Molecular Weight Peptides Research Articles

Effects of different iron saturations on biochemical properties of bovine lactoferrin (BLF) and on the gastrointestinal digestion of simulated in vitro feline model

Lactoferrin (LF) is an important bioactive protein widely found in biological secretions with excellent health benefits. The study investigated biochemical properties of bovine lactoferrin (BLF) with different iron levels, and the digestive properties of BLF in vitro feline digestion model. It was designed with 5 levels of iron saturation BLF, including Apo-BLF (<5%), Native-BLF (15–20%), 50%-BLF, 75%-BLF, Holo-BLF (>85%). As the iron levels of BLF increased, the zeta potential increased, particle size decreased, thermal stability increased, and the protein system became more stable. Changes in iron levels had little impact on the secondary structure, but had a significant effect on the protein's tertiary structure. Electrophoresis and peptide distribution results showed that the Holo-BLF group had better anti-digestive effects, followed by the 75%-BLF group. Holo-BLF group had higher peptide molecular weights compared to other groups. Higher iron saturation levels improved BLF's resistance to digestion in the stomach. The free amino acids and free amino produced during intestinal digestion were greater than those produced during gastric digestion, with the Apo-BLF and Native-BLF groups releasing more than other groups. This study emphasizes the biochemical properties of BLF with different iron levels and provides the essential digestive behavior information of degradation patterns and changes in functional properties at gastric and intestinal digestion stage in vitro feline model. These insights would further enhance the understanding of the information of BLF at different iron levels, which is essential for the future application of BLF in pet food.

Preservative Properties of Antimicrobial Peptides of Germinated Seeds of Pisum sativum (Garden Pea) On Roselle Flowers (Hibiscus sabdarifa L.) Drink

Study’s Excerpt This research presents the use of antimicrobial peptides derived from Pisum sativum (pea) seeds as natural preservatives for extending the shelf life of local African beverages. It demonstrated that peptides, particularly from germinated peas on day four, exhibits strong antimicrobial activity against bacteria. Plant-derived peptides could replace synthetic preservatives in the food and beverage industry. Full Abstract The demand for natural and effective food preservatives has increased due to the growing concern over the safety and quality of processed beverages. Antimicrobial peptides have gained significant attention as natural alternatives to synthetic preservatives, offering potential benefits such as broad-spectrum activity and lower toxicity. Germinating seeds of Pisum sativum were harvested on days 2, 4, 6 and 8 and peptides were extracted and purified using Ammonium Sulphate Precipitation at 70% saturation, Dialysis, Gel filtration and the peaks were obtained. The molecular weight of peptides was determined using SDS-PAGE while Six (6) Bacteria and Four (4) Fungal species were isolated and characterized using standard procedures. The highest protein concentration was obtained with germinated peas day four (P4) with a value of 1.7866mg/mL, while the highest zone diameter of inhibition was recorded with peptide of germinated pea seeds day six fraction three (P6:3) with a value of 17.000±0.577mm against B. cereus. The least MIC value, expressed in protein concentration (mg/mL), was obtained as 0.0253mg/mL with 100% concentration of the peptide of germinated peas on day four (P4). All four (4) fungal species recorded no activity/zone diameter of inhibition. Sanitizing activity of the peptides on the ‘Zobo’ drink adjusted to pH 2.3, at 0hr, 24hr, 48hr and 72hrs was carried out, and all the purified peptides extended the shelf life of the drink except for peptide of germinated peas on day two. Sensory evaluation equally indicated that the drink was preserved with 10% (P4:4) at pH 2.3, being the most preferred treatment by the judges with a percentage likeness of 72.22% after 72hrs of storage. Findings from this study highlight the potential of these peptides in developing innovative and safe preservation techniques for extending the shelf life of local African beverages. This study further offers promising insights into the application of Pisum sativum peptides in preserving traditional drinks like 'Zobo.'

Skin Improvement Effects of Ultrasound-Enzyme-Treated Collagen Peptide Extracts from Flatfish (Paralichthys olivaceus) Skin in an In Vitro Model.

