Peptide Cleavage Research Articles

Fast and accurate multi-bacterial identification using cleavable and FRET-based peptide nucleic acid probes

Fast and accurate identification of pathogenic microbes in patient samples is crucial for the timely treatment of acute infectious diseases such as sepsis. The fluorescence in situ hybridization (FISH) technique allows the rapid detection and identification of microbes based on their variation in genomic sequence without time-consuming culturing or sequencing. However, the recent explosion of microbial genomic data has made it challenging to design an appropriate set of probes for microbial mixtures. We developed a novel set of peptide nucleic acid (PNA)-based FISH probes with optimal target specificity by analyzing the variations in 16S ribosomal RNA sequence across all bacterial species. Owing to their superior penetration into bacteria and higher mismatch sensitivity, the PNA probes distinguished seven bacterial species commonly observed in bacteremia with 96–99.9% accuracy using our optimized FISH procedure. Detection based on Förster resonance energy transfer (FRET) between pairs of adjacent binding PNA probes eliminated crosstalk between species. Rapid sequential species identification was implemented, using chemically cleavable fluorophores, without compromising detection accuracy. Owing to their outstanding accuracy and enhanced speed, this set of techniques shows great potential for clinical use.

Alpha-1-antitrypsin as novel substrate for S. aureus' Spl proteases - implications for virulence.

The serine protease like (Spl) proteases of Staphylococcus aureus are a family of six proteases whose function and impact on virulence are poorly understood. Here we propose alpha-1-antitrypsin (AAT), an important immunomodulatory serine protease inhibitor as target of SplD, E and F. AAT is an acute phase protein, interacting with many proteases and crucial for prevention of excess tissue damage by neutrophil elastase during the innate immune response to infections. We used MALDI-TOF-MS to identify the cleavage site of Spl proteases within AAT's reactive center loop (RCL) and LC-MS/MS to quantify the resulting peptide cleavage product in in vitro digestions of AAT and heterologous expressed proteases or culture supernatants from different S. aureus strains. We further confirmed proteolytic cleavage and formation of a covalent complex with Western Blots, investigated AAT's inhibitory potential against Spls and examined the NETosis inhibitory activity of AAT-Spl-digestions. SplD, E and F, but not A or B, cleave AAT in its RCL, resulting in the release of a peptide consisting of AAT's C-terminal 36 amino acids (C36). Synthetic C36, as well as AAT-SplD/E/F-digestions exhibit NETosis inhibition. Only SplE, but not D or F, was partly inhibited by AAT, forming a covalent complex. We unraveled a new virulence trait of S. aureus, where SplD/E/F cleave and inactivate AAT while the cleavage product C36 inhibits NETosis.

Analysis and Prediction of Chymotrypsin Substrate Preferences through Large Data Acquisition with Target-Free mRNA Display.

Oral delivery of peptide therapeutics is limited by degradation by gut proteases like chymotrypsin. Existing databases of peptidases are limited in size and do not enable systematic analyses of protease substrate preferences, especially for non-natural amino acids. Thus, stability optimization of hit compounds is time and resource intensive. To accelerate the stability optimization of peptide ligands, we generated large datasets of chymotrypsin-resistant peptides via mRNA display to create a predictive model for chymotrypsin-resistant sequences. Through analysis of enriched motifs, we recapitulate known chymotrypsin cleavage sites, reveal positionally dependent effects of monomers on peptide cleavage, and report previously unidentified protective and destabilizing residues. We then developed a machine-learning-based modelpredicting peptide resistance to chymotrypsin cleavage and validated both model performance and the NGS experimental data by measuring chymotrypsin half-lives for a subset of peptides. Finally, we simulated stability predictions on non-natural amino acids through a leucine hold-out model and observed robust performance. Overall, we demonstrate the utility of mRNA display as a tool for big data generation and show that pairing mRNA display with machine learning yields valuable predictions for chymotrypsin cleavage. Expansion of this workflow to additional proteases could provide complementary predictive models that focus future peptide drug discovery efforts.

Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1’ position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities.

