Serum Protease Research Articles

Enhancing the Intrinsic Antiplasmodial Activity and Improving the Stability and Selectivity of a Tunable Peptide Scaffold Derived from Human Platelet Factor 4.

The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.

Association of Serum Proteases and Acute Phase Factors Levels with Survival Outcomes in Patients with Colorectal Cancer.

Colorectal cancer (CRC) represents a substantial burden on global healthcare, contributing to significant morbidity and mortality worldwide. Despite advances in screening methodologies, its incidence remains high, necessitating continued efforts in early detection and treatment. Neoplastic invasion and metastasis are primary determinants of CRC lethality, emphasizing the urgency of understanding underlying mechanisms to develop effective therapeutic strategies. This study aimed to explore the potential of serum biomarkers in predicting survival outcomes in CRC patients, with a focus on cathepsin B (CB), leukocytic elastase (LE), total sialic acid (TSA), lipid-associated sialic acid (LASA), antitrypsin activity (ATA), C-reactive protein (CRP), and cystatin C (CC). We recruited 185 CRC patients and 35 healthy controls, assessing demographic variables, tumor characteristics, and 7 serum biomarker levels, including (1) CB, (2) LE, (3) TSA, (4) LASA, (5) ATA, (6) CRP, and (7) CC. Statistical analyses included ANOVA with Tukey's post hoc tests and MANOVA for continuous variables. Student's t-test was used for dependent samples, while non-parametric tests like Mann-Whitney U and Wilcoxon signed-rank tests were applied for variables deviating from the normal distribution. Categorical variables were assessed using chi-square and Kruskal-Wallis tests. Spearman's rank correlation coefficient was utilized to examine variable correlations. Survival analysis employed the Kaplan-Meier method with a log-rank test for comparing survival times between groups. Significant associations were observed between CB (p = 0.04), LE (p = 0.01), and TSA (p = 0.008) levels and survival outcomes in CRC patients. Dukes' classification stages also showed a significant correlation with survival (p = 0.001). However, no significant associations were found for LASA, ATA, CRP, and CC. Multivariate analysis of LE, TSA, and ATA demonstrated a notable correlation with survival (p = 0.041), notwithstanding ATA's lack of significance in univariate analysis (p = 0.13). CB, LE, and TSA emerged as promising diagnostic markers with prognostic value in CRC, potentially aiding in early diagnosis and treatment planning. Further research is needed to validate these findings and explore additional prognostic indicators.

Dietary supplementation of Astragalus fermentation products improves the growth performance, immunological characteristics, and disease resistance of crucian carp (Carassius auratus)

The fermentation products of Astragalus have been acknowledged for their ability to enhance immune functions. This study assessed the impact of incorporating Astragalus, fermented with Lactobacillus plantarum and Bacillus coagulans, on crucian carp’s growth, disease resistance, and immunological characteristics. The experimental groups were fed with common feed (C), C + Astragalus (A), A + Lactobacillus plantarum (AL), A + Bacillus coagulans (AB), and AL + Bacillus coagulans (ALB). The fermented products were mixed with common feed at a 1:99 ratio, and crucian carp were fed 2% of their body weight for four weeks, with sampling conducted on days 3, 7, 14, 21, and 28. Disease resistance was evaluated using Aeromonas hydrophila (A. hydrophila) at a concentration of 0.2 mL (1.0×107 CFU/mL). The final weights in the AL, AB, and ALB groups significantly increased compared to the C group. The ALB group exhibited elevated serum albumin levels, alkaline phosphatase, intestinal lipase, protease enzyme, C3, and IgM gene expression compared to the C group. At the same time, alanine aminotransferase, aspartate aminotransferase, and glucose contents were significantly reduced. The survival rate significantly increased in all experimental groups after treatment with A. hydrophila. In conclusion, Astragalus products fermented with Lactobacillus plantarum and Bacillus coagulans could effectively improve crucian carp’s growth, disease resistance, and immune response.

