The role of the BTLA-HVEM complex in the pathogenesis of autoimmune diseases.

Autoimmune diseases constitute a heterogeneous group of disorders with one common feature - the loss of immune tolerance towards autoantigens. Due to the complexity of the pathogenesis of these diseases, there are still many open questions regarding their etiology. Therefore, scientists unceasingly search for new data hoping to detect dependable biomarkers and design safe and effective treatment. The research on immune checkpoints is in line with these scientific and clinical demands. Immune checkpoints may be the key to understanding the pathogenesis of many immunological disorders. BTLA-HVEM complex, the inhibitory immune checkpoint, has recently caught scientific attention as an important regulator in different immune contexts, including autoreactivity. So far, the BTLA-HVEM complex has been mainly studied in the context of cancer, but as numerous data show, it may also be a target in the treating of autoimmune diseases. In this review, we intend to focus on the mechanisms of BTLA-HVEM interactions in immune cells and summarize the available data in the context of autoimmunity.

Targeting the HVEM protein using a fragment of glycoprotein D to inhibit formation of the BTLA/HVEM complex.

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein. To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10C-D30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD(1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy.

Design, synthesis and biological evaluation of PD-1 derived peptides as inhibitors of PD-1/PD-L1 complex formation for cancer therapy.

Over the past few years, many molecules such as monoclonal antibodies, affibodies, nanobodies, and small compounds have been designed and tested as inhibitors of PD-1/PD-L1 complex formation. Some of them have been successfully implemented into clinical oncology practice. However, the majority of these compounds have disadvantages and limitations, such as high production price, potential for immunogenicity and/or prolonged clearance. Thus, new inhibitors of the PD-1/PD-L1 immune checkpoints are needed. Recently, peptides emerged as potential novel approach for blocking receptor/ligand interaction. In the presented studies we have designed, synthesised and tested peptides, which are potential inhibitors of the PD-1/PD-L1 axis. The amino acid sequences of the designed peptides were based on the binding sites of PD-1 to PD-L1, as determined by the crystal structure of the protein complex and also based on MM/GBSA analysis. Interactions of the peptides with PD-L1 protein were confirmed using SPR, while their inhibitory properties were studied using cell-based PD-1/PD-L1 immune checkpoint blockade assays. The characterization of the peptides has shown that the peptides PD-1(119-142)T120C-E141C, PD-1(119-142)C123-S137C and PD-1(122-138)C123-S137C strongly bind to PD-L1 protein and disrupt the interaction of the proteins. PD-1(122-138)C123-S137C peptide was shown to have the best inhibitory potential from the panel of peptides. Its 3D NMR structure was determined and the binding site to PD-L1 was established using molecular modelling methods. Our results indicate that the PD-1 derived peptides are able to mimic the PD-1 protein and inhibit PD-1/PD-L1 complex formation.

Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

Fragments of gD Protein as Inhibitors of BTLA/HVEM Complex Formation-Design, Synthesis, and Cellular Studies.

One of the major current trends in cancer immunotherapy is the blockade of immune checkpoint proteins that negatively regulate the immune response. This has been achieved through antibodies blocking PD-1/PD-L1 and CTLA-4/CD80/CD86 interactions. Such antibodies have revolutionized oncological therapy and shown a new way to fight cancer. Additional (negative) immune checkpoints are also promising targets in cancer therapy and there is a demand for inhibitors for these molecules. Our studies are focused on BTLA/HVEM complex, which inhibits T-cell proliferation and cytokine production and therefore has great potential as a new target for cancer treatment. The goal of the presented studies was the design and synthesis of compounds able to block BTLA/HVEM interactions. For that purpose, the N-terminal fragment of glycoprotein D (gD), which interacts with HVEM, was used. Based on the crystal structure of the gD/HVEM complex and MM/GBSA analysis performed on it, several peptides were designed and synthesized as potential inhibitors of the BTLA/HVEM interaction. Affinity tests, ELISA tests, and cellular-based reporter assays were performed on these compounds to check their ability to bind to HVEM and to inhibit BTLA/HVEM complex formation. For leading peptides candidates, all-atom and subsequent docking simulations with a coarse-grained force field were performed to determine their binding modes. To further evaluate their potential as drug candidates, their stability in plasma and their cytotoxicity effects on PBMCs were assessed. Our data indicate that the peptide gD(1-36)(K10C-T29C) is the best candidate as a future drug. It interacts with HVEM protein, blocks the BTLA/HVEM interaction, and is nontoxic to cells. The present study provides a new perspective on the development of BTLA/HVEM inhibitors that disrupt protein interactions.

