Protein turnover and extracellular proteolysis continuously generate diverse peptide fragments within biological systems, yet the metabolic and pharmacological implications of these peptides remain incompletely understood. Among these transporters, members of the solute carrier family 15 (SLC15), including peptide transporter 1 (PEPT1/SLC15A1) and peptide transporter 2 (PEPT2/SLC15A2), mediate the proton-coupled uptake of dipeptides, tripeptides, and structurally related compounds across cellular membranes. While these transporters have been extensively studied in the context of intestinal peptide absorption and drug delivery, their potential roles in cancer biology remain incompletely understood. Tumor microenvironments are characterized by extensive proteolysis and dynamic metabolic remodeling, processes that can generate diverse peptide fragments derived from extracellular matrix proteins and intracellular protein turnover. These peptides may accumulate locally and potentially serve as substrates for cellular peptide transport systems. Once internalized through peptide transporters, dipeptides are typically hydrolyzed into free amino acids that can support biosynthetic pathways, energy metabolism, and cellular growth. In addition to their potential metabolic roles, certain endogenous dipeptides have also been reported to influence cellular signaling pathways and redox homeostasis. The broad substrate specificity of peptide transporters has also attracted significant interest in pharmacology because numerous clinically used drugs exploit these transport systems for efficient cellular uptake. This property raises the possibility that peptide transporters may be utilized for transporter-mediated drug delivery strategies, including the development of peptide-modified prodrugs or dipeptide-drug conjugates. In this review, we summarize the molecular characteristics and physiological functions of dipeptide transport systems with a particular focus on the SLC15 transporter family. We then discuss emerging evidence linking peptide transporters to tumor metabolism and the tumor microenvironment. Finally, we highlight current progress and future perspectives in exploiting peptide transport systems for transporter-mediated drug delivery and therapeutic targeting in cancer.
Advances in mass spectrometry-based metabolomics have enabled the detection of numerous small molecules in biological systems, revealing complex metabolic alterations associated with cancer. Among these, dipeptides are consistently detected in plasma, serum, and tumor tissue metabolomic profiles, yet their biological significance is not fully understood. In most studies, circulating dipeptides are interpreted as nonspecific byproducts of protein degradation generated during increased proteolysis. However, accumulating evidence suggests that at least some endogenous dipeptides may have biological activities, including antioxidant effects, metabolic modulation, and potential signaling functions. In this review, we examine the possible origins, transport mechanisms, and biological implications of circulating dipeptides in cancer metabolomics. We discuss multiple sources of dipeptide generation, including intracellular proteolysis, autophagy, extracellular matrix remodeling, tumor cell death, host tissue catabolism, and microbiome metabolism. We also summarize current knowledge regarding peptide transport systems and intracellular dipeptide metabolism that may regulate the fate of these molecules within mammalian systems. In addition, evidence supporting the biological activities of certain endogenous dipeptides is reviewed to evaluate the possibility that some circulating dipeptides may function as bioactive metabolites. Finally, we propose conceptual frameworks for interpreting circulating dipeptides in cancer, including their potential roles as indicators of protein turnover, intermediates in amino acid recycling, stress-buffering molecules, metabolic signals, or components of tumor-host metabolic communication. A better understanding of circulating dipeptides may provide new insights into cancer metabolism and reveal previously overlooked metabolite classes with potential biomarker or functional significance.
Small, nonpolar cyclic peptides can both bind challenging targets and cross cell membranes, making them attractive for addressing currently undruggable targets such as many protein-protein interactions (PPIs). However, developing such compounds de novo without prior information about lead structures such as natural ligands or fragments remains a notable challenge. Here we show that functional screening of structurally highly diverse cyclic peptide libraries synthesized at nanomole scale allows identification of sub-kDa inhibitors of a PPI. By screening 15,360 fully random cyclic peptides, we were able to identify an inhibitor of the E3 ligase adaptor Keap1 and its substrate Nrf2. Optimization by rapid design-build-test cycles produced a membrane-permeable compound active in live cells. This study demonstrates that large, diverse cyclic peptide libraries can enable the discovery of cell-permeable PPI inhibitors from the ground up, providing a way to harness the powerful modality of small cyclic peptides to address often difficult-to-target intracellular interactions.