Collagen is considered to be an intercellular adhesive that prevents tissue stretching or damage. It is widely utilized in cosmetic skin solutions, drug delivery, vitreous substitutions, 3D cell cultures, and surgery. In this study, we report the development of a green technology for manufacturing collagen peptides from flatfish skin using ultrasound and enzymatic treatment and a subsequent assessment on skin functionality. First, flatfish skin was extracted using ultrasound in distilled water (DW) for 6 h at 80 °C. Molecular weight analysis via high-performance liquid chromatography (HPLC) after treatment with industrial enzymes (alcalase, papain, protamex, and flavourzyme) showed that the smallest molecular weight (3.56 kDa) was achieved by adding papain (0.5% for 2 h). To determine functionality based on peptide molecular weight, two fractions of 1100 Da and 468 Da were obtained through separation using Sephadex™ G-10. We evaluated the effects of these peptides on protection against oxidative stress in human keratinocytes (HaCaT) cells, inhibition of MMP-1 expression in human dermal fibroblast (HDF) cells, reduction in melanin content, and the inhibition of tyrosinase enzyme activity in murine melanoma (B16F10) cells. These results demonstrate that the isolated low-molecular-weight peptides exhibit superior skin anti-oxidant, anti-wrinkle, and whitening properties.

Functional Peptides from Yak Milk Casein: Biological Activities and Structural Characteristics.

The average content of casein in yak milk is 40.2 g/L. Casein can be degraded by enzymatic digestion or food processing to produce abundant degradation peptides. International researchers have studied the degradation peptides of yak milk casein by using multiple techniques and methods, such as in vitro activity tests, cellular experiments, proteomics, bioinformatics, etc., and found that the degradation peptides have a wide range of functional activities that are beneficial to the human body, such as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, anti-inflammatory, antidiabetic, antimicrobial, anticancer, and immunomodulatory activities, etc., and it has been proved that the types and strengths of functional activities are closely related to the structural characteristics of the peptides. This paper describes the characteristics of yak milk proteins, the functional activities, and mechanism of action of degraded peptides. Based on the types of functional activities of yak milk casein degradation peptides, we classified and elucidated the effects of structural factors, such as peptide molecular weight, peptide length, amino acid sequence, physicochemical properties, electrical charge, hydrophobicity, spatial conformation, chain length, and the type of enzyme on these activities. It reveals the great potential of yak milk casein degradation peptides as functional active peptide resources and as auxiliary treatments for diseases. It also provides important insights for analyzing yak casein degradation peptide activity and exploring high-value utilization.

Production of bioactive peptides during yogurt fermentation, their extraction and functional characterization

This study aimed to evaluate the antioxidant, antibacterial, antidiabetic and angiotensin-converting enzyme (ACE)-inhibitory effects of different fractions of bioactive peptides produced as a result of milk fermentation with yogurt starters and probiotics. In addition to yogurt starters, milk was fermented separately and together with Lacticaseibacillus rhamnosus GG and L. paracasei, then passed through 5 and 10 kDa (kilodalton) filters. A significant difference was observed in the fermentation time between the samples. The antioxidant, antibacterial, antidiabetic and ACE-inhibitory activities of the bioactive peptides were significantly affected by the probiotic strains and the molecular weight of the peptide (p < 0.05). ≤5 kDa fractions of Lactobacillus delbrueckii subsp. bulgaricus + Streptococcus thermophilus + L. rhamnosus GG + L. paracasei had the highest antioxidant activity. The addition of L. rhamnosus GG and L. paracasei to yogurt starter significantly increased ɑ-amylase activity, ɑ-glucosidase activity and ACE inhibitor activity. All samples showed antibacterial activity to Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium microorganisms and the molecular weight of peptides and sample types were significantly exhibited in antibacterial activity (p < 0.05). Cytotoxic activity showed that bioactive peptides had cytotoxic effect against Caco-2 adenocarcinoma cell and the presence of probiotics increased the cytotoxic effect. The results of the study suggest that the use of L. rhamnosus GG and L. paracasei, together with yogurt starters, could improve the bioactive properties of peptides formed in dairy products.