Distinct phenotypes of salivaricin-producing Ligilactobacillus salivarius isolated from the gastrointestinal tract of broiler chickens and laying hens

Ligilactobacillus salivarius harbors bacteriocin genes in its repA-type megaplasmid, specifically salivaricin P (salP), a class IIb bacteriocin. This study aimed to differentiate 25 salP-positive Lig. salivarius strains isolated from the gastrointestinal tract (GIT) of broilers and laying hens. Results showed that 12 isolates were classified as Type A, with active bacteriocins, while the rest were Type B, with no active bacteriocins. In vitro and in silico characterization of salP bacteriocins revealed narrow-spectrum antibacterial activity against Listeria monocytogenes and Enterococcus faecalis. SalP bacteriocins were predicted as positively charged, hydrophobic, small molecular weight (α, 4.097 kDa; ß, 4.285 kDa) bacteriocins with characteristic GXXXG motif. Investigation of the salP gene cluster based on genomic data revealed that Type B strains lacked the lanT and hlyD genes that encode export proteins dedicated to the modification and extracellular transport of mature salP peptides. However, two Type B strains (B4311 and B5258) showed inhibitory activity against L. monocytogenes ATCC19114. Multiplex PCR analysis and synteny mapping analysis revealed that B4311 and B5258 strains harbored the lanT gene, highlighting the importance of LanT protein in the cleavage of leader peptide and excretion of mature peptides. Further analysis revealed that the resistance of Type B strains to salP was attributable to the presence of a dedicated immunity protein, blurring the evolutionary significance of producing active bacteriocins for competitive advantage. Additionally, the loss of export proteins occurred in a polyphyletic manner, consistent with the genetic plasticity of the repA-type megaplasmid. This suggests that the loss of lanT and hlyD is likely in the presence of limited nutritional competitors. In conclusion, the observed differences in salivaricin production of Lig. salivarius exist independent of isolation host and that Type A and Type B strains can coexist in the same environment. Finally, the functional characterization of active salP allows for a better understanding of its potential to control specific bacteria in human food and animal production.

Perspectives for using serratiopeptidase in systemic enzyme therapy for low-intensity chronic inflammation and pain syndromes: from mechanisms of action to practical implementation (literature review)

Background. Recent experimental and clinical stu­dies have confirmed the effectiveness and safety of serratiopeptidase (SRP) as a powerful anti-inflammatory agent, highlighting its potential benefits across various fields of medicine. The ­purpose was to analyze current literature on the mechanisms of action of SRP as a means of systemic enzyme therapy for low-intensity chronic inflammation and pain syndromes, its clinical applications, and prospects for implementation in general medical practice. ­Materials and methods. To identify relevant literature sources, a comprehensive search was conducted in electronic databases, inclu­ding PubMed, Scopus, Web of Science, and the Cochrane Library. ­Results. According to modern literature data, SRP demonstrates quite powerful anti-inflammatory, analgesic, reparative, fibrinoly­tic, and mucolytic properties, and exhibits a certain antimicrobial activity, especially against biofilm-forming bacteria. The combination of this enzyme with traditional antibiotics provides a more effective treatment of infectious processes. SRP has significant potential in the treatment of conditions and diseases associated with the development of low-intensity chronic inflammation and pain syndromes (especially in comorbid ones) due to its anti-inflammatory, anti-edematous, antithrombotic, and analgesic properties associated with the inhibition of cyclooxygenase 1 and 2, 5-lipoxygenase activity, myeloperoxidase and elastase, suppression of the formation and/or release of bradykinin, biogenic amines, pro-inflammatory cytokines, cell adhesion molecules, cleavage of bradykinin-related peptides, limitation of oxidative-nitrosative stress. The effectiveness of the enzyme notably increases when it is combined with some prebiotics and/or probiotics. Conclusions. The development of new dosage forms of SRP, along with further preclinical and clinical trials, could lead to new strategies for the prevention and treatment of inflamatory diseases.

Artificial Zymogen Based on Protein-Polymer Hybrids.

This study explores the synthesis and application of artificial zymogens using protein-polymer hybrids to mimic the controlled enzyme activation observed in natural zymogens. Pro-trypsin (pro-TR) and pro-chymotrypsin (pro-CT) hybrids were engineered by modifying the surfaces of trypsin (TR) and chymotrypsin (CT) with cleavable peptide inhibitors utilizing surface-initiated atom transfer radical polymerization. These hybrids exhibited 70 and 90% reductions in catalytic efficiency for pro-TR and pro-CT, respectively, due to the inhibitory effect of the grafted peptide inhibitors. The activation of pro-TR by CT and pro-CT by TR resulted in 1.5- and 2.5-fold increases in enzymatic activity, respectively. Furthermore, the activated hybrids triggered an enzyme activation cascade, enabling amplification of activity through a dual pro-protease hybrid system. This study highlights the potential of artificial zymogens for therapeutic interventions and biodetection platforms by harnessing enzyme activation cascades for precise control of catalytic activity.

