Native Peptide Research Articles

Exploring the Potential of Cyclic Peptidyl Antitumor Agents Derived from Natural Macrocyclic Peptide Phakellistatin 13.

The exploration of novel anticancer compounds based on natural cyclopeptides has emerged as a pivotal paradigm in the contemporary advancement of macrocyclic pharmaceuticals. Phakellistatin 13 is a cycloheptapeptide derived from the brown snubby sponge and exhibits remarkable antitumor activity. In this study, we have designed and synthesized a series of chiral cyclopeptides incorporating the rigid isoindolinone moiety at various sites within the natural cycloheptapeptide Phakellistatin 13, with the aim of investigating conformationally constrained cyclopeptides as potential antitumor agents. Cyclopeptide 3, comprising alternating l-/d-amino acid residues, exhibited promising antihepatocellular carcinoma effects. Detailed biological experiments have revealed that Phakellistatin 13 analogs effectively inhibit the proliferation of tumor cells and induce apoptosis and autophagy, while also causing cell cycle arrest through the modulation of the p53 and mitogen-activated protein kinase (MAPK) signaling pathway. This study not only provides valuable insights into chemical structural modifications but also contributes to a deeper understanding of the biological mechanisms underlying the development of natural cyclopeptide-based drugs.

Design and Discovery of New Collagen V-Derived FGF2-Blocking Natural Peptides Inhibiting Lung Squamous Cell Carcinoma In Vitro and In Vivo.

Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.

Natural zinc-rich oyster peptides ameliorate DSS-induced colitis via antioxidation, anti-inflammation, intestinal barrier repair, microbiota modulation, and SCFA accumulation

Natural oyster zinc-rich peptide (OZP) was extracted and purified. The higher zinc concentration in natural OZP may be closely related to the high zinc affinity of amino acids containing Lys, His, Cys, Val, Arg and Ser residues. FTIR analysis indicated that amino, amide, carboxyl and carbonyl groups may contribute to the binding sites for zinc and peptides. Administration of OZP ameliorated the symptoms of DSS-induced ulcerative colitis (UC) in mice, including weight loss, colonic damage, mucosal edema, goblet cell exhaustion and crypt disappearance. Natural OZP suppressed oxidative stress by significantly reducing the level of NO, MDA and MPO as well as increasing SOD activity. The anti-inflammatory effects of OZP were associated with the inhibition of TLR4/NF-κB pathways by diminishing NF-κB, TLR4, COX-2, iNOS, TNF-α, IL-6 and IFN-γ and elevating IL-10. By promoting mucus secretion, repairing goblet cells and enhancing the expression of tight junction proteins including occludin, claudin-1 and ZO-1, natural OZP maintained the intestinal barrier function. Furthermore, natural OZP regulated the microbial composition by remarkedly enhancing the beneficial microbial abundance like Faecalibaculum, Odoribacter and Anaerostipes, which in turn led to a significant increase in SCFA production. Collectively, natural OZP exerted the synergistic capacity to protect against DSS-induced UC by improving antioxidation and anti-inflammation, repairing intestinal barrier and modulating intestinal microbiota and SCFA.

Effects of alloferon and its analogues on reproduction and development of the Tenebrio molitor beetle

As the most numerous group of animals on Earth, insects are found in almost every ecosystem. Their useful role in the environment is priceless; however, for humans, their presence may be considered negative or even harmful. For years, people have been trying to control the number of pests by using synthetic insecticides, which eventually causes an increased level of resistance to applied compounds. The effects of synthetic insecticides have encouraged researchers to search for alternatives and thus develop safe compounds with high specificity. Using knowledge about the physiology of insects and the functionality of compounds of insect origin, a new class of bioinsecticides called peptidomimetics, which are appropriately modified insect analogues, was created. One promising compound that might be successfully modified is the thirteen amino acid peptide alloferon (HGVSGHGQHGVHG), which is obtained from the hemolymph of the blue blowfly Calliphora vicinia. Our research aimed to understand the physiological properties of alloferon and the activity of its peptidomimetics, which will provide the possibility of using alloferon or its analogues in the pharmaceutical industry, as a drug or adjuvant, or in agriculture as a bioinsecticide. We used alloferon and its three peptidomimetics, which are conjugates of the native peptide with three unsaturated fatty acids with various chain lengths: caprylic, myristic, and palmitic. We tested their effects on the morphology and activity of the reproductive system and the embryogenesis of the Tenebrio molitor beetle. We found that the tested compounds influenced the growth and maturation of ovaries and the expression level of the vitellogenin gene. The tested compounds also influenced the process of egg laying, embryogenesis, and offspring hatching, showing that alloferon might be a good peptide for the synthesis of effective bioinsecticides or biopharmaceuticals.