Detection of Gnathostoma spinigerum larva in the brain with complete follow-up after surgical treatment of human neurognathostomiasis

Human gnathostomiasis is a food-borne zoonotic helminthic infection widely reported in Latin America, Asia, and Southeast Asia. Consuming raw, or under-cooked fresh-water fish is the leading cause of this helminthic infection, which is clinically characterized by signs of inflammation, itching sensation, or irritation with migratory swelling. Neurological symptoms resulting from neurognathostomiasis vary, and there is scant information due to the rareness of patient brain samples. This study aimed to demonstrate the first evidence of human neurognathostomiasis by the detection of Gnathostoma spinigerum larva in patient's brain during craniotomy, supported by histopathological, immunological and proteomic evidence. Clinical symptoms were obtained from medical history and physical examination with laboratory investigations, including magnetic resonance imaging (MRI), left temporal craniotomy, histopathology of brain tissue, and Western blot analysis, were performed to elucidate the causative pathogens for diagnosis. In addition, the host–parasite interaction of the parasite invading the patient's brain was characterized through proteomics. Histopathology revealed worms with the characteristic cuticular spines of G. spinigerum which were detected and identified. These histopathological findings were consistent with a positive Western blot showing a 24-kDa reactive-band for gnathostomiasis. Proteomic analysis revealed the presence of G. spinigerum serpin and serine protease in the patient's serum. Moreover, the leucine-rich alpha-2-glycoprotein was indicated as a systemic biomarker of early brain injury related to invasion by G. spinigerum. Therefore, our study provides the initial evidence of human neurognathostomiasis due to G. spinigerum larval invasion along with successful craniotomy and proven larval detection including complete follow-up, and the disease prognosis after surgical treatment.

Antimicrobial Activity of Human C-Type Lectin Domain Family 3 Member A (CLEC3A)

C-type lectins (CTLs) are a group of proteins that play a crucial role in immunological functions. With the rise of antibiotic-resistant bacteria, CTLs have emerged as a potential alternative to traditional antibiotics and antimicrobial peptides (AMPs), the latter exhibiting limited application due to their low biostability. In this study, we used viable count assays to investigate the antimicrobial activity of the human C-type Lectin Domain Family 3 Member A (CLEC3A) and its two protein domains, CLEC3A Ex23 and CLEC3A Ex3, against gram-positive and gram-negative bacteria. Additionally, using immunoblot analysis, we assessed the biostability of CLEC3A and its protein domains in bacterial supernatant and murine serum. Our findings demonstrate that CLEC3A, CLEC3A Ex23, and CLEC3A Ex3 possess antimicrobial activity against gram-positive Staphyloccocus aureus and gram-negative Pseudomonas aeruginosa. CLEC3A is more effective against P. aeruginosa than the well-investigated antimicrobial peptide LL-37. Furthermore, CLEC3A and its domains have low sensitivity to bacterial and serum proteases, making them more advantageous for systemic application than most AMPs. In conclusion, our research has demonstrated that CLEC3A is not only a precursor of AMPs but also an antimicrobial protein itself, with favorable characteristics for therapeutic applications.

Lactic acid bacteria with probiotic characteristics in fermented dairy products reduce cow milk allergy

The use of fermented dairy products containing lactic acid bacteria (LAB) with probiotic properties has been suggested as a suitable solution to reduce cow milk allergy (CMA). In our previous study, we screened four LAB strains with the ability to hydrolyze allergic milk protein. In this study, we further evaluated the in vivo allergenicity of fermented milk beverage (FMB) made with the compound of four strains. Our results showed that FMB administration significantly reduced the allergic responses induced by milk proteins compared to skimmed milk (SM) administration. Specifically, FMB modulated the T helper cell (Th) 1/Th2 and Th17/T regulatory cell (Treg) immune balance of BALB/c mice, reduced total IgG, total IgG1, and total IgE antibody levels, mast cell protease in serum, and histamine levels in plasma. Additionally, intestinal microbiota analysis demonstrated that FMB administration increased the abundance of Lachnospiraceae, a common immunomodulatory commensal bacteria, and down-regulated the abundance of Muribaculaceae and Prevotellaceae. Overall, FMB is a low-allergenicity dairy product that can alleviate allergy symptoms by regulating the immune response and intestinal microbiota composition. FMB has the potential to be developed as a low-allergenicity dairy product or allergy therapy as well.