Exo-Cleavable Linkers: Enhanced Stability and Therapeutic Efficacy in Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) combine cytotoxic payloads with monoclonal antibodies through chemical linkers. Finding linkers that both enhance circulatory stability and enable effective tumor payload release remains a challenge. The conventional valine-citrulline (Val-Cit) linker is associated with several inherent drawbacks, including hydrophobicity-induced aggregation, a limited drug-antibody ratio (DAR), and premature payload release. This study introduces an exolinker approach, repositioning the cleavable peptide linker at the exo position of the p-aminobenzylcarbamate moiety, as an advancement over conventional linear linkers. This design, which incorporates hydrophilic glutamic acid, addresses the limitations of the Val-Cit platform and improves the ADC in vivo profiles. In vitro and in vivo evaluations showed that exolinker ADCs reduced premature payload release, increased drug-to-antibody ratios, and avoided significant aggregation, even with hydrophobic payloads. Furthermore, the payload remained stably attached to the ADC even in the presence of enzymes like carboxylesterases and human neutrophil elastase, indicating the potential for a favorable safety profile.

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis.

Comparison of Protocols to Test Peptide Stability in Blood Plasma and Cell Culture Supernatants.

Due to their high specificity, peptides are promising candidates in drug development, but fast degradation often limits their biological activity. Thus, a short half-life is one of the major challenges in the development of new peptide therapeutics. Moreover, the enzymatic cleavage of peptides can be a reason for misleading results in biological assays. Peptide stability assays typically consist of incubation, precipitation, and detection steps. However, the current methods differ greatly regarding these three steps, thus limiting the compatibility. Here, we systematically evaluate different parameters of peptide stability assays. First, we quantified and compared the analyte loss during the precipitation of plasma proteins. Especially, broadly used precipitation by strong acids was found to be unsuitable, while mixtures of organic solvents preserved more peptides for further analysis. Next, the stability of four fluorescently labeled model peptides was analyzed in blood plasma and two different cell culture supernatants. Strong variation in the degradation dynamics and patterns was found. Finally, we evaluated the role of fluorescent labeling on peptide stability and compared results to peptides with isotopic labels, underlining the individual advantages of both methods. Altogether, the data provide important parameters for analyzing and comparing the peptide stability.

Allelic Diversity and Development of Breeder-Friendly Marker Specific to floury2 Gene Regulating the Accumulation of α-Zeins and Essential Amino Acids in Maize Kernel.

Maize zeins lack essential amino acids, such as methionine, lysine, and tryptophan. The floury2 (fl2) mutation reduces zein synthesis and increases methionine and lysine content in kernels. In this study, fl2 gene (1612bp) was sequenced in eight wild-type and two mutant inbreds and detected 218 SNPs and 18 InDels. Transversion of C to T at 343bp position caused the substitution of alanine by valine in the fl2 mutant. A PCR-based marker (FL-SNP-CT) was developed, which distinguished the favorable mutant fl2 allele (T) from the wild-type (C) Fl2 allele. Gene-based diversity analysis using seven gene-based InDel markers grouped 48 inbred lines into three major clusters, with an average genetic dissimilarity coefficient of 0.534. The average major allele frequency, gene diversity, heterozygosity, and polymorphism information content of the InDel markers were 0.701, 0.392, 0.039, and 0.318, respectively. Haplotype analysis revealed 29 haplotypes of fl2 gene among these 48 inbreds. Amino acid substitution (Ala-Val) at the signal peptide cleavage site produced unprocessed 24-kDa mutant protein instead of 22-kDa zein found in normal genotype. Eight paralogues of fl2 detected in the study showed variation in exon lengths (616-1170bp) and translation lengths (135-267 amino acids). Orthologue analysis among 15 accessions of Sorghum bicolor and two accessions of Saccharum spontaneum revealed a single exon in fl2 gene, ranging from 267 to 810bp. The study elucidated the molecular basis of fl2 mutation and reported a breeder-friendly marker for molecular breeding programs. This is the first study to characterize fl2 gene in a set of subtropically adapted inbreds.

A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins

Key messageAnalysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins.Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1,144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. In addition, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria, and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins.Graphical abstract

Self-Tumor Antigens in Solid Tumors Turned into Vaccines by α-gal Micelle Immunotherapy.