Worldwide, female reproductive organ cancers account for more than 1.5 million new cases and 700,000 deaths each year. Of these, ovarian cancer (OC) is the most fatal. Regrettably, OC is typically diagnosed at a late stage, after metastasis occur, leading to a 5-year survival rate of only ∼30%. Current treatment options of advanced OC include chemotherapy combined with cytoreduction surgery, however, more than 80% of patients that responded to initial treatment experience relapse within 18 months. A dire need exists therefore for new therapy options that could replace, or be used in combination with, current therapies. Here, we report on the development of a peptide that selectively kills OC cells but is non-toxic to healthy cells in vitro and in vivo in a human OC xenograft mice system. The peptide (3D-NAF-144-67-6K) is derived from the human protein CISD2/NAF-1. It permeates the plasma membrane of OC cells, without affecting healthy cells, and targets their mitochondria leading to selective cancer cell death. In vivo studies using mice carrying xenograft tumours of human SKOV-3 cells showed that the peptide significantly reduces the overall size and growth rate of both primary and metastatic OC tumours. Our study suggests that 3D-NAF-144-67-6K could be used alone, or in combination with existing therapies, to treat OC and improve patient survival. We further show that 3D-NAF-144-67-6K has a broad-spectrum anticancer activity and can target brain and pancreatic cancer cells that are also unmet cancers with a high death rate.
Protein-based gels are central structural elements in fermented foods, but their formation during lactic acid bacteria (LAB) fermentation cannot be adequately explained by acidification alone. Although pH reduction and isoelectric aggregation initiate gelation in many systems, the final network architecture and functionality are also governed by exopolysaccharide (EPS) production, proteolysis, ionic interactions and the initial colloidal state of the protein matrix. This review reinterprets LAB fermentation-driven protein gelation using a strain-metabolite-protein colloidal state-gel functionality framework. Within this framework, strain-specific traits determine acidification kinetics, EPS yield and structure, proteolytic activity and ionic microenvironment; these factors collectively modulate protein charge, conformational stability, hydrophobic exposure, peptide formation, ion bridging and protein-polysaccharide compatibility. Importantly, the same mechanism may produce opposite outcomes depending on the matrix: EPS can reinforce networks by bridging and pore filling but may also promote incompatibility or phase separation; controlled or limited proteolysis can expose reactive sites, generate crosslinkable peptides and enhance network formation, whereas excessive hydrolysis weakens network continuity; divalent ions can strengthen gels through bridging but may induce coarse aggregation when unbalanced. We further compare dairy, plant, meat and microbial protein systems to identify matrix-dependent control targets, including acidification rate, endpoint pH, EPS molecular features, degree of hydrolysis, ionic strength, fermentation temperature and fermentation duration. Finally, we highlight current knowledge gaps, particularly the lack of standardised quantitative reporting and predictive models linking microbial metabolism, colloidal transitions and gel functionality. This review provides a mechanistic basis for rational starter selection and process design in fermented protein gel systems.
Breast cancer (BC) exhibits substantial molecular heterogeneity, with HER2-Positive (HER2+) subtypes accounting for 15-20% of cases and associated with aggressive behavior. Current diagnostics rely on invasive biopsies and often fail to capture dynamic changes or enable longitudinal monitoring. Exosomes, nanoscale vesicles reflecting the molecular profiles of their parent cells, offer a promising liquid biopsy approach. Here, we present a peptide-based fluorescent probe platform for the sensitive detection of HER2-positive exosomes. HER2-binding peptides were rationally designed and optimized computationally, and their binding affinity was validated using molecular docking and isothermal titration calorimetry. Site-specific conjugation of C12 alkyl chains and fluorescent dyes enhanced interfacial localization and fluorescence output. The probes demonstrated preferential recognition of HER2-positive exosomes in cell-derived samples as well as in human plasma and urine. Multivariate analysis was applied to interpret the multiplexed fluorescence signals, illustrating the method's potential for distinguishing HER2-enriched exosomes from diverse biological backgrounds. This work establishes a modular and noninvasive sensing strategy for profiling exosomal membrane proteins.