Trifluoroacetic Acid Mediated Additive-Free Late-Stage Native Peptide Cyclization to Form Disulfide Mimetics via Thioketalization with Ketones.

Peptide cyclization is often used to introduce conformational rigidity and to enhance the physiological stability of the peptide. This study presents a novel late-stage cyclization method for creating thioketal cyclic peptides from bis-cysteine peptides and drugs. Symmetrical cyclic ketones and acetone were found to react with bis-cysteine unprotected peptides efficiently to form thioketal linkages in trifluoroacetic acid (TFA) without any other additive. The attractive features of this method include high chemoselectivity, operational simplicity, and robustness. In addition, TFA as the reaction solvent can dissolve any unprotected peptide. As a showcase, the dimethyl thioketal versions of lanreotide and octreotide were prepared and evaluated, both of which showed much improved reductive stability and comparable activity.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

Assessment of the impact of whey protein hydrolysate on myofibrillar proteins in surimi during repeated freeze–thaw cycles: Quality enhancement and antifreeze potential

The quality of surimi, widely used in processed seafood, is compromised by freeze–thaw cycles, leading to protein denaturation and oxidative degradation. The objective of this study is to explore the effects of adding natural whey peptide hydrolysate (WPH) on the myofibrillar proteins of repeatedly freeze–thawed surimi. Results indicated surimi treated with 15% WPH exhibited only a 128% increase in surface hydrophobicity and a maximum peroxide value of 7.84 μg/kg, significantly lower than the control group. Additionally, salt-soluble protein content, emulsification activity, and stability decreased with the increase in freeze–thaw cycles. With a 15% WPH offering the most significant protective effect, evidenced by reductions of only 25.02%, 42.52% and 37.02% in salt-soluble protein content, emulsification activity, and stability, respectively. These outcomes demonstrate that WPH effectively reduces protein denaturation during repeated freeze–thaw processes. Future research should explore the molecular mechanisms underlying WPH's protective effects and evaluate their applicability in other food systems.

A Natural Bioactive Peptide from Pinctada fucata Pearls Can Be Used as a Potential Inhibitor of the Interaction between SARS-CoV-2 and ACE2 against COVID-19.

The frequent occurrence of viral infections poses a serious threat to human life. Identifying effective antiviral components is urgent. In China, pearls have been important traditional medicinal ingredients since ancient times, exhibiting various therapeutic properties, including detoxification properties. In this study, a peptide, KKCH, which acts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was derived from Pinctada fucata pearls. Molecular docking showed that it bound to the same pocket of the SARS-CoV-2 S protein and cell surface target angiotensin-converting enzyme II (ACE2). The function of KKCH was analyzed through surface plasmon resonance (SPR), Enzyme-Linked Immunosorbent Assays, immunofluorescence, and simulation methods using the SARS-CoV-2 pseudovirus and live virus. The results showed that KKCH had a good affinity for ACE2 (KD = 6.24 × 10-7 M) and could inhibit the binding of the S1 protein to ACE2 via competitive binding. As a natural peptide, KKCH inhibited the binding of the SARS-CoV-2 S1 protein to the surface of human BEAS-2B and HEK293T cells. Moreover, viral experiments confirmed the antiviral activity of KKCH against both the SARS-CoV-2 spike pseudovirus and SARS-CoV-2 live virus, with half-maximal inhibitory concentration (IC50) values of 398.1 μM and 462.4 μM, respectively. This study provides new insights and potential avenues for the prevention and treatment of SARS-CoV-2 infections.