A major reason for the failure of the immune system to detect tumor antigens (TAs) is the insufficient uptake, processing, and presentation of TAs by antigen-presenting cells (APCs). The immunogenicity of TAs in the individual patient can be markedly increased by the in situ targeting of tumor cells for robust uptake by APCs, without the need to identify and characterize the TAs. This is feasible by the intra-tumoral injection of α-gal micelles comprised of glycolipids presenting the carbohydrate-antigen "α-gal epitope" (Galα1-3Galβ1-4GlcNAc-R). Humans produce a natural antibody called "anti-Gal" (constituting ~1% of immunoglobulins), which binds to α-gal epitopes. Tumor-injected α-gal micelles spontaneously insert into tumor cell membranes, so that multiple α-gal epitopes are presented on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in the killing of tumor cells, and the recruitment of multiple APCs (dendritic cells and macrophages) into treated tumors by the chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into a personalized TA vaccine, the recruited APC phagocytose anti-Gal opsonized tumor cells and cell membranes, process the internalized TAs and transport them to regional lymph-nodes. TA peptides presented on APCs activate TA-specific T cells to proliferate and destroy the metastatic tumor cells presenting the TAs. Studies in anti-Gal-producing mice demonstrated the induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic α-gal micelles into primary tumors. This treatment was further found to synergize with checkpoint inhibitor therapy by the anti-PD1 antibody. Phase-1 clinical trials indicated that α-gal micelle immunotherapy is safe and can induce the infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that, in addition to converting treated metastases into an autologous TA vaccine, this treatment should be considered as a neoadjuvant therapy, administering α-gal micelles into primary tumors immediately following their detection. Such an immunotherapy will convert tumors into a personalized anti-TA vaccine for the period prior to their resection.

Determinants of maturation of the Staphylococcus aureus autoinducing peptide.

The accessory gene regulatory (Agr) system is required for virulence factor gene expression and pathogenesis of Staphylococcus aureus. The Agr system is activated in response to the accumulation of a cyclic autoinducing peptide (AIP), which is matured and secreted by the bacterium. The precursor of AIP, AgrD, consists of the AIP flanked by an N-terminal [Formula: see text]-helical Leader and a charged C-terminal tail. AgrD is matured to AIP by the action of two proteases, AgrB and MroQ. AgrB cleaves the C-terminal tail and promotes the formation of a thiolactone ring, whereas MroQ cleaves the N-terminal Leader in a manner that depends on the four-amino acid linker immediately following a conserved IG helix breaker motif. However, the attributes of AgrD that dictate the sequence of events in peptide maturation are not fully defined. Here, we used engineered AgrD peptide intermediates to ascertain the sufficiency of MroQ for N-terminal peptide cleavage, peptide export, and generation of mature AIP. We found that MroQ promotes the removal of the N-terminal Leader peptide from both linear and cyclic peptide intermediates, while peptide cyclization remained essential for signaling. The expression of the Leader peptide in isolation was sufficient for MroQ-dependent cleavage proximal to the four-amino-acid linker. In addition, active site mutations within AgrB destabilized full-length AgrD and thiolactone-containing intermediates and prevented the release of the Leader peptide. Altogether, our data support a tandem peptide maturation event involving both MroQ and AgrB that appears to couple protease activity and export of bioactive AIP.IMPORTANCEThe accessory gene regulatory (Agr) system is important for S. aureus pathogenesis. Activation of the Agr system requires recognition of a cyclic peptide pheromone, which must be fully matured to exert its biological activity. The complete events in cyclic peptide maturation and export from the bacterial cell remain to be fully defined. We and others recently discovered that the membrane peptidase MroQ is required for pheromone maturation. This study builds off the identification of MroQ and considers the attributes of the pheromone pro-peptide that are required for MroQ-mediated processing as well as uncovers features important for peptide stability and export. Overall, the findings in this study have implications for understanding bacterial pheromone maturation and virulence.

Preclinical Evaluation of STI-8811, a Novel Antibody-Drug Conjugate Targeting BCMA for the Treatment of Multiple Myeloma.

