ABSTRACT Cecropins are a class of antimicrobial peptides (AMPs) expressed by insects that have broad‐spectrum antimicrobial activity and low cytotoxicity. However, cecropins are also long (40–45 amino acids) peptides, making their adaptation for therapeutic use challenging. Here, we present a series of 16 amino acid‐long peptides derived from cecropin B from Drosophila melanogaster . The native truncated peptide is cationic and expected to form an amphipathic α‐helix, but does not have antimicrobial activity. The incorporation of two Trp residues, depending on the position in the peptide backbone, results in antibacterial activity comparable to that of full‐length cecropin B against the gram‐negative bacteria Escherichia coli , Pseudomonas aeruginosa , Acinetobacter baumannii , and Klebsiella pneumoniae , and there is minimal improvement as additional Trp substitutions are made. The incorporation of three or four Trp residues results in antibacterial activity against Gram‐positive bacteria Staphylococcus aureus and Enterococcus faecalis , which are not affected by the full‐length peptide at tested concentrations. However, peptides containing three or four Trp residues also had increased levels of cytotoxicity and hemolysis. NMR studies revealed an intrinsically disordered peptide in water, with some residual secondary structure. Secondary structure analysis by circular dichroism spectroscopy found that the propensity for helicity increases with increasing numbers of Trp residues, although individual Trp interactions at the C‐terminal side of the peptide seem to have the biggest role in peptide structure. Similar to the full‐length cecropin peptides, the truncated AMPs cause membrane permeability, but differences in the rate of permeabilization and bacterial killing do not directly correlate with the minimum inhibitory concentration (MIC) for each peptide. We propose that the location of the Trp residues, as opposed to the number incorporated, determines the antibacterial activity, and that peptide activity depends on the membrane composition of the bacteria.

ABSTRACT The rapid increase in multidrug‐resistant microbial pathogens has emerged as a major global challenge, making it essential to develop and discover new, highly potent, broad‐spectrum therapeutic agents. The heterocyclic framework has drawn considerable interest due to its wide range of biological activities, and the incorporation of a peptide can further enhance the selectivity. Herein, we report the synthesis of peptide‐linked trifluoromethyl triazolo‐pyrazine derivatives ( 7a‐e and 10a‐e ), characterized using 1 H NMR, 13 C NMR, mass spectrometry, and IR spectroscopy. All the synthesized compounds' antibacterial and antifungal activities were evaluated using a minimum inhibitory concentration (MIC) assay. Biological efficacy results revealed that compounds 7a and 7b displayed MIC values (20–35 μg/mL) that are comparable to or better than chloramphenicol (16–26 μg/mL). Likewise, antifungal assays revealed that compound 10d exhibited activity (67–82 μg/mL) notably superior to griseofulvin (130–160 μg/mL). The obtained data clearly indicate that the synthesized compounds are potent against microbes with the inclusion of structural diversity of peptide–triazolopyrazine conjugates. Furthermore, molecular docking studies show strong binding interactions of the potent compounds with their targets, as evidenced by docking scores comparable to or surpassing the control compounds. These findings provide a promising foundation for the development of next‐generation antimicrobial agents.

ABSTRACT The global cancer burden data 2022 estimated 2.3 M newly diagnosed breast cancer cases with over 6 M mortalities. Moreover, the cases are expected to increase by 10% by 2045, raising health concerns worldwide. Triple‐negative breast cancer is a highly aggressive subtype of BC with a shorter survival rate and a higher likelihood of recurrence. EGFR overexpression is seen in 15%–45% of BC. It is associated with nearly 50% of TNBC and inflammatory breast cancer incidences, associated with motility, survival, and differentiation through mutation. This study investigates pyrrolo[2,1‐f][1,2,4]triazine analogs as potential inhibitors of both wild‐type and mutant (T790M) EGFR, utilizing computational approaches to address resistance and enhance breast cancer therapeutics. A dataset of pyrrolo[2,1‐f][1,2,4]triazine analogs was used to develop pharmacophore models and a predictive 3D‐QSAR model. The designed library underwent ADME screening, molecular docking with mutant and wild‐type EGFR, MM‐GBSA calculations, DFT, MD simulations, and post‐MD studies using Schrödinger 2025–2 software. Compound SK31 showed favorable docking (wild‐type: −10.8 kcal/mol; mutant: −9.4 kcal/mol), strong MM‐GBSA binding energies, and stable interactions in MD. DFT confirmed favorable electronic behavior. Post‐MD analyses revealed low‐energy conformational stability and strong residue correlations. This study provides a framework for designing pyrrolo[2,1‐f][1,2,4]triazine‐based EGFR inhibitors and offers valuable insights into future breast cancer therapeutic development.