LC-MS/MS-based peptidomics and metabolomics reveal different metabolic patterns of common spoilage bacteria in grass carp flesh

Grass carp are highly perishable under the action of spoilage bacteria including Aeromonas rivipollensis, Pseudomonas putida, and Shewanella putrefaciens. However, the metabolic characteristics of the three bacteria in degrading fish proteins and small molecular nutrients are unclear. In this study, we used LC-MS/MS-based peptidomics and metabolomics to detect peptides and small molecular metabolites in grass carp flesh inoculated with the three bacteria. The peptidomic results showed that A. rivipollensis and S. putrefaciens induced an overall increase in the molecular weight of peptides in grass carp flesh. S. putrefaciens tended to hydrolyze peptide bonds composed of at least one hydrophobic amino acid whereas A. rivipollensis and P. putida preferred hydrolyzing Xaa-serine bonds. The metabolomic results showed that amino acid metabolism and nucleotide metabolism are among the most enriched pathways of differential metabolites. P. putida and S. putrefaciens were active in decomposing amino acids and nucleosides, respectively. Besides, lipid metabolism also plays an important role in grass carp spoilage, representing as the degradation from phosphatidylcholine to lysophosphatidylcholine by the three bacteria. To conclude, this study revealed distinct metabolic characteristics of the three bacteria in degrading proteins and small molecular metabolites, and thus contribute to the understanding of fish spoilage mechanisms.

Antioxidant polypeptides derived from pigeon pea (Cajanus cajan (L) Mill sp.) by enzymatic hydrolysis

Pigeon pea (Cajanus cajan (L) Mill sp.) seeds are rich sources of protein in the legume family and their consumption has been associated with the prevention of noncommunicated diseases, which is attributable to their content of bioactive components. Antioxidant protein hydrolysates were produced from pigeon pea protein isolate (PPI) by enzymatic hydrolysis using pancreatin and flavourzyme. The hydrolysates were analyzed for their physicochemical, molecular weight, amino acid composition, and in vitro antioxidant activities. The molecular weights of polypeptides in the hydrolysates were 8, 20, 25 and 48 kDa, which were determined after pancreatin or flavourzyme hydrolysis of the protein isolate for 4 h. Pancreatin-hydrolyzed pigeon pea protein (PPHP) contained high hydrophobic amino acids, especially isoleucine, leucine and valine, which were related to the high content of aromatic amino acids. The hydrolysates obtained from flavourzyme hydrolysis of pigeon pea proteins (PPHF) presented significantly higher capacities to scavenge ABTS˙+ and reduce Fe3 + better than that of PPI, while the PPI exhibited strong DPPH scavenging (98.4 mg trolox equivalent antioxidant capacity). The results indicated that the partial hydrolysis for PPI provided medium to high molecular weight of peptides. Therefore, PPHF could be a promising source of bioactive peptides and a potential ingredient for the formulation of functional foods against oxidative stress

Advances in Research on the Activity Evaluation, Mechanism and Structure-Activity Relationships of Natural Antioxidant Peptides.

Antioxidant peptides are a class of biologically active peptides with low molecular weights and stable antioxidant properties that are isolated from proteins. In this review, the progress in research on the activity evaluation, action mechanism, and structure-activity relationships of natural antioxidant peptides are summarized. The methods used to evaluate antioxidant activity are mainly classified into three categories: in vitro chemical, in vitro cellular, and in vivo animal methods. Also, the biological effects produced by these three methods are listed: the scavenging of free radicals, chelation of metal ions, inhibition of lipid peroxidation, inhibition of oxidative enzyme activities, and activation of antioxidant enzymes and non-enzymatic systems. The antioxidant effects of natural peptides primarily consist of the regulation of redox signaling pathways, which includes activation of the Nrf2 pathway and the inhibition of the NF-κB pathway. The structure-activity relationships of the antioxidant peptides are investigated, including the effects of peptide molecular weight, amino acid composition and sequence, and secondary structure on antioxidant activity. In addition, four computer-assisted methods (molecular docking, molecular dynamics simulation, quantum chemical calculations, and the determination of quantitative structure-activity relationships) for analyzing the structure-activity effects of natural peptides are summarized. Thus, this review lays a theoretical foundation for the development of new antioxidants, nutraceuticals, and cosmetics.