Teaching Neuro Images : Granulomatosis with polyangiitis causing Collet-Sicard syndrome and refractory headache

A 52-year-old man presented for 4 months of progressive hypophonia and dysphagia with refractory headache and bilateral otitis media. Examination and MRI revealed abnormalities (figures 1 and 2). Serum proteinase 3 antibody level was 180 units (normal <21 units) and c-ANCA (antineutrophil cytoplasmic antibody) was 1:160, consistent with granulomatosis with polyangiitis (GPA; previously known as Wegener granulomatosis). Lung nodule biopsy confirmed GPA.

In Silico and In Vivo Studies of a Tumor-Penetrating and Interfering Peptide with Antitumoral Effect on Xenograft Models of Breast Cancer.

The combination of a tumor-penetrating peptide (TPP) with a peptide able to interfere with a given protein-protein interaction (IP) is a promising strategy with potential clinical application. Little is known about the impact of fusing a TPP with an IP, both in terms of internalization and functional effect. Here, we analyze these aspects in the context of breast cancer, targeting PP2A/SET interaction, using both in silico and in vivo approaches. Our results support the fact that state-of-the-art deep learning approaches developed for protein-peptide interaction modeling can reliably identify good candidate poses for the IP-TPP in interaction with the Neuropilin-1 receptor. The association of the IP with the TPP does not seem to affect the ability of the TPP to bind to Neuropilin-1. Molecular simulation results suggest that peptide IP-GG-LinTT1 in a cleaved form interacts with Neuropilin-1 in a more stable manner and has a more helical secondary structure than the cleaved IP-GG-iRGD. Surprisingly, in silico investigations also suggest that the non-cleaved TPPs can bind the Neuropilin-1 in a stable manner. The in vivo results using xenografts models show that both bifunctional peptides resulting from the combination of the IP and either LinTT1 or iRGD are effective against tumoral growth. The peptide iRGD-IP shows the highest stability to serum proteases degradation while having the same antitumoral effect as Lin TT1-IP, which is more sensitive to proteases degradation. Our results support the development of the TPP-IP strategy as therapeutic peptides against cancer.

Influence of a Physiologically Formed Blood Clot on Pre-Osteoblastic Cells Grown on a BMP-7-Coated Nanoporous Titanium Surface.

Titanium (Ti) nanotopography modulates the osteogenic response to exogenous bone morphogenetic protein 7 (BMP-7) in vitro, supporting enhanced alkaline phosphatase mRNA expression and activity, as well as higher osteopontin (OPN) mRNA and protein levels. As the biological effects of OPN protein are modulated by its proteolytic cleavage by serum proteases, this in vitro study evaluated the effects on osteogenic cells in the presence of a physiological blood clot previously formed on a BMP-7-coated nanostructured Ti surface obtained by chemical etching (Nano-Ti). Pre-osteoblastic MC3T3-E1 cells were cultured during 5 days on recombinant mouse (rm) BMP-7-coated Nano-Ti after it was implanted in adult female C57BI/6 mouse dorsal dermal tissue for 18 h. Nano-Ti without blood clot or with blood clot at time 0 were used as the controls. The presence of blood clots tended to inhibit the expression of key osteoblast markers, except for Opn, and rmBMP-7 functionalization resulted in a tendency towards relatively greater osteoblastic differentiation, which was corroborated by runt-related transcription factor 2 (RUNX2) amounts. Undetectable levels of OPN and phosphorylated suppressor of mothers against decapentaplegic (SMAD) 1/5/9 were noted in these groups, and the cleaved form of OPN was only detected in the blood clot immediately prior to cell plating. In conclusion, the strategy to mimic in vitro the initial interfacial in vivo events by forming a blood clot on a Ti nanoporous surface resulted in the inhibition of pre-osteoblastic differentiation, which was minimally reverted with an rmBMP-7 coating.

Glycosylation and Lipidation Strategies: Approaches for Improving Antimicrobial Peptide Efficacy.

