11-mer Peptide Research Articles

Picturins and Pictuseptins, two novel antimicrobial peptide families from the skin secretions of the Chachi treefrog, Boana picturata

The Imbabura treefrog (Boana picturata) is an underexplored source of bioactive peptides. The combination of molecular cloning and mass spectrometry allowed us to identify three new peptide families, named “Picturins” (PTR), “Pictuseptins” (PTS), and “Boanins” (BNS). PTR is composed of three 25-mer peptides, characterized by the N-terminal sequence: GVFKDALKQ and the C-terminal sequence: AANALKPK. The sequences of PTR-1, −2 and − 3 are highly conserved only showing two divergent sites: (L/F) in position 10 and (K/Q) in position 17. PTS gathers six peptides. PTS -1, −2 and − 4 have 22 amino acid residues in length, while PTS -3, −5 and − 6 are composed of 26 residues. Whereas BNS are four 28–37 mer peptides, showing two conserved regions: the N-terminal sequence FLGAL and the C-terminal sequence KALNP. PTR-1 to 3 and PTS -1 to −3 were chemically synthetized and their antimicrobial and haemolytic activity was assessed. PTR displayed moderate activity against Escherichia coli (MIC 24.80 to 48.95 μM), while PTS showed a broad antimicrobial and antifungal effect. PTS-1 was the most active peptide against E. coli (6.8 μM) followed by PTS-3 (11.7 μM) and PTS-2 (14.24 μM). These peptides also showed low haemolytic activity, pointing to a favorable selectivity. Overall, new unique non-hemolytic and cationic peptide sequences were characterized that could be valuable for the next-generation of anti-infective drugs. Future functional studies should explore the pharmacological potential of Boanins to include them as antimicrobial scaffolds. Biological significanceNature-inspired solutions have shown their importance mainly for the development of the pharmaceutical industry. Frog skin peptides are excellent examples of the biomedical potential of naturally evolved molecules for specific targets, including multi-resistant bacteria. The characterization of new chemical entities from poorly studied skin secretions of Ecuadorian biodiversity, such as B. picturata, represents an unprecedented opportunity to identify candidates to tackle global concerns, for instance, antibiotic resistance.

Supramolecular Detoxification of Macromolecular Biotoxin through the Complexation by a Large-Sized Macrocycle.

Biotoxins are diverse, complex, and hypertoxic, ultimately serving as grave and lasting menaces to humanity. Here, it is aimed to introduce a new detoxification methodology for macromolecular biotoxin through complexation by a very large macrocycle. A 25-mer peptide isolated from Lycosa erythrognatha spider venom (LyeTxI) is selected as the model macromolecular biotoxin. Quaterphen[4]arene, with a side length of ≈1.6nm, has a sufficient cavity to bind LyeTxI. Hence, the water-soluble derivative of Quaterphen[4]arene (H) is designed and synthesized. H exhibits an overall host-guest complexation toward LyeTxI, resulting in a considerably high association constant of (7.01±0.18)×107 m-1 . This encapsulation of peptide is interesting as traditional macrocycles can only engulf the amino acid residues of peptides due to their limited cavity size. In vitro assay verifies that complexation by H inhibits the interactions of LyeTxI with cell membranes, thereby reducing its cytotoxicity, suppressing hemolysis, and decreasing the release of lactate dehydrogenase. Notably, the intravenous administration of H has a significant therapeutic effect on LyeTxI-poisoned mice, alleviating inflammation and tissue damage, and markedly improving the survival rate from 10% to 80%. An efficient and potentially versatile approach is provided to detoxify macromolecular biotoxins, with giant macrocycle serving as an antidote.

Short peptide pharmacophores developed from protein phosphatase-1 disrupting peptides (PDPs)

PP1 is a major phosphoserine/threonine-specific phosphatase that is involved in diseases such as heart insufficiency and diabetes. PP1-disrupting peptides (PDPs) are selective modulators of PP1 activity that release its catalytic subunit, which then dephosphorylates nearby substrates. Recently, PDPs enabled the creation of phosphatase-recruiting chimeras, which are bifunctional molecules that guide PP1 to a kinase to dephosphorylate and inactivate it. However, PDPs are 23mer peptides, which is not optimal for their use in therapy due to potential stability and immunogenicity issues. Therefore, we present here the sequence optimization of the 23mer PDP to a 5mer peptide, involving several attempts considering structure-based virtual screening, high throughput screening and peptide sequence optimization. We provide here a strong pharmacophore as lead structure to enable PP1 targeting in therapy or its use in phosphatase-recruiting chimeras in the future.