Complementarity of two proteomic data analysis tools in the identification of drug-metabolising enzymes and transporters in human liver.

Several software packages are available for the analysis of proteomic LC-MS/MS data, including commercial (e.g. Mascot/Progenesis LC-MS) and open access software (e.g. MaxQuant). In this study, Progenesis and MaxQuant were used to analyse the same data set from human liver microsomes (n = 23). Comparison focussed on the total number of peptides and proteins identified by the two packages. For the peptides exclusively identified by each software package, distribution of peptide length, hydrophobicity, molecular weight, isoelectric point and score were compared. Using standard cut-off peptide scores, we found an average of only 65% overlap in detected peptides, with surprisingly little consistency in the characteristics of peptides exclusively detected by each package. Generally, MaxQuant detected more peptides than Progenesis, and the additional peptides were longer and had relatively lower scores. Progenesis-specific peptides tended to be more hydrophilic and basic relative to peptides detected only by MaxQuant. At the protein level, we focussed on drug-metabolising enzymes (DMEs) and transporters, by comparing the number of unique peptides detected by the two packages for these specific proteins of interest, and their abundance. The abundance of DMEs and SLC transporters showed good correlation between the two software tools, but ABC showed less consistency. In conclusion, in order to maximise the use of MS datasets, we recommend processing with more than one software package. Together, Progenesis and MaxQuant provided excellent coverage, with a core of common peptides identified in a very robust way.

The targeted development of collagen-active peptides based on composite enzyme hydrolysis: a study on the structure-activity relationship.

Fish collagen, derived from sustainable sources, offers a valuable substrate for generating peptides with diverse biofunctionalities. In this study, alkaline, papain, and ginger protease were used to enzymatically hydrolyze fish skin collagen. The peptide molecular weight distribution and sequence were measured using HPLC and ICP-MS-MS, with papain/alkaline protease (AP) and papain/alkaline/ginger protease (APG) hydrolyzed samples compared. As the results showed, the incorporation of ginger protease was useful for increasing the degree of hydrolysis, with the content of <400 Da peptides increasing from 49.82% to 58.56%. The identified peptide sequence in the APG sample had more proline at the C-terminal. The peptides were separated into two components (different in molecular weight) using gel column chromatography. The molecular weight distribution, amino acid composition, ACE inhibitory activity, and fibroblast proliferation activity of the collected components were measured. In comparison, the contents of proline and hydroxyproline in the larger peptides decreased obviously after combined hydrolysis by ginger protease, reflecting the formation of a peptide sequence of smaller molecular weight containing glycine and hydroxyproline. The combined hydrolysis of ginger protease was beneficial for the improvement of the ACE inhibitory activity of the sample. However, the fibroblast proliferation activity of AP was higher than that of APG, indicating that further hydrolysis by ginger protease may destroy the hydroxyproline at the end of the peptide sequence. This study proposed a creative directional hydrolysis method and provided practical guidance for the production of collagen peptides with enhanced functional activity.

Skin Anti-Aging Efficacy of Enzyme-Treated Supercritical Caviar Extract: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Oxidative stress in the skin, induced by an unhealthy lifestyle and exposure to UVB radiation, leads to skin aging, including reduced elasticity, formation of wrinkles, moisture loss, and inflammation. In a previous study, we revealed the photoaging effects of enzyme-treated caviar extract (CV) by regulating collagen and hyaluronic acid synthase, melanogenesis, anti-oxidant mechanisms, and inflammation in a UVB irradiation-induced mice model. HPLC and MALDI-TOF were performed to determine the effect of enzyme treatment on the free amino acid contents and peptide molecular weight in supercritical caviar extract. As results of the analysis, CV is mainly composed of low-molecular-weight peptides consisting of leucine, tyrosine, and phenylalanine. Based on our in vitro and in vivo study, we conducted a clinical trial to assess the skin anti-aging efficacy of CV. In this randomized, double-blind, placebo-controlled trial, we measured indicators related to elasticity, wrinkles, and skin hydration at 4 and 8 weeks after consumption of CV. The subjects were categorized into caviar, combination, and placebo groups. After 4 weeks, skin hydration, dermal hydration, and transepidermal water loss all showed significant improvement. Furthermore, after 8 weeks, skin elasticity indexes-R2 (total elasticity), R5 (net elasticity), and R7 (ratio of elastic recovery to total deformation)-exhibited significant increases. Improvement in wrinkle indicators (Rmax, Ra, and Rz) and the whitening indicator melanin pigment was also observed. This is the first report showing that CV has significant skin anti-aging efficacy on human skin. In conclusion, our study suggests that CV can be used as skin anti-aging nutraceuticals through positive effects on skin condition in clinical trials.