The growing support for noncanonical amino acids in structure prediction tools such as AlphaFold3 has been largely facilitated by the Chemical Component Dictionary (CCD). However, the limited coverage of modified residues in CCD continues to restrict the application of these models to many biologically and therapeutically relevant peptides. To address this gap, we present HighRes_Builder, a computational method for efficient residue search and automated construction of noncanonical amino acids not currently archived in CCD. We demonstrate the utility of our approach by predicting structures for 3179 noncanonical residues beyond the CCD using AlphaFold3. AlphaFold3 achieved 100% acceptance for both the noncanonical residue monomers and their corresponding 'GGXGG' motifs (where X denotes the noncanonical residue). Of these, 72.44% of the predicted residue monomer structures concurrently satisfy all five geometric criteria (d_N_C1, d_Ck_Ccarb, d_Ccarb_O_mean, ang_Ck_Ccarb_O_mean, and ang_O1_Ccarb_O2). Furthermore, among the generated motif structures, 85.78% exhibited favorable ω values for the embedded noncanonical residues. Furthermore, by integrating HighRes_Builder with structure prediction systems, AlphaFold3 for linear peptides and HighFold3 for cyclic peptides, we successfully model the conformation of the linear peptide drug Relamorelin and the cyclic therapeutic peptides LUNA18 and JNJ-77242113 in complex with their target proteins, elucidating structural determinants of their mechanism of action. This work establishes a scalable and accurate framework for structure prediction of diverse nonstandard peptides, highlighting its potential to accelerate rational design of peptide-based therapeutics.
Lentils (Lens culinaris; family: Fabaceae) are increasingly recognized as functional legumes with potential benefits for gut health because they provide bioactive peptides, resistant starch, and polyphenol-rich fractions within a shared food matrix. However, most existing studies have focused on individual lentil-derived compounds, and their matrix-dependent complementary interactions during digestion and fermentation remain insufficiently resolved. This review synthesizes current evidence on lentil-derived peptides, resistant starch, and polyphenols, with particular emphasis on their matrix-dependent complementary relationships, digestion-dependent transformation, microbial co-metabolism, and implications for intestinal barrier function. During gastrointestinal digestion and colonic fermentation, lentil proteins, resistant starch, and phenolic compounds undergo sequential transformation, yielding bioactive peptides, fermentable substrates, short-chain fatty acids (SCFAs), and phenolic metabolites that may collectively influence microbial composition and metabolic activity. Emerging evidence suggests that these interconnected processes may support gut health through microbiota-host crosstalk by modulating tight junction-related markers, reducing intestinal permeability, and maintaining epithelial homeostasis. Mechanistically, these effects have been associated with SCFA-mediated G protein-coupled receptor (GPCR) signaling, suppression of TLR4-NF-κB/MAPK inflammatory cascades, and activation of Keap1-Nrf2 antioxidant defenses, thereby attenuating oxidative stress and pro-inflammatory responses. Current evidence is more consistent with matrix-dependent complementary or convergent actions than with demonstrated synergy. At present, phenolic-rich fractions provide clear pathway-level evidence, whereas fermentation-linked carbohydrate effects are more strongly supported by microbiota- and in vivo-associated outcomes, and protein- or peptide-related mechanisms remain comparatively underdefined. Nevertheless, the evidence base remains limited by the scarcity of integrated studies, well-controlled human intervention trials, and factorial experimental designs capable of distinguishing complementary, additive, and truly synergistic effects among lentil bioactives. This review therefore highlights the need to move from describing coexisting beneficial effects toward formally testing interaction effects within physiologically relevant lentil matrices.