Disulfide-Linked Peptides for Blocking BTLA/HVEM Binding.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

Identification and characterization of antibodies elicited by human cystatin C fragment.

Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. One of rather atypical, occurring at a young age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real, lethal threat to some populations and any work on factors that can affect possible influencing hCC aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue 60-70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in rabbit immunization. As a result, specific anti-hCC antibodies were found in both rabbit sera. Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The obtained antibodies were characterized, and their influence on the aggregation propensity of the hCC molecules was evaluated. The antibodies turned out not to have any significant influence on the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other hCC fragments could lead to elaboration of effective treatment against HCCAA.

Characteristics of C-terminal, ß-amyloid peptide binding fragment of neuroprotective protease inhibitor, cystatin C.

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aß and the hCC-induced inhibition of Aß fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aß-binding region. The fragment of Aß peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aß(17-24). Structures and affinities of both Aß and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aß-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aß-fibril formation. Herein, we present further studies on molecular details of the hCC-Aß complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aß-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.

Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of ß2 and ß3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and ß sheet fragments make the hCC structure rigid and unable to undergo the swapping process.

Isolation and characterization of autoantibodies against human cystatin C.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a severe neurodegenerative disorder related to the point mutation in cystatin C gene resulting in human cystatin C (hCC) L68Q variant. One of the potential immunotherapeutic approaches to HCCAA treatment is based on naturally occurring antibodies against cystatin C. A recent growing interest in autoantibodies, especially in the context of neurodegenerative diseases, emerges from their potential use as valuable diagnostic markers and for controlling protein aggregation. In this work, we present characteristics of natural anti-hCC antibodies isolated from the IgG fraction of human serum by affinity chromatography. The electrophoresis (1-D and 2-D) results demonstrated that the isolated NAbs are a polyclonal mixture, but their electrophoretic properties did not allow to classify the new autoantibodies to any particular type of IgG. The Fc-glycan status of the studied autoantibodies was assessed using mass spectrometry analysis. For the isolated NAbs, the epitopic fragments in hCC sequence were identified by MS-assisted proteolytic excision of the immune complex and compared with the ones predicted theoretically. The knowledge of hCC fragments binding to NAbs and other ligands may contribute to the search for new diagnostic methods for amyloidosis of different types and the search for their treatment.

Structural studies of the C-terminal 19-peptide of serum amyloid A and its Pro → Ala variants interacting with human cystatin C.

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose concentration in serum increases markedly following a number of chronic inflammatory and neoplastic diseases. Prolonged high SAA level may give rise to reactive systemic amyloid A (AA) amyloidosis, where the N-terminal segment of SAA is deposited as amyloid fibrils. Besides, recently, well-documented association of SAA with high-density lipoprotein or glycosaminoglycans, in particular heparin/heparin sulfate (HS), and specific interaction between SAA and human cystatin C (hCC), the ubiquitous inhibitor of cysteine proteases, was proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, a hCC binding site in the SAA sequence was determined as SAA(86-104). The role of this SAA C-terminal fragment as a ligand-binding locus is still not clear. It was postulated important in native SAA folding and in pathogenesis of AA amyloidosis. In the search of conformational details of this SAA fragment, we did its structure and affinity studies, including its selected double/triple Pro → Ala variants. Our results clearly show that the SAA(86-104) 19-peptide has rather unordered structure with bends in its C-terminal part, which is consistent with the previous results relating to the whole protein. The results of affinity chromatography, fluorescent ELISA-like test, CD and NMR studies point to an importance of proline residues on structure of SAA(86-104). Conformational details of SAA fragment, responsible for hCC binding, may help to understand the objective of hCC-SAA complex formation and its importance for pathogenesis of reactive amyloid A amyloidosis.

