Peptide Cleavage Research Articles

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.

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.

SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.

Dynamic polyphenol nanoparticles boost cuproptosis-driven metalloimmunotherapy of breast cancer

Polyphenolic materials have been extensively explored for biomedical applications. However, most polymer-based polyphenols are synthesized by covalently grafting phenol groups on the backbone of polymers and more convenient synthetic strategy remains challenging. Herein, a general and robust strategy to synthesize polypeptide-based polyphenol via selective ortho-hydroxylation of poly(tyrosine) was developed to construct dynamic nanoparticles (GPCuD NPs) composed of Cu2+ and phenylboronic acid modified doxorubicin prodrug (DOX-PBA) via metal-phenol coordination interaction and pH-reversible phenylboronate ester bond, respectively. Furthermore, matrix metalloproteinase-2 (MMP-2)-cleavable GPLGLAG peptide was also included between the polyethylene glycol and polyphenol segments, endowing GPCuD NPs with tumor-specific accumulation and deep penetration through enzyme-triggered depegylation. Notably, Cu2+-chelated nanoparticles efficiently ameliorated the immunosuppressive tumor microenvironment via recruiting antitumor immune cells and repolarizing M2-type tumor-associated macrophages to M1 phenotype. The combination of cuproptosis-driven metalloimmunotherapy with DOX chemotherapy remarkably suppressed 4T1 orthotopic breast tumor growth by 81.9 % and established a long-term immune memory (effective memory T cells up to 30.8 %) to prevent lung metastasis. This study has demonstrated a generalizable polyphenol nanoplatform for tumor-targeted and cuproptosis-regulated combination cancer metalloimmunotherapy.

The associations between dysregulation of human blood metabolites and lung cancer risk: evidence from genetic data

BackgroundMetabolic dysregulation is recognized as a significant hallmark of cancer progression. Although numerous studies have linked specific metabolic pathways to cancer incidence, the causal relationship between blood metabolites and lung cancer risk remains unclear.MethodsGenomic data from 29,266 lung cancer patients and 56,450 control individuals from the Transdisciplinary Research in Cancer of the Lung and the International Lung Cancer Consortium (TRICL-ILCCO) were utilized, and findings were replicated using additional data from the FinnGen consortium. The analysis focused on the associations between 486 blood metabolites and the susceptibility to overall lung cancer and its three major clinical subtypes. Various Mendelian randomization methods, including inverse-variance weighting, weighted median estimation, and MR-Egger regression, were employed to ensure the robustness of our findings.ResultsA total of 19 blood metabolites were identified with significant associations with lung cancer risk. Specifically, oleate (OR per SD = 2.56, 95% CI: 1.51 to 4.36), 1-arachidonoylglyceropholine (OR = 1.79, 95% CI: 1.22 to 2.65), and arachidonate (OR = 1.67, 95% CI: 1.16 to 2.40) were associated with a higher risk of lung cancer. Conversely, 1-linoleoylglycerophosphoethanolamine (OR = 0.57, 95% CI: 0.40 to 0.82), ADpSGEGDFXAEGGGVR, a fibrinogen cleavage peptide (OR = 0.60, 95% CI: 0.47 to 0.77), and isovalerylcarnitine (OR = 0.62, 95% CI: 0.49 to 0.78) were associated with a lower risk of lung cancer. Notably, isoleucine (OR = 9.64, 95% CI: 2.55 to 36.38) was associated with a significantly higher risk of lung squamous cell cancer, while acetyl phosphate (OR = 0.11, 95% CI: 0.01 to 0.89) was associated with a significantly lower risk of small cell lung cancer.ConclusionThis study reveals the complex relationships between specific blood metabolites and lung cancer risk, highlighting their potential as biomarkers for lung cancer prevention, screening, and treatment. The findings not only deepen our understanding of the metabolic mechanisms of lung cancer but also provide new insights for future treatment strategies.