Antimicrobial peptides (AMPs) have recently gained attention as a viable solution for combatting antibiotic resistance due to their numerous advantages, including their broad-spectrum activity, low propensity for inducing resistance, and low cytotoxicity. Unfortunately, their clinical application is limited due to their short half-life and susceptibility to proteolytic cleavage by serum proteases. Indeed, several chemical strategies, such as peptide cyclization, N-methylation, PEGylation, glycosylation, and lipidation, are widely used for overcoming these issues. This review describes how lipidation and glycosylation are commonly used to increase AMPs' efficacy and engineer novel AMP-based delivery systems. The glycosylation of AMPs, which involves the conjugation of sugar moieties such as glucose and N-acetyl galactosamine, modulates their pharmacokinetic and pharmacodynamic properties, improves their antimicrobial activity, and reduces their interaction with mammalian cells, thereby increasing selectivity toward bacterial membranes. In the same way, lipidation of AMPs, which involves the covalent addition of fatty acids, has a significant impact on their therapeutic index by influencing their physicochemical properties and interaction with bacterial and mammalian membranes. This review highlights the possibility of using glycosylation and lipidation strategies to increase the efficacy and activity of conventional AMPs.

Introduction of a β-leucine residue instead of leucine9 and glycine10 residues in Temporin L for improved cell selectivity, stability and activity against planktonic and biofilm of methicillin resistant S. aureus

Leucine and glycine residues, at the 9th and 10th positions of helical domain of naturally occurring antimicrobial peptide (AMP), Temporin L were substituted with an unnatural amino acid, β-leucine (homovaline) to improve its serum protease stability, haemolytic/cytotoxic properties and reduce the size to some extent. The designed analogue, L9βl-TL showed either equal or improved antimicrobial activity to TL against different microorganisms including the resistant strains. Interestingly, L9βl-TL also exhibited lower haemolytic and cytotoxic activities against human red blood cells and 3T3 cells, respectively. Moreover, L9βl-TL showed antibacterial activity in presence of 25% (v/v) human serum and showed resistance against proteolytic cleavage in presence of it that suggested the serum protease stability of the TL-analogue. L9βl-TL exhibited un-ordered secondary structures in both bacterial and mammalian membrane mimetic lipid vesicles as compared to the helical structures of TL in these environments. However, tryptophan fluorescence studies demonstrated more selective interaction of L9βl-TL with bacterial membrane mimetic lipid vesicles in comparison to non-selective interactions of TL with both kinds of lipid vesicles. Membrane depolarization studies with live MRSA and bacterial membrane-mimetic lipid vesicles suggested a membrane-disrupting mode of action of L9βl-TL. L9βl-TL showed faster bactericidal mechanism compared to TL against MRSA. Interestingly, L9βl-TL was found as more potent than TL either in inhibiting biofilm formation or in eradicating the mature biofilm formed by MRSA. Overall, the present work demonstrates a simple and useful strategy to design of an analogue of TL, with minimal modifications while maintaining its antimicrobial activity with lesser toxicity and higher stability which could be attempted for other AMPs as well.

Ficoline-3 and MASP-2 gene variants in siberian arctic populations: SUMMARIZED Evidence of selective pressure for the high frequency of lectin complement pathway deficiency.

A total of 926 specimens of dried blood spots of the newborns were genotyped. The newborns represented four populations: Nenets, Dolgan-Nganasans, Mixed aboriginal population, and Russians (Caucasians) to study the prevalence of single nucleotide polymorphisms of FCN3 rs532781899 and MASP2 rs72550870. The prevalence of the deletion allele of the rs532781899 variant in the FCN3 gene associated with the decreased production of ficoline-3 was found to be increased in Russians compared to the Nenets aboriginal populations (p=0.002). The prevalence of the rs72550870*G allele in the MASP2 gene associated with low serum protease activity was found to be increased in Russians compared with Nenets and Dolgan-Nganasans (p < 0.001 and p=0.03, respectively). The results of the current study and our previous findings corroborate with a hypothesis that human evolution has been directed toward the accumulation of genotypes associated with low activity of the lectin complement activation pathway.