Improving the utilization efficiency of nitrogen source through co-culture of Lactobacillus strains with different nitrogen source metabolisms

The growth of lactic acid bacteria depends largely on a sufficient source of nitrogen as well as a wide variety of nitrogen sources. Nutrient dependence, especially in relation to nitrogen sources, plays a key role in controlling the positive interactions between microorganisms. However, little is known regarding the relationship between co-culture and nitrogen source dependence in current studies. This study aimed to obtain a higher proliferation of strains with the same nitrogen source content by co-cultivation of Lactobacillus. First, upon the detection of various commercial nitrogen sources, notable differences in peptide molecular weight distributions were observed. In addition, diverse Lactobacillus strains exhibit varying proficiencies in utilizing these nitrogen sources. Second, in the exploration of co-cultivation of distinct bacterial strains, three synergistic combinations were identified. These assemblies exhibited superior total viable cell counts, growth trajectories, and glucose consumption. Finally, strains possessing capsular proteinase genes adeptly hydrolyzed large peptides, yielding smaller peptides and amino acids for strains lacking analogous enzymatic and peptide transport mechanisms. This culminated in optimized utilization of the nitrogen source and provided a method for the direct production of multi-strain probiotics.

Antioxidant and Antibacterial Activities of Curd and Whey Kefir Produced from Etawa Goat Milk

Kefir is a fermented drink with many benefits. Kefir contains antioxidant compounds that can inhibit free radical activity.In addition, kefir contains lactic acid bacteria, which provide good antibacterial activity and improve the health of the digestive tract. This research aimed to determine the antioxidant and antibacterial activities of kefir made from Etawa goat milk. The methods used in this research included fermentation of Etawa goat milk with 10 % kefir seeds at 37 °C and a fermentation time of 2 days. Antioxidant activity was tested using 1,1-diphenyl-2-picrylhydrazyl (DPPH), and antibacterial activity was tested using agar diffusion. The peptide molecular weights in kefir were analyzed using SDS-PAGE. The results showed that the antibacterial and antioxidant activities in Etawa goat's milk curd kefir has a higher activity than whey kefir. The antioxidant activity of curd has an IC50 of 43.99 ppm, which is a powerful antioxidant. Curd's antibacterial activity forms a clear zone of 7 mm against Bacillus cereus and is included in the solid antibacterial category. In Escherichia coli, a clear zone of 6 mm was formed and included in the moderate antibacterial category. The results of SDS-PAGE gel visualization found three prominent bands measuring 10, 15, and 33 kDa, respectively.

Plastein reaction augments the metal chelating capabilities of silver carp (Hypophthalmichthys molitrix) hydrolysates: Unlocking the chemical modification mechanism

Plastein reaction mechanisms and the alteration of its product properties have been studied for decades. This study investigated the plastein-mediated modifications in silver carp protein hydrolysate (SCPH) from both mechanistic and functional perspectives. Unlike prior research, this investigation uncovered that hydrogen bonding supplemented the dominant hydrophobic interactions in plastein's mechanism for the first time, as supported by peptide concentrations, molecular weight, amino acids, chemical forces, and peptide sequence by LC-MS/MS. This innovative reaction mechanism cascaded into the enhancement of SCPH functional attributes. Plastein induced increased COOH in SCPH’s side-chain groups significantly enhanced Fe2+ (from 4.49 to 14.12 %) and Zn2+ (from 53.53 to 64.47 %) chelation. Moreover, the elevated DPPH (17.56 %–23.97 %) and hydroxyl radical (68.49 %–79.32 %) scavenging power indicated a broader improvement in SCPH with plastein. In SCPH, plastein elucidated reaction intricacies and enhanced its utility, propelling SCPH into a realm of extended potential.