A specific 25-mer peptide from the cysteine protease of P. gingivalis is the epitope of humoral immune response in rheumatoid arthritis

Abstract Objective Several lines of evidence suggest that the pathobiont Porphyromonas gingivalis is involved in the development and/or progression of rheumatoid arthritis. This bacterium produces cysteine proteases, such as gingipain RgpA, endowed with the potential to induce significant inflammation in patients with periodontitis and rheumatoid arthritis. Both diseases involve chronic inflammation with the production of pro-inflammatory cytokines, connective tissue breakdown and bone erosion. Methods A 25-peptide named BR1 from Porphyromonas gingivalis was synthesized for the study. Serum samples from patients with RA, early arthritis, other rheumatic diseases, and normal controls were used in the study. A monoclonal antibody named BR2mAb was derived from BALB/c mice immunized with the 25-mer peptide was produced as the control. Anti-BR1, ACPA and RF were detected by ELISA. Results Anti-BR1 were detected in 56.0% (59/106) of RA patients and were found in 63.8% (23/36) of RA patients without both RF and/or ACPA. The serological positive rates with the two tests of RF and ACPA and with the three tests of anti-BR1, RF, and ACPA were 66% and 84.2%, respectively. Anti-BR1 were detected in 60.0% (9/15) of patients with early arthritis, but neither RF nor ACPA was detected in this group of patients. The sensitivity and specificity for RA were 60.1% and 95%, respectively. The BR2 mAb reacted to Porphyromonas gingivalis as well. Conclusions A specific 25-mer peptide from the cysteine protease of P. gingivalis is the epitope for humoral immune response in rheumatoid arthritis. The anti-25-mer peptide antibody test is an excellent new test for the diagnosis of patients with RA and early arthritis. This study was supported by grants from the Ministry of Science and Technology, Taiwan (MOST-104-2314-B-039-045, MOST-105-2911-I-039-504, MOST-105-2314-B-039-047, MOST-106-2911-I-039-501, MOST-107-2314-B-039-050, MOST-108-2314-B-039-019), and from China Medical University Hospital (DMR-106-188, DMR-108-183, DMR-109-206).

CHARM: COVID-19 Health Action Response for Marines-Association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test.

SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses against S, N and M proteins in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were enrolled in an immune response sub-study. FluoroSpot interferon-gamma (IFN-γ) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-γ responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-γ responses were significantly (p = 0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic but not symptomatic subjects. Asymptomatic IFN-γ responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but not symptomatic participants. The frequencies of IFN-γ responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-γ and IL2 responses to the N+M pool were significantly correlated with IFN-γ and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-γ and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

Adiponectin-derived pentapeptide ameliorates psoriasiform skin inflammation by suppressing IL-17 production in γδT cells

BackgroundPsoriasis is a common immunologic chronic skin disease that affects at least 100 million individuals worldwide. Adiponectin is associated with psoriasis and suppresses psoriasiform inflammation. Recently, a small-sized transdermally deliverable 5-mer peptide (GLYYF; P5) was discovered as a potential adiponectin receptor 1 agonist. ObjectivesTo confirm reduction in adiponectin protein level in the human skin and investigate whether functional adiponectin replenishment by topical P5 application improves psoriasiform skin inflammation. MethodsAdiponectin protein expression in the skin of individuals with psoriasis and normal skin was examined by immunofluorescence staining. Imiquimod-induced psoriasis-like skin inflammation was induced in wild-type (WT) and adiponectin-deficient (Adipoq−/−) mice. Vehicle and P5 were topically applied to the back skin and ears of mice. Histological study, reverse-transcription quantitative polymerase chain reaction, multiplex-bead array assay, and flow cytometric analysis were performed. ResultsAdiponectin protein expression was downregulated both in the epidermis and dermis of psoriatic lesions as compared to that in the normal skin. Topically applied P5 attenuated the severity of imiquimod-induced psoriatic dermatitis in both WT and Adipoq−/− mice by decreasing the expression of psoriasis-related cytokines (Il17a, Il1b, Il6, and Tnfa). P5 application significantly reduced the proportion of interleukin-17A–producing γδT cells. ConclusionTransdermally deliverable adiponectin receptor 1 agonist, P5, can be a potential peptide drug to manage psoriasis by mediating the anti-psoriatic effect of adiponectin

In Vivo Evaluation of ECP Peptide Analogues for the Treatment of Acinetobacter baumannii Infection.