Substituting fish meal with a bacteria protein (Methylococcus capsulatus, Bath) grown on natural gas: Effects on growth non-specific immunity and gut health of spotted seabass (Lateolabrax maculatus)

This study aimed to evaluate effects of substituting fish meal (FM) by methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®, FK) on growth performance, non-specific immunity and gut health of Lateolabrax maculatus. Six iso-nitrogenous diets were formulated with 0, 3.5%, 7%, 10.5%, 14% and 17.5% FK to substitute 0 (control group, FM content: 35%), 10%, 20%, 30%, 40% and 50% fish meal, referred to as FM, FK3.5, FK7, FK10.5, FK14, and FK17.5 respectively. Fish (initial body weight: 11.00 ± 0.02 g) were fed experimental diets for 56 days. Results showed that growth of fish fed the FK3.5 diets was significantly increased (P < 0.05) but that there was no significant difference (P > 0.05) in growth of the other groups (FK7-FK17.5) compared with control group. Although survival of fish in the 50% substitution group was significantly decreased (P < 0.05) compared with the other groups, there was no overall trend in the survival data. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), gut lipase and protease and the contents of serum total protein and albumin in the FK3.5 group were significantly higher than those of control group (P < 0.05). In contrast, the activities/contents of serum ACP, AKP, lysozyme and complement 4 in the FK10.5, FK14 and FK17.5 groups were significantly decreased compared with control group. There was no significant difference in gut mucosal structure among all groups. The analysis of gut flora showed that the relative abundance of Lactobacillus in the FK7 and FK10.5 groups was significantly higher than other groups. In conclusion, dietary FK inclusion level up to 14% of total diet to replace 40% FM is possible without negatively affecting the growth performance of spotted seabass. Furthermore, the replacement of FM with 3.5% FK enhances the growth and immune, and substituting 20–30% of FM by 7–10.5% FK increased the abundance of beneficial bacteria in intestine of fish.

Design of Three Residues Peptides against SARS-CoV-2 Infection.

The continuous and rapid spread of the COVID-19 pandemic has emphasized the need to seek new therapeutic and prophylactic treatments. Peptide inhibitors are a valid alternative approach for the treatment of emerging viral infections, mainly due to their low toxicity and high efficiency. Recently, two small nucleotide signatures were identified in the genome of some members of the Coronaviridae family and many other human pathogens. In this study, we investigated whether the corresponding amino acid sequences of such nucleotide sequences could have effects on the viral infection of two representative human coronaviruses: HCoV-OC43 and SARS-CoV-2. Our results showed that the synthetic peptides analyzed inhibit the infection of both coronaviruses in a dose-dependent manner by binding the RBD of the Spike protein, as suggested by molecular docking and validated by biochemical studies. The peptides tested do not provide toxicity on cultured cells or human erythrocytes and are resistant to human serum proteases, indicating that they may be very promising antiviral peptides.

Synthesis and Systematic Study on the Effect of Different PEG Units on Stability of PEGylated, Integrin-αvβ6-Specific A20FMDV2 Analogues in Rat Serum and Human Plasma.

A20FMDV2 is a 20-mer peptide that exhibits high selectivity and affinity for the tumour-related αvβ6 integrin that can compete with extracellular ligands for the crucial RGD binding site, playing a role as a promising αvβ6-specific inhibitor for anti-cancer therapies. Unfortunately, the clinical value of A20FMDV2 is limited by its poor half-life in blood caused by rapid renal excretion and its reported high susceptibility to serum proteases. The incorporation of poly (ethylene glycol) chains, coined PEGylation, is a well-established approach to improve the pharmacokinetic properties of drug molecules. Here, we report a systematic study on the incorporation of a varying number of ethylene glycol units (1–20) into the A20FMDV2 peptide to establish the effects of PEGylation size on the peptide stability in both rat serum and human plasma. In addition, the effect of acetyl and propionyl PEGylation handles on peptide stability is also described. Selected peptide analogues were assessed for integrin-αvβ6-targeted binding, showing good specificity and activity in vitro. Stability studies in rat serum established that all of the PEGylated peptides displayed good stability, and an A20FMDV2 peptide containing twenty ethylene glycol units (PEG20) was the most stable. Surprisingly, the stability testing in human plasma identified shorter PEGs (PEG2 and PEG5) as more resistant to degradation than longer PEGs, a trend which was also observed with affinity binding to integrin αvβ6.