Impact of Wooden Breast myopathy on in vitro protein digestibility, metabolomic profile, and cell cytotoxicity of cooked chicken breast meat

This study investigated the impacts of Wooden Breast (WB) abnormality on in vitro protein digestibility and cytotoxicity of cooked chicken breast meat. Chicken breasts without (non-WB, n = 6) or with severe WB condition (WB, n = 6) were cooked and subjected to static in vitro protein digestion. The results showed no significant differences in free-NH2, degree of hydrolysis and distribution of peptide molecular weight between non-WB and WB samples at late intestinal digestion (P5), suggesting no adverse effects of WB on protein digestibility. Based on peptidomic analysis, P5 fraction of WB showed greater content of peptides with oxidative modification than that of non-WB. Untargeted metabolomics did not find any metabolites with potential toxicity either in non-WB and WB. Hydrolyzed non-WB and WB (1.56 to 100 µg/mL) did not affect viability of Caco-2 and Vero cells but addition of WB samples reduced Caco-2 cell viability compared with non-WB.

Biological activity evaluation and identification of different molecular weight peptides from wheat germ albumin

The hydrolyzate of wheat germ albumin (WGA) has high biological activity. Under the action of proteases, WGA can release bioactive peptides such as antioxidant, antihypertensive, anti fatigue, antibacterial, etc. Ultrafiltration was used to separate different molecular weight peptides from WGA. The effects of proteases on hydrolysis of WGA and biological activities of different molecular weight peptides were comparatively studied and identified target bioactive peptides. When WGA was hydrolyzed by the enzymatic hydrolysis with papain, alcalase, acid protease, and neutral protease, the results showed that papain had the highest DPPH• scavenging rate. The scavenging rate of O2− radical for peptides with MW (<3 KDa) was higher than that of the other two components (WGA and >3 KDa), with an IC50 of 4.59 mg/mL, and its reducing power was significantly higher than that of WGA and MW (>3 KDa). The speculated identification results showed the screened amino acid (AA) sequences (<3 KDa), which mainly consisted of four types: NIYLNFDALHGDKEHGGVR, TGGDGGPGKPTT, DVFWIPRFFVQ, and EEEDKNDKWHRVE. The molecular weight of the dominant AA sequence was concentrated between 1000 and 2200 Da, and almost all functional peptides had high biological activity AA, such as His, Leu, Cys, Lys, and Tyr.

Saltiness enhancing peptides isolated from enzymolysis extract of Lentinula edodes and their taste enhancing action mechanisms

The purpose of this study was to identify saltiness-enhancing peptides from continuous enzymolysis extract of Lentinula edodes by gel filtration chromatography (GFC), LC-MS/MS, virtual screening, molecular docking, molecular dynamics simulation, electronic tongue analysis, and sensory evaluation. The results showed that the molecular weight of peptides in GFC P2 fraction was between 647 and 2110 Da, and heptapeptides-decapeptides were the most important. Four potential saltiness peptides, ADHDLPF, DIQPEER, DEPLIVW, and LPDEPSR were screened by BIOPEP-UWM and further verified by sensory evaluation. Molecular docking and molecular dynamics simulations indicated that four peptides could interact with TMC4 receptors, Arg583, Arg294, Ala584 and Glu161 of TMC4 pocket1 and Arg437, Gln527, Arg580 and Lys412 of TMC4 pocket2 may play a key role in saltiness binding. In addition, the electronic tongue results showed that saltiness peptides had a synergistic effect on improving the saltiness of NaCl solutions and could replace at least 25%–50% of NaCl. The results showed that the saltiness-enhancing peptides of L.edodes could be obtained by enzymolysis, which laid a foundation for developing salt-reducing condiment from L.edodes.

