32, with high binding activity to glycoprotein E of TBEV, was selected and examined in post exposure prophylaxis in a mouse model of TBEV infection. BALB/c mice were injected mAb FVN-32 at doses of 200 μg, 50 μg, and 12.5 μg per mouse one day after a TBEV challenge. mAb FVN-32 showed 37.5% protective efficacy when administered at doses of 200 μg and 50 μg per mouse. The epitope for protective mAb FVN-32 was localized in TBEV glycoprotein E domain I+II, using a set of truncated fragments of glycoprotein E. Additionally, the target site recognized by mAb FVN-32 was defined using combinatorial libraries of peptides. Three-dimensional modeling revealed that the site is dspatially close to the fusion loop, but does not come into contact with it, and is localized in a region between 247 and 254 amino acid residues on the envelope protein. This region is conserved among TBEV-like orthoflaviviruses.

Abstract PR08: TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development

Abstract Background: KRAS mutations drive tumorigenesis in adenocarcinomas of the lung (30-40%), colon (40-50%) and pancreas (&amp;gt;90%). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with KRAS-mutated advanced solid tumors. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. Here, we describe the pre-clinical development of TCR1020, a human TCRab specific for KRAS G12V restricted to HLA-A*11:01. Methods: In vitro stimulation assays were used to generate CD8+ T cell responses specific for a decamer epitope of KRAS G12V (VVVGAVGVGK) using blood specimens collected from A*11:01+ healthy donors. TCR1020 was identified by TCRab RNA sequencing of flow cytometrically sorted peptide/HLA multimer+ CD8 T cells. Transgenic expression of TCR1020 was attained via lentiviral transduction of reporter cell lines and gene-edited (TCRabnull) primary T cells. TCR1020 antigen specificity, affinity and cross-reactivity was assessed using in vitro coculture, limiting peptide dilution, and combinatorial peptide scanning assays. TCR1020 reactivity to G12V/A*11:01+ cell lines was assessed using in vitro cytokine release and cytotoxicity assays, as well as mouse xenograft tumor models. Results: TCR1020 exhibits specificity to the cognate KRAS G12V epitope with cross-reactivity to G12C but not wild-type KRAS. TCR1020 has a measured functional avidity (EC50) of 262 pM. TCR1020-redirected primary CD8+ T cells promote cytolysis of A*11:01+ tumor cell lines of lung, colonic or pancreatic origin harboring endogenous KRAS G12V mutations. The adoptive transfer of TCR1020-engineered primary CD8+ T cells exhibits potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. TCR1020 demonstrates CD8 co-receptor independence, and CD4+ T cells expressing TCR1020 express cytokines and lyse G12V/A*11:01+ tumor cell lines. In-depth characterization of the TCR1020 peptide recognition motif using alanine/glycine scanning libraries reveals a unique antigen binding motif with 5 critical amino acid residues excluding peptide/HLA anchor residues. Combinatorial peptide library scanning identifies five A*11:01-restricted peptide candidates within the human proteome with potential cross-reactivity to TCR1020, none of which activate TCR1020-engineered T cells over a wide range of concentrations. TCR1020 exhibits no evidence of reactivity against cell lines derived from healthy human tissues nor alloreactivity against a panel of lymphoblastoid cell lines encompassing a wide array of HLA class I alleles. Conclusion: TCR1020 exhibits high potency and exquisite antigen cognate specificity, and it is under clinical development for the treatment of A*11:01+ patients with KRAS G12V+ advanced solid tumors. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR08.

Targeting Protein-Protein Interfaces with Peptides: The Contribution of Chemical Combinatorial Peptide Library Approaches.

Protein-protein interfaces play fundamental roles in the molecular mechanisms underlying pathophysiological pathways and are important targets for the design of compounds of therapeutic interest. However, the identification of binding sites on protein surfaces and the development of modulators of protein-protein interactions still represent a major challenge due to their highly dynamic and extensive interfacial areas. Over the years, multiple strategies including structural, computational, and combinatorial approaches have been developed to characterize PPI and to date, several successful examples of small molecules, antibodies, peptides, and aptamers able to modulate these interfaces have been determined. Notably, peptides are a particularly useful tool for inhibiting PPIs due to their exquisite potency, specificity, and selectivity. Here, after an overview of PPIs and of the commonly used approaches to identify and characterize them, we describe and evaluate the impact of chemical peptide libraries in medicinal chemistry with a special focus on the results achieved through recent applications of this methodology. Finally, we also discuss the role that this methodology can have in the framework of the opportunities, and challenges that the application of new predictive approaches based on artificial intelligence is generating in structural biology.

Screening for Primordial RNA–Peptide Interactions Using High-Density Peptide Arrays

RNA-peptide interactions are an important factor in the origin of the modern mechanism of translation and the genetic code. Despite great progress in the bioinformatics of RNA-peptide interactions due to the rapid growth in the number of known RNA-protein complexes, there is no comprehensive experimental method to take into account the influence of individual amino acids on non-covalent RNA-peptide bonds. First, we designed the combinatorial libraries of primordial peptides according to the combinatorial fusion rules based on Watson-Crick mutations. Next, we used high-density peptide arrays to investigate the interaction of primordial peptides with their cognate homo-oligonucleotides. We calculated the interaction scores of individual peptide fragments and evaluated the influence of the peptide length and its composition on the strength of RNA binding. The analysis shows that the amino acids phenylalanine, tyrosine, and proline contribute significantly to the strong binding between peptides and homo-oligonucleotides, while the sum charge of the peptide does not have a significant effect. We discuss the physicochemical implications of the combinatorial fusion cascade, a hypothesis that follows from the amino acid partition used in the work.

The cyclic peptide G4CP2 enables the modulation of galactose metabolism in yeast by interfering with GAL4 transcriptional activity.

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.