Treatment for patients with multiple myeloma has experienced rapid development and improvement in recent years; however, patients continue to experience relapse, and multiple myeloma remains largely incurable. B-cell maturation antigen (BCMA) has been widely recognized as a promising target for treatment of multiple myeloma due to its exclusive expression in B-cell linage cells and its critical role in the growth and survival of malignant plasma cells. Here, we introduce STI-8811, a BCMA-targeting antibody-drug conjugate (ADC) linked to an auristatin-derived duostatin payload via an enzymatically cleavable peptide linker, using our proprietary C-lock technology. STI-8811 exhibits target-specific binding activity and rapid internalization, leading to G2/M cell-cycle arrest, caspase 3/7 activation, and apoptosis in BCMA-expressing tumor cells in vitro. Soluble BCMA (sBCMA) is shed by multiple myeloma cells into the blood and increases with disease progression, competing for ADC binding and reducing its efficacy. We report enhanced cytotoxic activity in the presence of high levels of sBCMA compared with a belantamab mafodotin biosimilar (J6M0-mcMMAF). STI-8811 demonstrated greater in vivo activity than J6M0-mcMMAF in solid and disseminated multiple myeloma models, including tumor models with low BCMA expression and/or in large solid tumors representing soft-tissue plasmacytomas. In cynomolgus monkeys, STI-8811 was well tolerated, with toxicities consistent with other BCMA-targeting ADCs with auristatin payloads in clinical studies. STI-8811 has the potential to outperform current clinical candidates with lower toxicity and higher activity under conditions found in patients with advanced disease. Significance: STI-8811 is a BCMA-targeting ADC carrying a potent auristatin derivative. We report unique binding properties which maintain potent cytotoxic activity under sBCMA-high conditions that hinder the clinical efficacy of current BCMA-targeting ADC candidates. Beyond disseminated models of multiple myeloma, we observed efficacy in solid tumor models of plasmacytomas with low and heterogenous BCMA expressions at a magnitude and duration of response exceeding that of clinical comparators.

Mass Spectrometry-Based Top-Down Proteomics in Nanomedicine: Proteoform-Specific Measurement of Protein Corona.

Conventional mass spectrometry (MS)-based bottom-up proteomics (BUP) analysis of the protein corona [i.e., an evolving layer of biomolecules, mostly proteins, formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids] enabled the nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails to pinpoint the specific proteoforms─distinct molecular variants of proteins in the protein corona. The proteoform-level information could potentially advance the prediction of the biological fate and pharmacokinetics of nanomedicines. Recognizing this limitation, this study pioneers a robust and reproducible MS-based top-down proteomics (TDP) technique for characterizing proteoforms in the protein corona. Our TDP approach has successfully identified about 900 proteoforms in the protein corona of polystyrene NPs, ranging from 2 to 70 kDa, revealing proteoforms of 48 protein biomarkers with combinations of post-translational modifications, signal peptide cleavages, and/or truncations─details that BUP could not fully discern. This advancement in MS-based TDP offers a more advanced approach to characterize NP protein coronas, deepening our understanding of NPs' biological identities. We, therefore, propose using both TDP and BUP strategies to obtain more comprehensive information about the protein corona, which, in turn, can further enhance the diagnostic and therapeutic efficacy of nanomedicine technologies.

Integrating 3,4-Dihydro-2H-1,4-oxazine into Peptides as a Modification: Silver Triflate-Catalyzed Cyclization of N-Propargyl N-Sulfonyl Amino Alcohols for SPPS Applications.

We present a methodology yielding 3,4-dihydro-2H-1,4-oxazine by cyclization of N-propargyl N-sulfonyl amino alcohols using silver triflate as a catalyst at ambient temperature. Additionally, we showcase the applicability of this methodology in solid phase peptide synthesis (SPPS) to introduce the oxazine heterocyclic ring into short peptides containing serine and threonine. Notably, Rink amide resin supported the on-resin formation of 3,4-dihydro-2H-1,4-oxazine, while 2-CTC resin facilitated the oxazine formation in a one-pot process involving peptide cleavage, deprotection, and subsequent C-O ring formation, thus offering a versatile method for the late-stage modification of peptides.