Gene polymorphism of mannose-binding lectin-associated serine protease (MASP2) in indigenous populations of the Russian Arctic Territories

Mannose-binding lectin-associated serine proteases (MASP) are among of the key components in the lectin pathway (LP) of the complement activation. MASP-2 is the most studied agent among specific enzymes activating both mannose-binding lectin (MBL) and ficolins, pattern-recognition proteins involved in the elimination of pathogenic microorganisms through LP complement activation. There are some mutations in MASP2, with the most significant identified as rs72550870 (p.D120G). The homozygous GG rs72550870 is associated with congenital MASP-2 deficiency and characterized by a total lack of serum protease activity, which leads to impaired binding to lectins. This, in turn, results in severe course of infectious diseases with a high risk of adverse outcome. There seem to be some marked populational differences in the genotype and haplotype prevalence in MASP2 gene polymorphisms. To date, no data are available on the genotype distribution for the MASP2 gene in the indigenous populations of the Russian Arctic regions. The aim of the work was to study the prevalence and ethnic specificity in the distribution of allelic variants of MASP2 rs72550870 in the populations of the Taymyr Dolgan-Nenets District of the Krasnoyarsk Territory (Nenets, Dolgans, Nganasans) as well as the city of Krasnoyarsk (Russians). MASP2 genotyping was performed by using real-time PCR. The frequencies of the AG genotype associated with low MASP-2 level was 6.6% for ethnic Russian newborns in the Eastern Siberia. The prevalence of the AG genotype was significantly lower in newborns of the Arctic populations than in the Russians, being 0.3% and 0.9% for the Nenets and the Dolgan-Nganasans, respectively, which is close to the prevalence values identified for Asian and African populations (0%). No homozygous GG rs72550870 associated with congenital MASP-2 deficiency in newborns of the indigenous populations of the Taymyr Dolgan-Nenets region of Krasnoyarsk Territory (Nenets and Dolgan-Nganasans) and ethnic Caucasian subjects of the Krasnoyarsk city was detected. The frequency of the rare allelic variant G rs72550870 in ethnic Russian subjects was 3.3%, being close to the frequencies in the European populations of the world (4.0%), whereas it was 0.5% in the indigenous inhabitants of the Arctic Region. We have suggested that isolated Arctic populations encounter some intracellular infections historically later and, as contrasted with Caucasoid populations, retained a high activity in the lectin pathway of the complement activation established at the early stage of human evolution.

Abstract 3599: Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas

Abstract The overall 5-year survival for pancreatic ductal adenocarcinoma (PDAC) has changed little over the past few decades, and PDAC is predicted to become the second leading cause of cancer-related mortality in the next decade in Western countries. Low grade serous ovarian cancer (LGSOC) is a slow-growing but generally chemo-resistant cancer with few effective treatments for recurrent disease. More than 90% of PDAC and up to 40% of LGSOC express mutationally activated KRAS that drives persistent cell division, anti-apoptosis, cell migration and metastasis. Preventing signaling from the KRAS oncoprotein has been challenging. While recent studies have shown promising results by targeting the binding pocket in Switch II for development of KRAS mutant-specific inhibitors for the G12C mutant and G12D mutant, targeting other mutations remains a work in progress. We have discovered that DIRAS3, a novel endogenous physiological RAS inhibitor, blocks KRAS activity by directly binding KRAS with high affinity, inhibiting KRAS dimerization/nanoclustering and blocking effector activation. In this study we have developed drug-like, helical 10-mer stapled peptides derived from DIRAS3 α5 domain. Preliminary studies show that these lead compounds associate with KRAS with low nanomolar affinity, are largely resistant to serum proteases, and rapidly cross the cellular membrane. Functionally, DIRAS3 peptides—but not control peptides—block KRAS homodimers in ReBiL split luciferase assays of KRAS dimerization and disrupt nanoclustering by TEM analysis of GFP-KRAS(G12V)-labeled gold nanoparticles on the inner leaflet of the plasma membrane of cells. Moreover, DIRAS3 peptides significantly inhibit phospho-ERK, decrease cell viability and induce apoptosis in pancreatic and low-grade ovarian cancer cells with KRAS mutations. Finally, daily treatment with stapled DIRAS3-derived peptides inhibited growth of ASPC-1 (KRAS G12D) PDAC xenografts and improved survival. Thus, our study suggests that development of DIRAS3 stapled peptides may provide a novel therapeutic approach to target mutant KRAS-driven cancers. Citation Format: Joshua P. Gray, Gamze E. Bildik, Margie N. Sutton, Yong Zhou, Steven Millward, John F. Hancock, Zhen Lu, Robert C. Bast. Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3599.