Structural analysis of peptide identified from the 2KRR domain of the nucleolin protein with a c-Myc G4 structure using biophysical and biochemical methods.

For the first time, the c-Myc G4 structure is reported to be stabilized by binding of the peptide (derived from the 2KRR domain of the nucleolin protein called the Nu peptide) in the loop region of the G-quadruplex structure by stacking interactions. CD results showed the formation of parallel G4 structure in the presence of 100 mM Na+ or 100 mM K+ with the appearance of two isodichroic points at 229 nm, 254 nm and 252 nm in the presence of 100 mM Na+ or 100 mM K+, respectively. In addition, in UV thermal and CD melting studies, we observed drastic changes with an increase in the hyperchromicity at a DNA : peptide ratio of 1 : 50. On titrating the Nu peptide with c-Myc G4, we calculated the value of binding constant (K a) by plotting fluorescence intensity and DNA concentration as 0.1369 ± 0.008 μM and 0.1277 ± 0.073 μM in Na+ and K+, respectively, which confirms the strong association of Nu peptide with c-Myc G4. The Nu peptide showed preferential cytotoxicity against MDA-MB-231 cells with IC50 values of 5.020 μM and 5.501 μM after 72 and 96 hours. This approach suggests a novel strategy to target G4 structure using natural key peptide segments derived from G4 stabilizing protein.

Unlocking the Potential: PEGylation and Molecular Weight Reduction of Ionenes for Enhanced Antifungal Activity and Biocompatibility.

Numerous synthetic polymers, imitating natural antimicrobial peptides, have demonstrated potent antimicrobial activity, positioning them as potential candidates for new antimicrobial drugs. However, the high activity of these molecules often comes at the cost of elevated toxicity against eukaryotic organisms. In this study, a series of cationic ionenes with varying molecular weights to assess the influence of polymer chain length on ionene activity is investigated. To enhance polymer antimicrobial activity and limit toxicity a PEG side chain is introduced into the repeating unit. The resulting molecules consistently exhibited high activity against three model organisms: E. coli, S. aureus and C. albicans. The incorporation of side PEG chain improves antifungal properties and biocompatibility, regardless of molecular weight. The most important finding of this work is that the reduction of polymer molecular mass led to increased antifungal activity and reduced cytotoxicity against HMF and MRC-5 cell lines simultaneously. As a result, the best-performing molecules reported herein displayed minimal inhibitory concentrations (MIC) as low as 2 and 0.0625µgmL1 for C. albicans and C. tropicalis respectively, demonstrating exceptional selectivity. It is plausible that some of described herein molecules can serve as potential lead candidates for new antifungal drugs.

Novel antioxidant peptides from soybean protein by employing computational and experimental methods and their mechanisms of oxidative stress resistance

Antioxidant peptides have the potential to play a crucial role in mitigating and managing a variety range of diseases associated with oxidative stress. Soybean protein is an important source of antioxidant peptides. Here we employ computational and experimental methods to screen and identify five antioxidant peptides (Gly-Ala, Gly-Val-Pro-Tyr, Pro-His, Pro-Lys, and Ser-Gly-Pro) derived from soybean protein, and explore their potential targets and mechanisms of action. All five peptides demonstrated potent antioxidant activity, effectively scavenging superoxide radicals and protecting cells from H₂O₂-induced oxidative damage. These peptides exhibited multi-target effects on pathways implicated in oxidative stress, cardiovascular disease, and cancer. Notably, our results reveal that these peptides can stably interact with the key target nitric oxide synthase (NOS2) through Glu377, a critical residue of NOS2, thereby inhibiting its activity and potentially mitigating oxidative stress and inflammation. This study confirms the function of soybean peptide sequence that has not been previously disclosed, and provides a more specific theoretical basis for the development of natural antioxidant peptides.