African Viper Poly-His Tag Peptide Fragment Efficiently Binds Metal Ions and Is Folded into an α-Helical Structure.

Snake venoms are complex mixtures of toxic and often spectacularly biologically active components. Some African vipers contain polyhistidine and polyglycine peptides, which play a crucial role in the interaction with metal ions during the inhibition of snake metalloproteases. Polyhistidine peptide fragments, known as poly-His tags, play many important functions, e.g., in metal ion transport in bacterial chaperon proteins. In this paper, we report a detailed characterization of Cu(2+), Ni(2+), and Zn(2+) complexes with the EDDHHHHHHHHHG peptide fragment (pHG) derived from the venom of the rough scale bush viper (Atheris squamigera). In order to determine the thermodynamic properties, stoichiometry, binding sites, and structures of the metal-pHG complexes, we used a combination of experimental techniques (potentiometric titrations, electrospray ionization mass spectrometry, UV-vis spectroscopy, circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy) and extensive computational tools (molecular dynamics simulations and density functional theory calculations). The results showed that pHG has a high affinity toward metal ions. The numerous histidine residues located along this sequence are efficient metal ion chelators with high affinities toward Cu(2+), Ni(2+), and Zn(2+) ions. The formation of an α-helical structure induced by metal ion coordination and the occurrence of polymorphic binding states were observed. It is proposed that metal ions can "move along" the poly-His tag, which serves as a metal ion transport pathway. The coordination of Cu(2+), Ni(2+), and Zn(2+) ions to the histidine tag is very effective in comparison with other histidine-rich peptides. The stabilities of the metal-pHG complexes increase in the order Zn(2+) < Ni(2+)≪ Cu(2+).

The BTLA-HVEM complex - The future of cancer immunotherapy.

The BTLA-HVEM complex plays a pivotal role in cancer and cancer immunotherapy by regulating immune responses. Dysregulation of BTLA and HVEM expression contributes to immunosuppression and tumor progression across various cancer types. Targeting the interaction between BTLA and HVEM holds promise for enhancing anti-tumor immune responses. Disruption of this complex presents a valuable avenue for advancing cancer immunotherapy strategies. Aberrant expression of BTLA and HVEM adversely affects immune cell function, particularly T cells, exacerbating tumor evasion mechanisms. Understanding and modulating the BTLA-HVEM axis represents a crucial aspect of designing effective immunotherapeutic interventions against cancer. Here, we summarize the current knowledge regarding the structure and function of BTLA and HVEM, along with their interaction with each other and various immune partners. Moreover, the expression of soluble and transmembrane forms of BTLA and HVEM in different types of cancer and their impact on the prognosis of patients is also discussed. Additionally, inhibitors of the proteins binding that might be used to block BTLA-HVEM interaction are reviewed. All the presented data highlight the plausible clinical application of BTLA-HVEM targeted therapies in cancer and autoimmune disease management. However, further studies are required to confirm the practical use of this concept. Despite the increasing number of reports on the BTLA-HVEM complex, many aspects of its biology and function still need to be elucidated. This review can be regarded as an encouragement and a guide to follow the path of BTLA-HVEM research.

The effect of gD-derived peptides on T cell immune response mediated by BTLA-HVEM protein complex in melanoma patients.