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

Mucin-fused myeloid-derived growth factor (MYDGF164) exhibits a prolonged serum half-life and alleviates fibrosis in chronic kidney disease.

Although no effective therapy is available to stop or reverse CKD progression targeting its key feature, the loss of peritubular capillaries (PTCs) leading to interstitial fibrosis, myeloid-derived growth factor (MYDGF) with tissue-repairing activities enlightens its therapeutic potential for CKD. However, the extremely short circulatory lifetime (15 min) restricts its application. We selected a tandem repeated (TR) region of human CD164 as a carrier to fuse with MYDGF and then investigated for biophysical and pharmacokinetic changes. The MYDGF164 bioactivities were validated in HUVECs and then assessed in HK-2 cells. We also investigated its efficacy in unilateral ureteral obstruction (UUO)-treated mice and in adenine-induced CKD rats. MYDGF164 was modified with sialoglycans, improving its resistance to serum proteases and increasing its hydrodynamic radius. The half-life of MYDGF164 was significantly prolonged but retained its original cell proliferation, anti-apoptosis, and tubulogenesis activities. It selectively stimulated the proliferation in endothelial and epithelial cells through phosphorylating MAPK1/3. MYDGF164 alleviated capillary rarefaction, hypoxia, renal fibrosis, and tubular atrophy in UUO mice and in adenine-induced CKD rats. MYDGF164 restored renal function, with normalized creatinine and urea levels in adenine-induced CKD rats. Histopathology and immunohistochemistry revealed that MYDGF164 protection was related to its cell-proliferative, anti-apoptosis, and angiogenesis activities. This study is the first successful example of using a tandem repeated region of hCD164 as a cargo protein for the pharmacokinetic improvement of therapeutic proteins. Our findings highlight the potential of MYDGF164 in alleviating renal fibrosis in CKD.

Interaction of Synthetic Pyrethroid Insecticide Deltamethrin with Human Alpha-2-Macroglobulin: Spectroscopic and Molecular Docking Studies.

Deltamethrin (DLM) is a commercial insecticide of the synthetic pyrethroid family that is used to control disease-causing insects and vectors. When humans are exposed to the fumes or aerosols of DLM, it enters the body via cuticular absorption and reacts with proteins and other biomolecules. Alpha-2-macroglobulin (α2M) is a serum proteinase inhibitor that also carries out receptor- mediated endocytosis of extracellular substances. This study was done to decipher the structural and functional alterations of α2M by DLM. Various spectroscopic techniques, including UV absorption and fluorescence spectroscopy, binding studies, and molecular docking, were used to characterize the interaction of DLM with α2M. The affinity constant was calculated from the Stern-Volmer equation using fluorescence data. The UV-Vis and fluorescence spectral studies indicated the formation of a complex between α2M and DLM. Thermodynamically, the interaction was found to be spontaneous with ΔG = -4.23 kcal/mol. CD spectra suggested a change in the secondary structure of the protein from β to α helical content with increasing concentration of DLM. The molecular docking study by Autodock Vina established the interaction of DLM with Glu-926, Ala-1103, Ala-1108, Val-1116, Asn-1159, Glu-1220, Leu-1261, Thr-1272, Ile-1390, Pro-1391, Lys-1393, Val-1396, Lys-1397, Thr-1408, Glu-1409, Val-1410, Ser-1411, Ser-1412, and Asn-1413 with an improved docking score of -6.191 kcal/mol. The binding was carried out in the vicinity of the receptor-binding domain at the C-terminal of α2M. The decrease in the functional activity and structural changes of protein after binding with DLM has a significant effect on human α2M. The information may be useful for exploring the role of DLM in a clinical chemistry laboratory.