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative species with lipopolysaccharides (LPS) binding and endotoxin-neutralization activities. Next, analogues that contain non-natural amino acids were designed to increase serum stability. Here, two analogues were selected for in vivo assays: the all-D version (ECPep-D) and the Arg to Orn version that incorporates a D-amino acid at position 2 (ECPep-2D-Orn). The peptide analogues retained high LPS-binding and anti-endotoxin activities. The peptides efficacy was tested in a murine acute infection model of Acinetobacter baumannii. Results highlighted a survival rate above 70% following a 3-day supervision with a single administration of ECPep-D. Moreover, in both ECPep-D and ECPep-2D-Orn peptide-treated groups, clinical symptoms improved significantly and the tissue infection was reduced to equivalent levels to mice treated with colistin, used as a last resort in the clinics. Moreover, treatment drastically reduced serum levels of TNF-α inflammation marker within the first 8 h. The present results support ECP-derived peptides as alternative candidates for the treatment of acute infections caused by Gram-negative bacteria.

Luminescent gold-peptide spheric aggregates: selective and effective cellular targeting

Although the restriction of intramolecular motion has been well recognized as the fundamental of aggregation induced emission enhancement (AIEE), the regulation mechanism of gold nanoclusters (AuNCs) based AIEE system are still unclear. In this paper, we have investigated the Zn2+-induced AIEE process of thiolate ligands (i.e., cysteine, glutathione and an 8-mer peptide) protected AuNCs, which shows a pH-dependent evolution from single AuNCs to spheric aggregates to irregular network. Using photoluminescent enhancement ratio as an index, the concept of “mid-pH” is proposed to indicate the optimal pH for the formation of spheric AuNCs aggregates. Importantly, the surface ligands allow the formation of spheric AuNCs aggregates at tunable mid-pH between 5.7 and 7.5. Owing to the appropriate size and surface peptide targetability, the spheric AuNCs aggregates can be successfully screened for targeted tumor cell uptake and imaging at physiological pH. The cell uptake mechanism study showed that AuNCs aggregates was specifically recognized by arginine-glycine-aspartic acid (RGD) sequence on the ligand and integrin αvβ3 on the cell surface, thus mainly through clathrin-mediated endocytosis. This work provides new sight to artificially regulate the construction of efficient cellular imaging probes.