Systemic amyloidosis is a chronic, devastating illness caused by the build-up of misfolded proteins, leading to abdominothoracic organ dysfunction. Currently approved treatment options focus on preventing further amyloid accumulation. As such, there is a clinically unmet need for therapeutics that can opsonize tissue-deposited amyloid for clearance by phagocytic cells. We have developed a class of polybasic peptides that bind conserved properties of amyloid and amyloid-associated hypersulfated heparan sulfate, with high specificity. We have generated a novel structural class of polybasic peptides (termed PxR peptides) based on a proline-rich repeat strategy that demonstrates potency against amyloid. Amyloid-reactivity and specificity were probed using amyloid-laden and control tissue sections from mice and humans. Amyloid binding to synthetic fibrils and human patient-derived amyloid extracts was quantified using immunosorbent assays. PxR peptide stability in mouse and human serum was quantified using a bioactivity assay. PxR peptides were predicted to form a linear face of positive charges, which can bind with high specificity and potency to synthetic fibrils and amyloid extracts, and are resistant to serum proteases. Polybasic PxR peptides offer additional resources as pan-amyloid binding peptides for effective delivery of bioactive and amyloid-clearing therapeutics to amyloid.
Skin injury disrupts the body's primary barrier against pathogens and environmental insults, often leading to infection, delayed healing, and pathological scarring. Therefore, the development of effective wound-healing therapies remains a major clinical priority. Amphibian-derived peptides have attracted growing interest as a diverse group of bioactive molecules for wound repair. In addition to their antimicrobial effects, increasing evidence suggests that these peptides can regulate multiple stages of healing by modulating inflammation, promoting keratinocyte and fibroblast migration and proliferation, enhancing angiogenesis, and supporting extracellular matrix remodeling. This review summarizes current knowledge on the discovery and distribution of amphibian-derived wound-healing peptides and discusses recent findings on their biological activities, mechanisms of action, and potential applications. Rather than simply cataloguing reported peptides, we also assess the strength of the current evidence and discuss major translational challenges in the field, including insufficient mechanistic validation for some candidates, limited use of clinically relevant wound models, peptide instability, toxicity, and barriers related to formulation and delivery. We also discuss strategies that may improve peptide developability, including rational structural modification, biomaterial-based delivery systems, and translation-oriented preclinical evaluation. Overall, amphibian-derived peptides are a valuable source of multifunctional wound-healing agents, but their clinical development will require stronger mechanistic evidence, standardized efficacy assessment, and better translational design.
Background/Objectives: BPC-157 (body protection compound 157) is a synthetic pentadecapeptide derived from a gastric protein fragment with reported cytoprotective and regenerative properties across multiple organ systems. Despite over three decades of preclinical research demonstrating consistent biological activity, its pharmaceutical development remains rudimentary, with no approved formulation, no validated dosing regimen, and no completed Phase II clinical trial. This review critically evaluates BPC-157 from a biopharmaceutical and drug development perspective, examining its physicochemical and pharmacokinetic properties, formulation challenges across routes of administration, the pharmacokinetic-pharmacodynamic disconnect that characterizes its preclinical profile, and the regulatory and translational barriers that currently preclude clinical advancement. Methods: A narrative review of the literature was conducted using PubMed/MEDLINE, Embase, and Cochrane Library from database inception to April 2026. Search terms included "BPC-157", "BPC157", "body protection compound 157", "pentadecapeptide", and "GEPPPGKPADDAGLV", each combined with "pharmacokinetics", "formulation", "biopharmaceutics", "drug delivery", "clinical trial", "toxicology", and "regulatory". Patent databases (Espacenet, Google Patents) and regulatory agency websites (FDA, EMA, WADA) were searched independently. Searches were supplemented by forward and backward citation tracking of key references. Articles were selected based on relevance to biopharmaceutical characterization, pharmacokinetics, formulation science, clinical evidence, and regulatory status; pharmacodynamic studies were included insofar as they inform translational development. Evidence was synthesized with emphasis on pharmaceutical characterization, formulation science, and translational feasibility; no formal quality assessment instrument was applied, consistent with the narrative review design. Results: BPC-157 exhibits unusual stability in gastric juice and demonstrates activity via oral, parenteral, and topical routes, yet its human pharmacokinetic profile remains critically undercharacterized despite a recently published formal preclinical ADME study in two species confirming a sub-30-min plasma half-life, linear dose-proportional kinetics, and intramuscular bioavailability of 14-51% depending on species. A plasma half-life of under 30 min-confirmed preclinically and in a preliminary two-subject human pilot-contrasts with prolonged biological effects lasting hours to days-a disconnect with significant implications for dosing strategy and formulation design. No pharmaceutical-grade formulation has been developed or validated. The peptide lacks bcs classification data, permeability characterization, and formal excipient compatibility studies. Available clinical data derive from fewer than 30 subjects across three uncontrolled pilot studies, none of which employed standardized pharmaceutical preparations. Conclusions: BPC-157 presents a compelling but pharmaceutically underdeveloped profile. The primary barrier to clinical translation is not the absence of biological activity, but the absence of fundamental pharmaceutical science: characterized formulations, validated pharmacokinetics, and a coherent drug development strategy. Addressing these biopharmaceutical gaps is a prerequisite for any meaningful clinical program.