Introduction: The effector function of T cells is regulated via immune checkpoints, activating or inhibiting the immune response. The BTLA-HVEM complex, the inhibitory immune checkpoint, may act as one of the tumor immune escape mechanisms. Therefore, interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA, thus disrupting the BTLA-HVEM interaction. These peptides' structure and amino acid sequences are based on the gD protein, the ligand of HVEM. Here, we investigated their immunomodulatory potential in melanoma patients. Methods: Flow cytometry analyses of activation, proliferation, and apoptosis of T cells from patients were performed. Additionally, we evaluated changes within the T cell memory compartment. Results: The most promising compound - Pep(2), increased the percentages of activated T cells and promoted their proliferation. Additionally, this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells. Discussion: We conclude that the examined peptide may act as a booster for the immune system. Moreover, the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds.

Multilayered Computational Framework for Designing Peptide Inhibitors of HVEM-LIGHT Interaction.

The herpesvirus entry mediator (HVEM) and its ligand LIGHT play crucial roles in immune system regulation, including T-cell proliferation, B-cell differentiation, and immunoglobulin secretion. However, excessive T-cell activation can lead to chronic inflammation and autoimmune diseases. Thus, inhibiting the HVEM-LIGHT interaction emerges as a promising therapeutic strategy for these conditions and in preventing adverse reactions in organ transplantation. This study focused on designing peptide inhibitors, targeting the HVEM-LIGHT interaction, using molecular dynamics (MD) simulations of 65 peptides derived from HVEM. These peptides varied in length and disulfide-bond configurations, crucial for their interaction with the LIGHT trimer. By simulating 31 HVEM domain variants, including the full-length protein, we assessed conformational changes upon LIGHT binding to understand the influence of HVEM segments and disulfide bonds on the binding mechanism. Employing multitrajectory microsecond-scale, all-atom MD simulations and molecular mechanics with generalized Born and surface area (MM-GBSA) binding energy estimation, we identified promising CRD2 domain variants with high LIGHT affinity. Notably, point mutations in these variants led to a peptide with a single disulfide bond (C58-C73) and a K54E substitution, exhibiting the highest binding affinity. The importance of the CRD2 domain and Cys58-Cys73 disulfide bond for interrupting HVEM-LIGHT interaction was further supported by analyzing truncated CRD2 variants, demonstrating similar binding strengths and mechanisms. Further investigations into the binding mechanism utilized steered MD simulations at various pulling speeds and umbrella sampling to estimate the energy profile of HVEM-based inhibitors with LIGHT. These comprehensive analyses revealed key interactions and different binding mechanisms, highlighting the increased binding affinity of selected peptide variants. Experimental circular dichroism techniques confirmed the structural properties of these variants. This study not only advances our understanding of the molecular basis of HVEM-LIGHT interactions but also provides a foundation for developing novel therapeutic strategies for immune-related disorders. Furthermore, it sets a gold standard for peptide inhibitor design in drug development due to its systematic approach.

Peptides targeting the BTLA-HVEM complex can modulate T cell immune response.

Immune checkpoints secure the proper function of the immune system and the maintenance of the BTLA-HVEM complex, an inhibitory immune checkpoint, is one of the pathways vital for T cell responsiveness to various stimuli. The present study reports the immunomodulatory potential of five peptides targeting the BTLA-HVEM complex on the activity of human T cells. Isolated T cells were exposed to the peptides alone or combined with CD3/CD28 mAb for 72 h or 120 h. The flow cytometry was used to evaluate the activation markers (CD69, CD62L, CD25), changes within the T cell memory compartment, proliferation rate, and apoptosis of T cells. The immunomodulatory effect of the peptides was visible as an increase in the percentage of CD4+ and CD8+ T cells expressing CD69 or CD25, a boost in T cell proliferation, and shifts in the T cell memory compartment. Pep(2) and Pep(5) were the most promising compounds, displaying a putative immune-restoring function.

Targeting BTLA with the peptide inhibitor HVEM(14-39) - A new way to restore the activity of T cells in melanoma.