Molecular docking and proteomics approaches for the identification of neuroprotective effects of IL15.5 peptide against oxidative stress-induced apoptosis in SH-SY5Y neurons

Amyloid-β (Aβ) aggregation is central to Alzheimer’s disease (AD), causing oxidative and synaptic damage. We developed IL15.5, a peptide-base inhibitor derived from IL15, a natural peptide from Siamese crocodile hemoglobin. Computational and molecular assays confirm IL15.5 effectively inhibits Aβ42 aggregation, evident in thioflavin T assay results. IL15.5 also demonstrates acetylcholinesterase inhibition and antioxidant activity. It protects SH-SY5Y cells from oxidative stress and apoptosis, influencing caspase 3/MAPK pathways. Proteomic analysis reveals the role of IL15.5 in oxidative stress mitigation, affecting proteins linked to DNA repair, inflammation, and cell migration, with a significant association with Ubiquitin C. These findings suggest the potential of IL15.5 as an anti-amyloidogenic agent for AD treatment, offering neuronal protection and highlighting its therapeutic promise.

Clickable tryptophan modification for late-stage diversification of native peptides.

As the least abundant residue in proteins, tryptophan widely exists in peptide drugs and bioactive natural products and contributes to drug-target interactions in multiple ways. We report here a clickable tryptophan modification for late-stage diversification of native peptides, via catalyst-free C2-sulfenylation with 8-quinoline thiosulfonate reagents in trifluoroacetic acid (TFA). A wide range of groups including trifluoromethylthio (SCF3), difluoromethylthio (SCF2H), (ethoxycarbonyl)difluoromethylthio (SCF2CO2Et), alkylthio, and arylthio were readily incorporated. The rapid reaction kinetics of Trp modification and full tolerance with other 19 proteinogenic amino acids, as well as the super dissolving capability of TFA, render this method suitable for all kinds of Trp-containing peptides without limitations from sequences, hydrophobicity, and aggregation propensity. The late-stage modification of 15 therapeutic peptides (1.0 to 7.6 kilodaltons) and the improved bioactivity and serum stability of SCF3- and SCF2H-modified melittin analogs illustrated the effectiveness of this method and its potential in pharmacokinetic property improvement.

Differential potassium channel inhibitory activities of a novel thermostable degradation peptide BmKcug1a-P1 from scorpion medicinal material and its N-terminal truncated/restored peptides

Thermally stable full-length scorpion toxin peptides and partially degraded peptides with complete disulfide bond pairing are valuable natural peptide resources in traditional Chinese scorpion medicinal material. However, their pharmacological activities are largely unknown. This study discovered BmKcug1a-P1, a novel N-terminal degraded peptide, in this medicinal material. BmKcug1a-P1 inhibited hKv1.2 and hKv1.3 potassium channels with IC50 values of 2.12 ± 0.27 μM and 1.54 ± 0.28 μM, respectively. To investigate the influence of N-terminal amino acid loss on the potassium channel inhibiting activities, three analogs (i.e., full-length BmKcug1a, BmKcug1a-P1-D2 and BmKcug1a-P1-D4) of BmKcug1a-P1 were prepared, and their potassium channel inhibiting activities on hKv1.3 channel were verified by whole-cell patch clamp technique. Interestingly, the potassium channel inhibiting activity of full-length BmKcug1a on the hKv1.3 channel was significantly improved compared to its N-terminal degraded form (BmKcug1a-P1), while the activities of two truncated analogs (i.e., BmKcug1a-P1-D2 and BmKcug1a-P1-D4) were similar to that of BmKcug1a-P1. Extensive alanine-scanning experiments identified the bonding interface (including two key functional residues, Asn30 and Arg34) of BmKcug1a-P1. Structural and functional dissection further elucidated whether N-terminal residues of the peptide are located at the bonding interface is important in determining whether the N-terminus significantly influences the potassium channel inhibiting activity of the peptide. Altogether, this research identified a novel N-terminal degraded active peptide, BmKcug1a-P1, from traditional Chinese scorpion medicinal material and elucidated how the N-terminus of peptides influences their potassium channel inhibiting activity, contributing to the functional identification and molecular truncation optimization of full-length and degraded peptides from traditional Chinese scorpion medicinal material Buthus martensii Karsch.

EThcD as a Unique Tool for the Top-Down De Novo Sequencing of Intact Natural Ranid Amphibian Peptides.