ABSTRACTExcessive inflammatory responses and ferroptosis‐associated dysfunction of chondrocytes are significant pathological features of osteoarthritis (OA). Relaxin‐2, a well‐known hormone involved in metabolic regulation across various tissues and cells, has not been extensively studied in the context of chondrocyte dysfunction and OA. T/C‐28a2 cells were stimulated with tumor necrosis factor α (TNF‐α) (10 ng/mL) with or without recombinant human relaxin‐2 (rh relaxin‐2) (25, 50 nM) for 48 h. Various techniques, including 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining, real‐time PCR, western blot analysis, enzyme‐linked immunosorbent assay (ELISA), and luciferase activity assays, were employed. We discovered that exposure to TNF‐α decreased the expression of Relaxin‐2 at both the mRNA and protein levels. Further investigation revealed that treatment with rh relaxin‐2 mitigated lipid peroxidation by reducing levels of reactive oxygen species (ROS), malondialdehyde (MDA), and 4‐hydroxy‐2‐nonenal (4‐HNE), while increasing superoxide dismutase (SOD) activity and glutathione (GSH) content. Notably, rh relaxin‐2 restored iron metabolism balance disrupted by TNF‐α in human T/C‐28a2 chondrocytes by decreasing Fe2+ levels, downregulating transferrin receptor 1 (TFR1) gene expression, and upregulating ferritin gene expression. Additionally, rh relaxin‐2 alleviated ferroptosis induced by TNF‐α in these cells by increasing glutathione peroxidase 4 (GPX4) expression, decreasing acyl‐CoA synthetase long‐chain family member 4 (ACSL4) expression, and reducing lactate dehydrogenase (LDH) release. Rh relaxin‐2 also inhibited the TNF‐α‐induced expression of pro‐inflammatory cytokines, interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6). Interestingly, rh relaxin‐2 attenuated TNF‐α‐induced chondrocyte degeneration by suppressing matrix metalloproteinase‐13 (MMP‐13) expression and preventing degradation of collagen type II alpha1 chain (Col2α). Mechanistically, these effects of rh relaxin‐2 were found to be mediated through the nuclear factor erythroid 2‐related factor 2 (Nrf2)/NF‐κB signaling pathway. Our findings suggest that rh relaxin‐2 could be a potential therapeutic strategy for OA.

ABSTRACTOral squamous cell carcinoma (OSCC) is a major public health concern, accounting for more than 90% of all oral malignancies. Pathogenic bacteria, such as Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, are closely linked to OSCC and promote chronic inflammation, a key factor in the tumor microenvironment. The increasing incidence of oral malignancies necessitates the development of antimicrobial peptides (AMPs) with enhanced anticancer activity as targeted therapeutics against bacteria to mitigate their impact on OSCC. A total of 182 Streptomyces genus‐derived AMPs were collected and analyzed in silico to determine their potent antibacterial and anticancer characteristics. Fourteen AMPs were tested for 3D structural alignment prediction and validation, and the most efficient peptide was determined to be sAMP2. A genome‐based analysis of the peptide sourced from Streptomyces globisporus C‐1027 was conducted to explore its evolutionary significance. The cell‐penetrating peptide activity of sAMP2 was optimized to make it more stable and better able to target OSCC host cellular receptors. The peptide Mut_sAMP2 was found to interact with the bacterial virulence proteins DEX of S. mutans, LPS of F. nucleatum, and KGP of P. gingivalis and with OSCC‐associated proteins (TNFα, IL‐6, IL‐8, p38, p53, E‐cadherin, Jak‐1, and PAR2) with greater binding affinity. The peptide Mut_sAMP2 treated OECM‐1 cells showed reduced cell survival by the cytotoxicity activity of IC50 5.4 μg/mL. To the best of our knowledge, our computational study is the first to significantly impact the development of new and effective cancer therapeutics from Streptomyces sp. derived AMPs with a broad spectrum of activity and potential evaluating to combat OSCC progression linked with oral oncobacteria.

ABSTRACTCeruloplasmin, a multicopper oxidase essential for copper metabolism and oxidative defense, from blood serum of domestic goats (Capra aegagrus hircus) has been purified to homogeneity by using three steps of column chromatography. The purified goat ceruloplasmin (gCP) protein has an estimated molecular weight of approximately 141 kDa as determined by SDS‐PAGE. ICP‐AES analysis indicates that each molecule of the protein contains 5.6 ± 0.2 atoms of copper. The optimum pH of catalysis for gCP was found to be pH 5.5. Using the Hill equation, the Km and Vmax values for gCP are estimated to be 0.41 ± 0.01 mM and 0.35 ± 0.01 μmol/min/mg, respectively. The enzyme has an average of 3.3 ± 0.33 substrate binding sites per molecule. Both, circular dichroism (CD) spectroscopy studies and computational analysis using PDBsum reveals that gCP is primarily composed of β‐strands distinguishing it from the α‐helix‐rich human ceruloplasmin. Also, computational analysis of gCP reveals six copper‐binding sites, composed of histidine (his), methionine (Met), and cysteine (cys). Cyclic voltammetry studies of gCP immobilized on a glassy carbon electrode indicate the presence of Type 1 and Type 2/3 copper centers within the protein identified by two unique redox potential values of +294 mV and −250 mV versus the normal hydrogen electrode (NHE), respectively. The positive redox potential site that probably acts as the electron‐transfer center had a slightly lower redox potential compared to that reported in other ceruloplasmin. The negative redox potential site of the protein possibly corresponds to the catalytic site containing trinuclear copper center proposed earlier. The redox potential of the Type 1 center aligns closely with azurin, suggesting evolutionary homology and suitability for direct electron transfer (DET). The distinct electrochemical properties of gCP may enhance applications in biofuel cells, biosensors, and other bioelectronic devices due to its efficient electron‐transfer capabilities. This study provides valuable insights into the functional versatility of ceruloplasmin across species and highlights the biotechnological potential of this unique variant.