MTADV 5-MER PEPTIDE SUPPRESSES IBD PATHOLOGY AND UNVEILS A NEW POTENTIAL TARGET-SERUM AMYLOID A

Abstract We reported in the previous Crohn’s and Colitis Congress and published in Refs 1 and 2 ,that a segment of 5 aa of a CD44 variant -Methionine, Threonine, Alanine, Aspartic Acid, Valine (MTADV), called 5-MER peptide (5MP), inhibits the inflammation in DSS and TNBS mouse models of IBD, when subcutaneously injected or orally delivered. Similarly, 5MP inhibited the inflammation in mouse models of Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA; Ref 2). As IBD, MS and RA share up-regulation of pathological Serum Amyloid A (SAA) (which fuels chronic inflammations), we predicted and then showed that 5-MP targets and hampers the pathological SAA, thus ameliorating SAA-associated inflammatory diseases by inhibiting the release of pro-inflammatory cytokines from SAA-activated innate-immunity cells. Now we focus on deciphering 5MP mechanism of action. To this end, we show that 1. Rats with Collagen-Induced Arthritis (CIA; the animal model of RA), in contrast to mice or man, do not produce under inflammatory conditions SAA. Therefore, 5MP, in the absence of the SAA target, cannot inhibits the rat CIA, that is almost identical to mouse CIA. This fact supports our prediction that SAA is a 5MP target. 2. SAA binds 5MP, as indicated by mass spectrometry. 3. Polymerized and aggregated forms of SAA are the pathological forms of SAA, generating inflammation and amyloidosis, the last could be a severe complication of Crohn’s (Ref 3). Co-Incubation with 5MP interferes with SAA polymerization and aggregation (Figure 1), providing mechanistic explanation for 5MP therapeutic effect in IBD. 4. Human Fibroblasts, monocytes and Peripheral Blood Mononuclear Cells (PBMCs), stimulated with human SAA, release the pro-inflammatory cytokines IL-6, IL-1β and TNFα. Co-incubation of SAA with 5MP (but not with scrambled peptide TMVAD) suppresses the cytokines release from the cells, providing evidence on the 5-MP potency to interfere with SAA-cytokine destructive cross-talk. In support, mice with IBD reduce inflammation and alleviate IL-6 and IL-1β expression in colonic tissue after 5-MP injection (Figure 2). 5. Normal immune responses, such as DTH or antibody response to foreign antigen, are not inhibited by 5MP. 6. 5-MP is stable in room temperature for at least 5 months. 7. Dogs can tolerate (without signs of harmful effects) 5MP doses, which are much higher than the 5MP therapeutic dose, and finally, a finding which merits a special attention 8. Gene Set Enrichment Analysis reveals that 5-MP activates genes, inducing resistance to IBD, e.g., Batf2, Fgl2, Ifitm3, Pyrin (Nefv), TNFR2 and Tnfrsf9. 1. Nedvetzki, S., and Naor, D. J. Clin. Invest. 111, 1211-1220, 2003. 2. Hemed-Shaked M. and Naor D. J Autoimmun. Aug 12;124:102713, 2021. 3. Denis MA. Eur J Clin Invest. 2013;43:292-301

MTADV 5-MER PEPTIDE SUPPRESSES IBD PATHOLOGY AND UNVEILS A NEW POTENTIAL TARGET-SERUM AMYLOID A

We reported in the previous Crohn’s and Colitis Congress and published in Refs 1 and 2 ,that a segment of 5 aa of a CD44 variant -Methionine, Threonine, Alanine, Aspartic Acid, Valine (MTADV), called 5-MER peptide (5MP), inhibits the inflammation in DSS and TNBS mouse models of IBD, when subcutaneously injected or orally delivered. Similarly, 5MP inhibited the inflammation in mouse models of Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA; Ref 2). As IBD, MS and RA share up-regulation of pathological Serum Amyloid A (SAA) (which fuels chronic inflammations), we predicted and then showed that 5-MP targets and hampers the pathological SAA, thus ameliorating SAA-associated inflammatory diseases by inhibiting the release of pro-inflammatory cytokines from SAA-activated innate-immunity cells.

Conformational ensemble of the TNF-derived peptide solnatide in solution

Tumor necrosis factor (TNF) is a homotrimer that has two spatially distinct binding regions, three lectin-like domains (LLD) at the TIP of the protein and three basolaterally located receptor-binding sites, the latter of which are responsible for the inflammatory and cell death-inducing properties of the cytokine. Solnatide (a.k.a. TIP peptide, AP301) is a 17-mer cyclic peptide that mimics the LLD of human TNF which activates the amiloride-sensitive epithelial sodium channel (ENaC) and, as such, recapitulates the capacity of TNF to enhance alveolar fluid clearance, as demonstrated in numerous preclinical studies. TNF and solnatide interact with glycoproteins and these interactions are necessary for their trypanolytic and ENaC-activating activities. In view of the crucial role of ENaC in lung liquid clearance, solnatide is currently being evaluated as a novel therapeutic agent to treat pulmonary edema in patients with moderate-to-severe acute respiratory distress syndrome (ARDS), as well as severe COVID-19 patients with ARDS. To facilitate the description of the functional properties of solnatide in detail, as well as to further target-docking studies, we have analyzed its folding properties by NMR. In solution, solnatide populates a set of conformations characterized by a small hydrophobic core and two electrostatically charged poles. Using the structural information determined here and also that available for the ENaC protein, we propose a model to describe solnatide interaction with the C-terminal domain of the ENaCα subunit. This model may serve to guide future experiments to validate specific interactions with ENaCα and the design of new solnatide analogs with unexplored functionalities.

Immunohistochemical detection of enteroviruses in pancreatic tissues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus.