The NLRP3 inflammasome responds to chemically and biologically diverse stimuli, yet growing evidence indicates that this apparent diversity converges on a limited set of structural and spatial licensing steps. Here, we argue that NLRP3 regulation is best understood through an assembly-centered framework rather than as another stimulus-centered catalog of activators. Cryo-electron microscopy (Cryo-EM), biochemical, and cell-biological studies support a model in which NLRP3 is maintained in inactive cage-like assemblies, undergoes nucleotide-dependent conformational rearrangements, engages NEK7, and nucleates ordered supramolecular complexes containing apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). We synthesize current evidence for structural licensing, interface-level restraint, subcellular trafficking, phase-separation-linked organization, and post-translational and proteostatic control of NLRP3 assembly. We then examine a less explored question with translational implications: whether peptide-scale regulators, particularly endogenous microproteins, may control defined assembly transitions. Available evidence supports the existence of synthetic peptides that inhibit inflammasome interfaces and of endogenous microproteins that intersect with inflammatory signaling. However, direct evidence that endogenous microproteins act as dedicated interface-mimic inhibitors of NLRP3 assembly remains lacking. This review integrates mature structural models of NLRP3 regulation with the emerging microprotein field while clearly distinguishing established mechanisms from plausible but unproven hypotheses. This perspective defines a mechanistically explicit agenda for future work, including rigorous validation of translated small open reading frames (smORFs), direct interaction mapping to defined NLRP3 surfaces, and quantitative testing of effects on assembly, signaling output, and cellular context.
As a distinctive and sustainable raw material for pharmaceutical excipients, microalgae have emerged as a significant source of biopolymer compounds with immense functional and medicinal value. The pharmacoeconomic importance of key biopolymer compounds isolated from microalgae, such as fucoidan, ulvan, laminarin, and paramylon, having mucoadhesive, bioactive, and controlled release properties that meet the standards of advanced drug delivery systems, has been discussed in this review. Due to the compact cultivation requirements of microalgae that fix significant quantities of carbon dioxide from the environment, biopolymer compounds play an important role in the development of ecologically sustainable production practices. Also critically evaluated in this investigation are the advantages and limitations of industrial-scale cultivation practices of microalgae, which include closed bioreactor cultivation and open-pond cultivation procedures, for biopolymer applications. This study also discusses industrial applications that currently hinder the application of biopolymer compounds of microalgae to the pharmaceutical industry. Moreover, the ability of such compounds to be applied in formulation science has been underscored by their multifaceted properties, from thickening, gelation, and film-forming to immunomodulatory and antioxidant properties. The increased adoption of safer and more ecofriendly alternatives by the pharmaceutical industry to replace synthetic excipients has been facilitated by such compounds' ability to either complement or replace them. In order to incorporate microalgae-derived excipients into future pharmaceutical formulations, research on such compounds has been underscored as needed.