Ice Recrystallization Inhibition Activity of Soy Protein Hydrolysates.

Identifying and developing ice recrystallization inhibitors from sustainable food proteins such as soy protein isolate (SPI) can lead to practical applications in both pharmaceutical and food industries. The objective of this study was to investigate the ice recrystallization inhibition (IRI) activity of SPI hydrolysates, and this was achieved by using an IRI activity-guided fractionation approach and relating IRI activity to interfacial molecular activity measured by vibrational sum frequency generation (VSFG). In addition, the impact of molecular weight (MW) and enzyme specificity was analyzed using three different proteases (Alcalase, trypsin, and pancreatin) and varying hydrolysis times. Using preparative chromatography, hydrolysates from each enzyme treatment were fractionated into five different MW fractions (F1-F5), which were then characterized by high-performance liquid chromatography (HPLC). All SPI hydrolysates had IRI activity, resulting in a 57-29% ice crystal diameter reduction when compared to native SPI. The F1 fraction (of 4-14 kDa) was most effective among all tested hydrolysates, while the lower MW peptide fractions lacked activity. One sample (SPI-ALC 20-F1) had a 52% reduction of ice crystal size at a lower concentration of 2% compared to the typical 4% used. SFG showed a difference in H-bonding and hydrophobic interactions of the molecules on the water/air interface, which may be linked to IRI activity. This study demonstrates for the first time the ability of SPI hydrolysates to inhibit ice crystal growth and the potential application of SFG to study molecular interaction at the interface that may help illustrate the mechanism of action.

Identification of the Stapled α-Helical Peptide ATSP-7041 as a Substrate and Strong Inhibitor of OATP1B1 In Vitro.

ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein-protein interactions. As peptides of molecular weights over 1000 Da are not usually evaluated, data on the drug-drug interaction (DDI) potential of stapled α-helical peptides remain scarce. Here, we evaluate the interaction of ATSP-7041 with hepatic cytochrome P450s (CYPs; CYP1A2, CYP2C9, CYP2C19, CYP3A4, and CYP2D6) and transporters (organic anion transporting polypeptides (OATPs; OATP1B1 and OATP1B3), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP)). ATSP-7041 demonstrated negligible metabolism in human liver S9 fraction and a limited inhibition of CYP activities in yeast microsomes or S9 fractions. On the contrary, a substantial uptake by OATPs in HEK 293 cells, a strong inhibition of OATP activities in the cells, and an inhibition of P-gp and BCRP activities in reversed membrane vesicles were observed for ATSP-7041. A recent report describes that ALRN-6924, an ATSP-7041 analog, inhibited OATP activities in vivo; therefore, we focused on the interaction between ATSP-7041 and OATP1B1 to demonstrate that ATSP-7041, as a higher molecular weight stapled peptide, is a substrate and strong inhibitor of OATP1B1 activity. Our findings demonstrated the possibility of transporter-mediated DDI potential by high molecular weight stapled peptides and the necessity of their evaluation for drug development.

An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules.

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides an easy analytical tool for different stages of biopharmaceutical development. The protein's identity is confirmed when the experimental mass from MS is within a pre-defined mass error range of the theoretical mass. While several computational tools exist for the calculation of protein and peptide molecular weights, they either were not designed for direct application to biotherapeutic entities, have access limitations due to paid licenses, or require uploading protein sequences to host servers. We have developed a modular mass calculation routine that enables the easy determination of the average or monoisotopic masses and elemental compositions of therapeutic glycoproteins, including monoclonal antibodies (mAb), bispecific antibodies (bsAb), and antibody-drug conjugates (ADC). The modular nature of this Python-based calculation framework will allow the extension of this platform to other modalities such as vaccines, fusion proteins, and oligonucleotides in the future, and this framework could also be useful for the interrogation of top-down mass spectrometry data. By creating an open-source standalone desktop application with a graphical user interface (GUI), we hope to overcome the restrictions around use in environments where proprietary information cannot be uploaded to web-based tools. This article describes the algorithms and application of this tool, mAbScale, to different antibody-based therapeutic modalities.