ABSTRACTAims/IntroductionThe need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde‐fixed and paraffin‐embedded samples is increasing. The gold standard monoclonal antibody (clone 5D8/1) against EV‐envelope protein (VP1) was proven to cross‐react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro), which is encoded by the EV gene and translated by the host cells during EV replication, and participates processing proproteins to viral capsid proteins.Materials and MethodsWe raised polyclonal antiserum by immunizing a rabbit with an 18‐mer peptide of Coxsackievirus B1 (CVB1)‐2Apro, and examined the specificity and sensitivity for EV on formaldehyde‐fixed and paraffin‐embedded tissue samples.ResultsEnzyme‐linked immunosorbent assay study showed a high titer of antibody for 18‐mer peptide of CVB1‐2Apro, cross‐reacting with CVB3‐2Apro peptide. IHC showed that antiserum against 2Apro reacted with CVB1‐infected and VP1‐positive Vero cells. Confocal laser scanning microscopy showed that antigen stained by the 2Apro antibody located in the same cell with VP1 stained by 5D8/1. IHC using 2Apro antiserum showed dense staining in the islets of EV‐associated fulminant type 1 diabetes pancreas and that located in the same cell stained positive for VP1 (5D8/1). Specificity of 2Apro antiserum by IHC staining was confirmed by negative 2Apro in 14 VP1‐negative non‐diabetes control pancreases.ConclusionsOur study provides a new polyclonal antiserum against CVB1‐2Apro, which might be useful for IHC of EV‐infected human tissues stored as archive of formaldehyde‐fixed and paraffin‐embedded tissue samples.

Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6.

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared “classical” fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the “classical” method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the “classical” SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a “working day” (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.

MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1β from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent.

Computational design of SARS-CoV-2 peptide binders with better predicted binding affinities than human ACE2 receptor

SARS-CoV-2 is coronavirus causing COVID-19 pandemic. To enter human cells, receptor binding domain of S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) binds to peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptor. Employing peptides to inhibit binding between SARS-CoV-2-RBD and ACE2-PD is a therapeutic solution for COVID-19. Previous experimental study found that 23-mer peptide (SBP1) bound to SARS-CoV-2-RBD with lower affinity than ACE2. To increase SBP1 affinity, our previous study used residues 21–45 of α1 helix of ACE2-PD (SPB25) to design peptides with predicted affinity better than SBP1 and SPB25 by increasing interactions of residues that do not form favorable interactions with SARS-CoV-2-RBD. To design SPB25 with better affinity than ACE2, we employed computational protein design to increase interactions of residues reported to form favorable interactions with SARS-CoV-2-RBD and combine newly designed mutations with the best single mutations from our previous study. Molecular dynamics show that predicted binding affinities of three peptides (SPB25Q22R, SPB25F8R/K11W/L25R and SPB25F8R/K11F/Q22R/L25R) are better than ACE2. Moreover, their predicted stabilities may be slightly higher than SBP1 as suggested by their helicities. This study developed an approach to design SARS-CoV-2 peptide binders with predicted binding affinities better than ACE2. These designed peptides are promising candidates as SARS-CoV-2 inhibitors.

New insights into the mechanism of Keap1-Nrf2 interaction based on cancer-associated mutations

AimsKeap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer. Main methodsWe have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2Thr80 and Nrf2Pro85 on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1G364C, Keap1D422N, Keap1R470C, Keap1G480W, Keap1E493Q and Keap1R601L on binding with Nrf2 through ITC experiments. Key findingsNrf2Thr80 and Nrf2Pro85 play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2Thr80 and Nrf2Pro85 mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keap1, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keap1 mutants can activate Nrf2 without disrupting the binding to Nrf2. SignificanceThe study provides new insight into Keap1/Nrf2 signaling pathway and cancer.