De novo sequencing of any novel peptide/protein is a difficult task. Full sequence coverage, isomeric amino acid residues, inter- and intramolecular S-S bonds, and numerous other post-translational modifications make the investigators employ various chemical modifications, providing a variety of specific fragmentation MSn patterns. The chemical processes are time-consuming, and their yields never reach 100%, while the subsequent purification often leads to the loss of minor components of the initial peptide mixture. Here, we present the advantages of the EThcD method that enables establishing the full sequence of natural intact peptides of ranid frogs in de novo top-down mode without any chemical modifications. The method provides complete sequence coverage, including the cyclic disulfide section, and reliable identification of isomeric leucine/isoleucine residues. The proposed approach demonstrated its efficiency in the analysis of peptidomes of ranid frogs from several populations of Rana arvalis, Rana temporaria, and Pelophylax esculentus complexes.

The transcriptomic landscape of Botrytis cinerea infection on postharvest grapes sheds light on the biological function of the Bcnrps1 gene

Botrytis cinerea, a prevalent necrotrophic fungal pathogen, causes significant postharvest infections including fruits. This pathogen exploits senescent tissues, resulting in significant economic losses. During the Botrytis-plant interaction, a molecular cross-talk is established between the host and the pathogen, mediated by the interplay of the plant's defense pathways and the phytopathogenic fungus's virulence pathways, which are modulated by abiotic and biotic factors.The Bcnrps1 genes is secondary metabolims gene that encodes a non-ribosomal peptide synthetase. This family enzyme is responsible for the synthesis of natural peptides with a wide range of biological activities. Although the function of the majority of these peptides remains elusive, they could evolve in plant-pathogen interaction during infection process. Deletion of the Bcnrps1 gene increases the virulence of the pathogen otherwise, a reduction in sensitivity to toxic compounds such as spermidine and the fungicide pyrimethanil is observed. A transcriptome analysis was performed in this study to comprehensively elucidate the behavior of this mutant and to assess the molecular dynamics both in Bcnrps1 mutant and wild-type strains during the infection process of harvested white grapes. The number of differentially expressed genes (DEGs) during infection differs significantly between the B. cinerea B05.10 strain and the ΔBcnrps1 strain. The results demonstrate that, during white grape infection, the mutant induces genes related to five main functional groups: detoxification, transcription factors, CAZymes, virulence factors, and secondary metabolism. These findings underscore the crucial role of the Bcnrps1 gene in mediating the fungal response to environmental stressors and plant-derived substances during plant-pathogen interactions, as supported by observed gene expression patterns.

Boosting the anti-tumor activity of natural killer cells by caripe 8 – A Carapichea ipecacuanha isolated cyclotide

Cyclotides are head-to-tail cyclized peptides with a unique cystine-knot motif. Their structure provides exceptional resistance against enzymatic, chemical, or thermal degradation compared to other peptides. Peptide-based therapeutics promise high specificity, selectivity and lower immunogenicity, making them safer alternatives to small molecules or large biologicals. Cyclotides were researched due to their anti-cancer properties by inducing apoptosis in tumor cells in the past, but the impact of cyclotides on cytotoxic immune cells was poorly studied. Natural Killer (NK) cells are cytotoxic innate lymphoid cells and play an important role in the defense against infected, stressed and transformed cells. NK cells do not need prior sensitization and act in an antigen independent manner, holding promising potential in the field of immunotherapy. To investigate the effect of immunomodulatory cyclotides on NK cells, we evaluated several peptide-enriched plant extracts on NK cell mediated cytotoxicity. We observed that the extract samples derived from Carapichea ipecacuanha (Brot.) L. Andersson augments the killing potential of mouse NK cells against different tumor targets in vitro. Subsequent isolation of cyclotides from C. ipecacuanha extracts led to the identification of a primary candidate that enhances cytotoxicity of both mouse and human NK cells. The augmented killing is facilitated by the increased degranulation capacity of NK cells. In addition, we noted a direct toxic effect of caripe 8 on tumor cells, suggesting a dual therapeutic potential in cancer treatment. This study offers novel insights how natural peptides can influence NK cell cytotoxicity. These pre-clinical findings hold significant promise for advancing current immunotherapeutic approaches.