The complex of B- and T-lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) plays a critical role in immune regulation and has emerged as a promising therapeutic target for cancer treatment. In this study, we investigated the potential of the peptide inhibitor HVEM(14-39) to restore peripheral T cell activity in patients with advanced melanoma. In these patients, CD8+ T cells downregulated BTLA expression and increased HVEM expression upon activation. The addition of HVEM(14-39) reduced the percentage of BTLA+ CD8+ T cells and increased the subpopulation of HVEM+ CD8+ T cells. Additionally, HVEM(14-39) enhanced T cell activation, proliferation, and the shift toward effector memory T cell subpopulations. Finally, this peptide affected the proliferation rate and late apoptosis of melanoma cell line in co-culture with T cells. These findings suggest that HVEM(14-39) can overcome T cell exhaustion and improve antitumor responses. Peptide-based immunotherapy targeting the BTLA-HVEM complex offers a promising alternative to monoclonal antibody-based therapies, with the potential for fewer side effects and higher treatment efficacy.

Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer.

The PD-1/PD-L1 complex belongs to the group of inhibitory immune checkpoints and plays a critical role in immune regulation. The PD-1/PD-L1 axis is also responsible for immune evasion of cancer cells, and this complex is one of the main targets of immunotherapies used in oncology. Treatment using immune checkpoint inhibitors is mainly based on antibodies. This approach has great therapeutic potential; however, it also has major drawbacks and can induce immune-related adverse events. Thus, there is a strong need for alternative, non-antibody-based therapies using small molecules, peptides, or peptidomimetics. In the present study, we designed, synthesized, and evaluated a set of PD-1-targeting peptides based on the sequence and structure of PD-L1. The binding of these peptides to PD-1 was investigated using SPR and ELISA. We also assessed their ability to compete with PD-L1 for binding to PD-1 and their inhibitory properties against the PD-1/PD-L1 complex at the cellular level. The best results were obtained for the peptide PD-L1(111-127)(Y112C-I126C), named (L11), which displaced PD-L1 from binding to PD-1 in the competitive assay and inhibited the formation of the PD-1/PD-L1 complex. The (L11) peptide also exhibited strong affinity for PD-1. NMR studies revealed that (L11) does not form a well-defined secondary structure; however, MD simulation indicated that (L11) binds to PD-1 at the same place as PD-L1. After further optimization of the structure, the peptide inhibitor obtained in this study could also be used as a potential therapeutic compound targeting the PD-1/PD-L1 axis.

Interaction of serum amyloid A with human cystatin C--assessment of amino acid residues crucial for hCC-SAA formation (part II).

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life-threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute-phase-reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases--human cystatin C (hCC)--has been proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86-104) and hCC(96-102), respectively. Here, we report further details concerning the hCC-SAA interaction. With the use of affinity tests and florescent ELISA-like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser-98 and Tyr-102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC-SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins.

Exploring the inhibitory potential of in silico-designed small peptides on Helicobacter pylori Hp0231 (DsbK), a periplasmic oxidoreductase involved in disulfide bond formation.

Introduction: Helicobacter pylori is a bacterium that colonizes the gastric epithelium, which affects millions of people worldwide. H. pylori infection can lead to various gastrointestinal diseases, including gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Conventional antibiotic therapies face challenges due to increasing antibiotic resistance and patient non-compliance, necessitating the exploration of alternative treatment approaches. In this study, we focused on Hp0231 (DsbK), an essential component of the H. pylori Dsb (disulfide bond) oxidative pathway, and investigated peptide-based inhibition as a potential therapeutic strategy. Methods: Three inhibitory peptides designed by computational modeling were evaluated for their effectiveness using a time-resolved fluorescence assay. We also examined the binding affinity between Hp0231 and the peptides using microscale thermophoresis. Results and discussion: Our findings demonstrate that in silico-designed synthetic peptides can effectively inhibit Hp0231-mediated peptide oxidation. Targeting Hp0231 oxidase activity could attenuate H. pylori virulence without compromising bacterial viability. Therefore, peptide-based inhibitors of Hp0231 could be candidates for the development of new targeted strategy, which does not influence the composition of the natural human microbiome, but deprive the bacterium of its pathogenic properties.