Identification of Leukemia Associated Minor Histocompatibility Antigens through Computational Prediction and Targeted Mass Spectrometry

Background and Rationale: T-cell responses to minor histocompatibility antigens (mHA) mediate both anti-tumor immunity (GvL) and graft versus host disease (GvHD) in allogeneic stem cell transplantation. Clinical trials of cellular immunotherapies that target one or more of these mHA are ongoing, however current laboratory and GWAS methods have thus far produced a limited number of confirmed mHA (Oostvogels et al. BMT 2016, PMID: 26501766). Identifying mHA with tight binding affinity to common HLA alleles, narrow tissue restriction, and high population prevalence (GvL mHA) could provide promising immunotherapy targets against leukemia as therapeutic strategies that target these mHA would maximize the number of candidate recipients and may drive a beneficial GvL effect without causing GvHD.Methods: To evaluate a custom GvL mHA prediction pipeline (Figure 1) based on neoantigen prediction software developed in the Vincent lab (Kardos et al. JCI Insight 2016, PMID: 27699256), we predicted mHA for HLA-matched allogeneic stem cell transplant (SCT) donor-recipient pairs (DRPs) from a cohort of 101 patients with myeloid hematologic diseases who had been genotyped at 13,917 non-synonyomous coding polymorphisms (cSNPs) by Illumina NS-12 microarrays (Armistead et al . PLoS One 2011, PMID: 21858034). We computationally predicted GvL mHA by first eliciting in silico all possible 8, 9, 10, 11, 16, 20, and 24-mer peptides arising from DRP genetic variation, then estimating peptide/HLA binding with NetMHCpan and tissue restriction based on Human Protein Atlas and in-house AML sample RNAseq data. We validated our computational approach to mHA prediction by assessing our capacity to predict previously published mHA and by performing two complementary biological validations of a novel GvL mHA we discovered. We purified HLA-restricted peptide epitopes from HLA-A*02:01 expressing U937 cells and used targeted differential ion mobility spectrometry/tandem mass spectrometry (DIMS/MS/MS) (Dharmasiri et al. J. Proteome Res. 2014, PMID: 25184817) to test for HLA-A*02:01 presentation of a predicted novel GvL mHA (UNC-GVL-1). We also tested for T cell responses to UNC-GVL-1 using peptide/HLA tetramers on post-SCT AML patient samples.Results: We identified 18 previously published mHA that were potentially discoverable in our patient cohort because at least one DRP expressed the appropriate HLA and the mHA arose from a cSNP contained in the Illumina NS-12 microarray. Of these 18 possible mHA, we successfully predicted 14 in our analysis (Figure 2A), with the 4 missed mHA resulting from inadequate cohort size (n=2), non-canonical translation of a mHA (n=1), and poor predicted HLA binding (n=1). Of our 101 DRPs, 60 (59%) were predicted to have at least one of these 14 mHA, with T4A the most common, identified in 21 DRPs (21%), and UNC-GVL-1 identified in 13 DRPs (13%). All previously published Class I mHA predicted in our cohort as well as UNC-GVL-1 had similar or tighter predicted binding affinity for the recipient peptide/HLA as compared to the donor (i.e. alternate allele) peptide/HLA (Figure 2B). In addition to predicting previously discovered mHA, we predicted 102 novel public GvL mHA. By optimizing DIMS settings to filter out other peptide epitopes, we confirmed that the novel GvL mHA UNC-GVL-1 was presented on HLA-A*02:01 by HLA-A*02:01 expressing U937 cells. Using UNC-GVL-1/HLA-A*02:01 tetramers, we identified T cell responses in 4 of 9 HLA-A*02:01 expressing post-SCT samples.Conclusions: Computational prediction of GvL mHA with tight predicted binding affinity to common HLA alleles, narrow tissue restriction, and high population prevalence can provide novel public immunotherapy targets. UNC-GVL-1 is a promising novel target for immunotherapy in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A 8-mer Peptide of PGLYRP1/Tag7 Innate Immunity Protein Binds to TNFR1 Receptor and Inhibits TNFα-Induced Cytotoxic Effect and Inflammation.

Search for novel regulatory protein fragments with potential functional roles is required both for understanding the immune response mechanisms and the development of targeted immunotherapy. Earlier we demonstrated that the PGLYRP1/Tag7 innate immunity protein can be regarded as an inhibitor of TNFα cytotoxic activity via the interaction with its TNF receptor 1 (TNFR1). A C-terminal peptide fragment 17.1 of the molecule is responsible for this function. In this study we have identified a minimal 8-mer region of this peptide (hereinafter – 17.1A) capable to bind to TNFR1. As a result of such interaction, the cytotoxic signals induced by this receptor are blocked. Also, this peptide demonstrates an anti-inflammatory activity in vivo in the complete Freund’s adjuvant (CFA)-induced arthritis model in laboratory mice. Peptide 17.1A is capable to reduce periarticular inflammation, inhibit the development of synovitis and exhibit a protective effect on cartilage and bone tissues. This peptide can turn out to be a promising medicinal agent for autoimmune arthritis and other diseases.