Design of Cyborg Proteins by Loop Region Replacement with Oligo(ethylene glycol): Exploring Suitable Mutations for Cyborg Protein Construction Using Machine Learning

Abstract We synthesized a “cyborg protein,” wherein a synthetic molecule partially substitutes the main peptide chain by linking two protein domains with a synthetic oligomer. Green fluorescent protein (GFP) served as the model for constructing the cyborg proteins. We prepared circularly permuted GFP (cpGFP) with new termini between β10 and β11, where the original N- and C-termini were linked by a cleavable peptide loop. The cyborg GFP was constructed from cpGFP by linking the β10 and β11 with oligo(ethylene glycol) using maleimide-cysteine couplings, followed by the enzymatic cleavage of the N- and C-termini linking loop by thrombin. With the help of machine learning, we were able to obtain the cpGFP mutants that significantly alter the fluorescence activity (53% increase) by thrombin treatment, which splits cpGFP into two fragments (fragmented-GFP), and by heat shock. When the cyborg GFP was constructed using this mutant, the fluorescence intensity increased by 13% after heat treatment, similar to cpGFP (33% increase), and the behavior was significantly different from that of the fragmented-GFP. This result suggests the possibility that the oligo(ethylene glycol) chain in the cyborg protein plays a similar role to the peptide in the main chain of the protein.

P109 AMINOPEPTIDASE A: A POTENTIAL NOVEL THERAPY FOR ANGIOTENSIN-DEPENDENT HYPERTENSION?

Background: We have shown that mice with a genetic deficiency in Aminopeptidase A (APA) have elevatedblood pressure. APA is an enzyme that removes a single aspartate residue from the amino-terminus of several peptides in the renin-angiotensin systems (RAS), including angiotensin (Ang) II. In this study, we investigated the impact of a recombinant APA on the cleavage of various RAS peptides and its effect on blood pressure. Methods: An in vitro assay was used to identify Ang peptides as substrates for APA. Ang peptides identified as APA substrates using this assay were then injected to anesthetized WT and APA-deficient mice to assess their pressor effect by measuring systolic blood pressure (SBP). Subsequently, murine recombinant (r)APA was used to evaluate its action on the acute SBP elevation elicited by Ang peptides that were identified to trigger pressor response. Results: In the in vitro assay, Ang II (22427±3353, n=12), Ang I (21883±1692, n=3), Ang1-7 (15833±3395,n=3), Ang1-9 (15773±3395, n=3) and Ang1-12 (13170±220, n=3), were identified as a substrate for rAPA as indicated by a strong aspartate-driven fluorescence formation. When these five Ang peptides were administered to WT mice, only Ang I, Ang II and Ang 1-12 caused a significant increase in SBP. This effect was exaggerated in APA deficient mice. The SBP increase elicited by Ang II in WT mice was markedly reduced by the pre-administration of rAPA (Fig). Conclusion: Aminopeptidase A can cleave the N-terminal aspartate from Ang II as well as from other RASpeptides that elicit (Ang 1-12, Ang I) and do not elicit (Ang 1-9, Ang 1-7) pressor response in mice. APA deficiency is associated with an exaggerated SBP response to Ang I, Ang II and Ang 1-12 while the recombinant enzyme mitigates the acute hypertensive effect. rAPA markedly reduced the increase in SBP caused by AngII infusion suggesting that it may be used to treat hypertension caused by excess of AngII.

Light-Triggered PROTAC Nanoassemblies for Photodynamic IDO Proteolysis in Cancer Immunotherapy.

While proteolysis-targeting chimeras (PROTACs) hold great potential for persistently reprogramming the immunosuppressive tumor microenvironment via targeted protein degradation, precisely activating them in tumor tissues and preventing uncontrolled proteolysis at off-target sites remain challenging. Herein, a light-triggered PROTAC nanoassembly (LPN) for photodynamic indoleamine 2,3-dioxygenase (IDO) proteolysis is reported. The LPN is derived from the self-assembly of prodrug conjugates, which comprise a PROTAC, cathepsin B-specific cleavable peptide linker, and photosensitizer, without any additional carrier materials. In colon tumor models, intravenously injected LPNs initially silence the activity of PROTACs and accumulate significantly in targeted tumor tissues due to an enhanced permeability and retention effect. Subsequently, the cancer biomarker cathepsin B begins to trigger the release of active PROTACs from the LPNs through enzymatic cleavage of the linkers. Upon light irradiation, tumor cells undergo immunogenic cell death induced by photodynamic therapy to promote the activation of effector T cells, while the continuous IDO degradation of PROTAC simultaneously blocks tryptophan metabolite-regulated regulatory-T-cell-mediated immunosuppression. Such LPN-mediated combinatorial photodynamic IDO proteolysis effectively inhibits tumor growth, metastasis, and recurrence. Collectively, this study presents a promising nanomedicine, designed to synergize PROTACs with other immunotherapeutic modalities, for more effective and safer cancer immunotherapy.