Stable peptide-assembled nanozyme mimicking dual antifungal actions

Natural antimicrobial peptides (AMPs) and enzymes (AMEs) are promising non-antibiotic candidates against antimicrobial resistance but suffer from low efficiency and poor stability. Here, we develop peptide nanozymes which mimic the mode of action of AMPs and AMEs through de novo design and peptide assembly. Through modelling a minimal building block of IHIHICI is proposed by combining critical amino acids in AMPs and AMEs and hydrophobic isoleucine to conduct assembly. Experimental validations reveal that IHIHICI assemble into helical β-sheet nanotubes with acetate modulation and perform phospholipase C-like and peroxidase-like activities with Ni coordination, demonstrating high thermostability and resistance to enzymatic degradation. The assembled nanotubes demonstrate cascade antifungal actions including outer mannan docking, wall disruption, lipid peroxidation and subsequent ferroptotic death, synergistically killing >90% Candida albicans within 10 min on disinfection pad. These findings demonstrate an effective de novo design strategy for developing materials with multi-antimicrobial mode of actions.

Prevention of premature birth in female patients with bacterial vaginosis using a complex of natural antimicrobial peptides and cytokines

Introduction. The prevalence of bacterial vaginosis (BV) comprises 23–29 %, which in pregnant women is a known risk factor for premature birth (PB) that rates increases by 2.9-fold. BV treatment with antibiotics has no effect PB incidence, therefore stressing a need to search for alternative remedies.Aim: to evaluate the effectiveness of treatment, including antibacterial therapy and a complex preparation containing natural antimicrobial peptides and cytokines, to reduce the incidence of birth defects in pregnant women with BV.Materials and Methods. Design: a prospective open comparative cohort study in parallel groups was conducted with 101 pregnant women: Group I (n = 69) received the antibiotic Metronidazole, 500 mg tablets orally twice a day for 7 days, and a complex preparation containing exogenous natural antimicrobial peptides and cytokines (Superlymph®) suppositories per 25 IU once a day vaginally in the evening for 20 days; Group II (n = 32) received Metronidazole alone (the same regimen). Patient examination was carried out using approaches included clinical methods, accepted in obstetrics, and laboratory tests – microscopy of vaginal content smears, real-time polymerase chain reaction (PCR).Results. The PB (within 240–366 weeks) incidence in Group I was significantly lower than in Group II and comprised 2.9 % vs. 21.9 %, respectively (p = 0.004), with an 8-fold decline in developing PB risk (relative risk (RR) = 0.13; 95 % confidence interval (CI) = 0.03–0.60), whereas inter-group percentage of pregnant women with high risk (PB history) was comparable (p = 0.39) so that PB incidence did not differ from pregnant women without former PB. Microbiological recovery after treatment for BV was achieved in 85.5 % of patients from Group I vs. 56.3 % in Group II (RR = 1.52; 95 % CI = 1.10–2.10; p = 0.002) based on real-time PCR data. The persistence of anaerobic flora after treatment was significantly lower in Group I vs. Group II reaching 7.2 and 34.4 % (p &lt; 0.001), respectively, with a 5-fold lower PB risk (RR = 0.21; 95 % CI = 0.08–0.56). Cessation of viral shedding compared to the number of patients with initial viral shedding was achieved in 94.6 % vs. 8.3 % of patients, respectively, with a 50-fold decline in risk (RR = 0.02; 95 % CI = 0.005–0.08; p &lt; 0.001). The number of newborns weighing less than 2500 g was significantly lower from paired mothers who received Superlymph® + Metronidazole comprising 2.9 % vs. 15.6 % treated with Metronidazole alone (p = 0.03), whereas a risk of low birth weight neonates was decreased by 6-fold (RR = 0.16; 95 % CI = 0.03–0.88). The condition of the neonates assessed by birth Apgar score was comparable.Conclusion. The use of a complex preparation Superlymph® (suppositories per 25 IU once an day vaginally, for 20 days) along with oral antibiotic Мetronidazole in pregnant women with BV facilitates a decline in PB incidence down to 2.9 % at gestational age of 240–366 weeks lowering a risk of PB exceeding that of antibacterial therapy by 8-fold, including patients with former PB.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.