Hyperpigmentation disorders remain challenging to treat because most available agents mainly act on downstream melanogenic enzymes rather than upstream regulatory mechanisms. In this study, we identified AOP-P1, an amphibian skin-derived nonapeptide, as a potent anti-melanogenic peptide with nanomolar activity and no detectable cytotoxicity. By integrating in vitro and in vivo functional assays with transcriptomic profiling, receptor-binding studies, genetic perturbation, and pathway validation, the natriuretic peptide receptor 2 (NPR2) was identified as a unrecognized upstream regulator of melanogenesis in previous and also a mechanistically relevant target of AOP-P1. We further showed that AOP-P1 suppresses melanin production by inhibiting the NPR2/cGMP/MITF signaling axis, thereby attenuating UVB-induced hyperpigmentation. Collectively, our findings define AOP-P1 as a naturally derived bioactive peptide with receptor-linked anti-melanogenic activity and establish an NPR2-centered molecular basis for peptide-mediated regulation of pigmentation. These results expand current understanding of melanogenesis control and support the value of natural peptides as bioactive molecular scaffolds for pigmentation-related research.
The global population is aging at an accelerating pace, and sarcopenia has emerged as a central challenge to elderly health. Food-derived bioactive peptides, as natural functional compounds, can interact significantly with the gut microbiota, thereby indirectly influencing muscle metabolism and function. This review systematically summarizes the pathological mechanisms of sarcopenia and its associated complications. Moreover, it reveals the complex interactions between food-derived bioactive peptides and the gut microbiome, and innovatively summarizes the multi-level mechanisms by which these peptides regulate the gut-muscle axis. Furthermore, we discuss current research limitations, including the limited translational potential of animal models, insufficient precision of detection techniques, and lack of clinical validation. Future research directions are proposed, including leveraging multi-omics and artificial intelligence approaches for peptide-microbiota-metabolite functional prediction, employing organoid and organ-on-a-chip platforms for mechanistic validation, and advancing systematic translation through high-quality clinical trials. This review aims to provide a comprehensive theoretical framework and offer direction for the application of food-derived bioactive peptides based on gut-muscle axis interventions.
Antimicrobial peptides (AMPs), as key targets for novel anti-infection therapy, have made their efficient and precise identification technology a critical breakthrough in biomedical research. Currently, the high cost of experimental validation severely restricts the development process of antimicrobial peptides, while computational biology methods demonstrate significant advantages due to their cost-effectiveness and efficiency. This review comprehensively examines antimicrobial peptide database resources, with a focus on analyzing the characteristics and application value of mainstream databases such as APD3, DRAMP, and DBAASP, and systematically summarizes the latest advances in feature encoding techniques in recent years. At the methodological level, we discuss in detail the innovative prediction methods that have emerged in the past five years (2020-2024): from traditional structural feature analysis and machine learning algorithms to cutting-edge deep learning and generative model technologies, providing an in-depth comparison of the predictive performance of various methods on different datasets. Finally, based on current research bottlenecks, we prospectively propose future development trends in antimicrobial peptide prediction research, providing direction for scientific research efforts in related fields.
With an aging population and the prevalence of oxidative damage-related ailments, the quest for effective antioxidant and anti-aging agents has emerged as a focal point of biomedical research. Within this domain, peptides have garnered considerable attention for their potential activities. Hence, the current study investigates the antioxidant and anti-aging properties of a scorpion venom-derived peptide M6. We demonstrated that this peptide decreases H2O2-dependent apoptosis by binding to the TNFR1 receptor, which activates the NF-κB signaling pathway, thereby enhancing the capacity of cells to mitigate oxidative damage induced by H2O2 treatment. Moreover, M6 prolonged the lifespan of C. elegans and enhanced their thermal stress resistance. Further studies demonstrated that M6 inhibits the IIS/PI3K/AKT signaling pathway and activates daf-16 and skn-1 (homologous to Nrf-2), thereby enhancing the antioxidant capacity of nematodes and alleviating oxidative damage. Furthermore, M6 also alleviated D-galactose-induced oxidative damage in mice by enhancing the activity of catalase and superoxide dismutase. We also showed that M6 treatment protects liver and kidney tissues from oxidative damage induced by D-galactose. These results highlight the beneficial antioxidant and anti-aging properties of the peptide M6, which appear to be a promising lead for addressing related diseases and mitigating the effects of aging.