The Immunophilin Protein FKBPL and its Peptide Derivatives are Novel Regulators of Vascular Integrity and Inflammation via Nf-κB Signaling

A breakdown in vascular integrity and excessive inflammation are hallmarks of serious pathological conditions including sepsis, acute respiratory distress syndrome (ARDs) and most recently, severe COVID-19. FK506 binding protein like (FKBPL) is a member of the immunophilin protein superfamily with potent anti tumor activity through inhibition of angiogenesis and cancer stemness. An FKBPL based 23mer peptide, ALM201, displayed a good safety and pharmacokinetic profile in a Phase 1a oncology clinical trial and was subsequently designated orphan drug status by the FDA in ovarian cancer. Here we describe a novel role for FKBPL and its peptides in regulating vascular integrity and cytokine production though modulating NFκB signaling. FKBPL knockdown promoted endothelial cell barrier permeability, which was further exacerbated upon stimulation with lipopolysaccharide (LPS) and accompanied by increased expression of TNF mRNA and phosphorylation of p65(RelA). Whilst treatment with the FKBPL based pre-clinical peptide, AD-01, increased VE-cadherin endothelial tight junctions following LPS stimulation. Bone marrow derived macrophages (BMDM) from FKBPL haploinsufficient mice (Fkbpl+/- ) also demonstrated increased phosphorylation of p65(RelA) in response to LPS stimulation compared to wild-type mice. Furthermore, treatment with AD-01 inhibited p65(RelA) phosphorylation following LPS stimulation resulting in reduced NFκB target gene expression and proinflammatory cytokine production. In an in vivo LPS survival model, Fkbpl+/- mice have reduced survival compared to wild-type mice. Moreover, treatment of wild-type mice with the clinical FKBPL-based peptide, ALM201, following LPS injection resulted in a 100% survival rate in mice at experimental endpoint, as well as an abrogation of production of proinflammatory cytokines, TNF and IL-6, in peritoneal lavage washings. Analysis of human genetic biobanks found an association between common genetic variants associated with FKBPL and traits associated with inflammatory disorders such as psoriasis, rheumatoid arthritis and high lymphocyte count. In summary, for the first time, we describe a novel role for FKBPL as a regulator of inflammation and vascular integrity through modulating NFκB signaling and FKBPL based therapies demonstrate potent anti inflammatory activity.

Synthesis and Analysis of Properties of an Immunogenic Fragment from NS4A Polypeptide of Hepatitis C Virus

—The work is aimed at the synthesis and analysis from NS4A of hepatitis C virus (HCV) antigen peptide fragment that contains a conserved B-cell and T-helper epitopes. The 24-mer peptide VIVGRIILSGRPAVIPDREVLYRK-NH2, which contains the main immunogenic site 24–46 of HCV NS4A antigen (corresponding to the 1681–1703 amino acid residues of the HCV polypeptide), subtype 1b, has been prepared via solid-phase synthesis according to the Fmoc-protocol. Particles with diameters of 73 ± 10 nm (30%) and 236 ± 5 nm (70%) have been detected in the water solution of the highly purified peptide (0.5 mg/mL) by dynamic light scattering. The polydispersity index of 0.377 ± 0.012 implies the existence of heterogeneity because of the aggregation of the peptide molecules. The ζ-potential of the peptide aggregates has been determined as 7.0 ± 0.5 mV by means of electrophoretic light scattering. These data confirm the possibility for the development of a nanoscale liposome form of the peptide preparation. Immunoreactivity of the synthesized highly purified peptide has been studied with the use of blood sera of patients with chronic hepatitis C. Antipeptide immunoglobulins G have been detected in 41.7% of serum samples. Thus, this peptide has been shown to reproduce at least one B-epitope, to which antibodies are raised during natural HCV infection. The synthesized 24-mer peptide is a promising candidate for further research and for use as a potential immunogen for the design of a nanoscale therapeutic immunogenic liposomal peptide composition with synthetic lipids as an adjuvant.