Scalable preparation of green C-terminal amidation peptide-synthesis TAGs and the optimized TAG-assisted liquid-phase synthesis of eptifibatide

Eptifibatide is a cycloheptapeptide with C-terminal amidation and head-to-tail disulfide bridging, which has been approved as an antithrombotic drug. Currently, both at the laboratory scale and industrial level, the synthesis of eptifibatide predominantly relies on solid-phase peptide synthesis (SPPS), which is not regarded as a green sustainable and cost-effective approach to prepare eptifibatide. Herein, we have investigated the strategy employing tag-assisted liquid-phase peptide synthesis (TAG-Assisted LPPS) technology that is scalable to multi-gram scale manufacturing for eptifibatide preparation. We successfully obtained soluble Rink-Amide TAGs by esterifying organophosphorus TAG-OH with Rink-Amide, thereby facilitating the transition from synthesizing C-terminal carboxyl peptides to C-terminal amidation peptides. The products obtained during the tag-assisted coupling and Fmoc deprotection of eptifibatide intermediates can be purified through precipitation processes, eliminating the need for complex chromatographic operations. Accordingly, we developed an optimized laboratory-scale process (300 mmol) for the preparation of TAG-Rink Amide TAG, which was subsequently upscaled successfully to a 50 mmol batch synthesis of eptifibatide. The subsequent work also explores the processes of peptide cleavage and disulfide bond oxidative cyclization in eptifibatide preparation.

HER2-targeted, enzyme-activated liposomes show superior in vivo efficacy in an ovarian cancer model

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.

Charge-solvated versus protonated salt forms of cyclodepsipeptide toxins in electrospray: Dissociation of alkali-cationized forms enables straightforward sequencing of cereulide.

Bacillus cereus is responsible for foodborne outbreaks worldwide. Among the produced toxins, cereulide induces nausea and vomiting after 30 min to 6h following the consumption of contaminated foods. Cereulide, a cyclodepsipeptide, is an ionophore selective to K+ in solution. In electrospray, the selectivity is reduced as [M + Li]+; [M + Na]+ and [M + NH4]+ can also be detected without adding corresponding salts. Two forms are possible for alkali-cationized ions: charge-solvated (CS) that exclusively dissociates by releasing a bare alkali ion and protonated salt (PS), yielding alkali product ions by covalent bond cleavages (CBC) promoted by mobile proton. Based on a modified peptide cleavage nomenclature, the PS product ion series (b, a, [b + H2O] and [b + CnH2nO] [n = 4, 5]) are produced by Na+/Li+/K+-cationized cereulide species that specifically open at ester linkages followed by proton mobilization promoting competitive ester CBC as evidenced under resonant collision activation. What is more, unlike the sodiated or lithiated cereulide, which regenerates little or no alkali cation, the potassiated forms lead to an abundant K+ regeneration. This occurs by splitting of (i) the potassiated CS forms with an appearance threshold close to that of the PS first fragment ion generation and (ii) eight to four potassiated residue product ions from the PS forms. Since from Na+/Li+-cationized cereulide, (i) the negligible Na+/Li+ regeneration results in a higher sensibility than that of potassiated forms that abundantly releasing K+, and (ii) a better sequence recovering, the use of Na+ (or Li+) should be more pertinent to sequence isocereulides and other cyclodepsipeptides.

A Polyplatin with Hands-Holding Near-Infrared-II Fluorophores and Prodrugs at a Precise Ratio for Tracking Drug Fate with Realtime Readout and Treatment Feedback.

The in vivo fate of chemotherapeutic drugs plays a vital role in understanding the therapeutic outcome, side effects, and the mechanism. However, the lack of imaging abilities of drugs, tedious labeling processes, and premature leakage of imaging agents result in loss of fidelity between the drugs and imaging signals. Herein, an amphiphilic polymer is created by copolymerization of a near-infrared-II (NIR-II) fluorophore tracer (T) and an anticancer Pt(IV) prodrug (D) of cisplatin in a hand-holding manner into one polymer chain for the first time. The obtained PolyplatinDT is capable of delivering the drugs and the fluorophores concomitantly at a precise D/T ratio, thereby resulting in tracking the platinum drugs and even readout of them in real-time via NIR-II imaging. PolyplatinDT can self-assemble into nanoparticles, referred to as NanoplatinDT. Furthermore, a caspase-3 cleavable peptide that serves as an apoptosis reporter is attached to NanoplatinDT, resulting in NanoplatinDTR that are capable of simultaneously tracking platinum drugs and evaluating the therapeutic efficacy. Overall, it is reported here the design of the first theranostic polymer with anticancer drugs, drug tracers, and drug efficacy reporters that can work in concert to provide insight into the drug fate and mechanism of action.