Photodynamic therapy (PDT) is an innovative treatment option for cancer, but current approaches are limited by poor tumor selectivity and low uptake. Here, we introduce a novel concept for a targeted phototoxic peptide, in which a lysosomally activatable payload is delivered selectively into the cell by receptor-mediated endocytosis. For the phototoxic payload, 6-carboxytetramethylrhodamine (TMR) was attached to a cell-penetrating peptide (CPP), which is specifically activated after internalization in the endosome. The activity of the CPP was blocked by electrostatic interactions with a poly-glutamate sequence but could be restored through cleavage by the lysosomal protease cathepsin B, both in vitro and in cells. The unmasked CPP binds to the negatively charged lysosomal membrane and, upon irradiation, TMR generates reactive oxygen species (ROS) that disrupt the integrity of the membrane. This leads to a release of lysosomal contents into the cytosol, which subsequently induces cell death. To achieve targeted delivery, the activatable payload was additionally conjugated to chemerin-9, a high-affinity ligand for the chemokine-like receptor 1 (CMKLR1), a G protein-coupled receptor overexpressed in various cancers. Through this receptor-targeted approach, the peptide accumulates only in CMKLR1-expressing cells while the lysosomal activation completely prevented off-target toxicity. Notably, this strategy enables even a weak photosensitizer like TMR to achieve potent cytotoxicity through lysosomal targeting. Thus, this approach represents an advancement in the selectivity and efficacy of PDT and holds promise for the development of novel cancer therapies.
Measurements of the peptide epitope CA125 are crucial in ovarian cancer care. Despite its value as a tool in disease management, CA125 is an imperfect biomarker, with rates of false positive and false negative response that preclude its use as a screening tool in the general population. The monoclonal antibodies that perform capture and recognition in the CA125 test-OC125 and M11-were developed using complex targets as immunogens and recognize epitopes that have been located on mucin16 (MUC16) but otherwise remain undefined at the molecular level. We hypothesized that new antibodies recognizing MUC16 peptides of known sequence could enable the development of assay platforms that overcome the limitations of the current CA125 test. Here, we report the development and characterization of three sets of polyclonal antibodies that recognize known MUC16 peptides. Peptides that appear several times within MUC16's highly conserved tandem repeat region were used to immunize two sets of rabbits. Affinity-isolated antibodies were characterized by enzyme-linked immunosorbent assay (ELISA) and Western blot. All three peptides were successful antigens, as indicated by the ability of the resulting polyclonal antibodies to bind individually expressed proteins from the MUC16 tandem repeat region. In particular, the polyclonal antibodies raised against peptide 2 (ELGPYTLDRNSLYV) bound to all tandem repeats tested. Peptide 2 antibodies were able to detect intact MUC16 from ovarian cancer cells in a surface plasmon resonance (SPR) assay. In flow cytometry experiments, peptide 2 antibodies bind to MUC16-positive cells (OVCAR3) and do not bind to MUC16-negative cells (OVCAR8). The binding pattern of these polyclonal antibodies opens the possibility of developing new monoclonal antibodies recognizing known epitopes within the tandem repeat region. These reagents may eventually complement or replace the monoclonal antibodies used in the current clinical CA125 test.
Hydrophobic and aggregation-prone proteins still present obstacles for protein chemical synthesis and engineering. The chemical synthesis of Interleukin-15 (IL-15) is a formidable challenge due to inherent sequence hydrophobicity and severe peptide aggregation, which impedes downstream protein engineering and chemical biology studies. Here, we report the first total synthesis of IL-15 using a versatile solubilizing strategy (RST-2.0), which enables the preparation and ligation of aggregation-prone segments while being readily removable during folding. Remarkably, this strategy is fully compatible with glycopeptide synthesis, allowing for the synthesis of homogeneously N79-glycosylated IL-15. The effectiveness of RST-2.0 was further demonstrated through the efficient synthesis of wild-type and azide-labeled Interleukin-2 (IL-2) analogs. Moreover, the bioactivity of IL-15 and IL-2 analogs was validated by CTLL-2 proliferation assays and microscale thermophoresis (MST). This work provides a de novo synthesis approach to elucidate the role of N-glycosylation on IL-15-mediated immune regulation and lays the foundation for developing next-generation cancer immunotherapies based on synthetic IL-15 variants.