Search Results (691)

The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil and a single novel zinc finger with four cysteines and two histidines, from which the protein obtains its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using synthetic peptide fragments that the zinc finger of ZC4H2 is genuine and folds upon binding a zinc ion with picomolar affinity. NMR pH titration of histidines and UV–Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc, while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger are likely to perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy, it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative functions. Full article

Phospholipase A1 (Ves a 1), a major toxin from Vespa affinis venom, poses significant risks to allergic individuals. Nevertheless, the epitope determinants of Ves a 1 have not been characterized. Thus, identifying its linear B-cell epitopes is crucial for understanding envenomation mechanisms. In this study, we predicted and identified B-cell epitopes EP5 and EP6 as potential candidates. EP5 formed an α-helix at the active site of Ves a 1, whereas EP6 adopted an extended loop conformation. Both synthetic peptides were synthesized and evaluated for their inhibitory effects using immune-inhibitory assays with polyclonal antibodies (pAbs) targeting both native (nVes a 1) and recombinant (rVes a 1) forms. The Ves a 1 polyclonal antibodies (pAb-nVes a 1 and pAb-Ves a 1) were produced, and their specificity binding to Ves a 1 was confirmed by Western blot. Next, ELISA inhibition assays showed that EP5 and EP6 significantly blocked pAb binding to both nVes a 1 and rVes a 1. Dot blot and Western blot assays supported these findings, particularly with stronger inhibition toward rVes a 1. Furthermore, enzymatic assays indicated that nVes a 1 and rVes a 1 retained phospholipase activity. Immunoinformatics docking showed that EP5 and EP6 specifically bind to a single-chain variable fragment antibody (scFv) targeting Naja naja PLA2. Molecular analysis revealed similar amino acid interactions to the template, suggesting effective paratope–epitope binding. These results support the potential of EP5 and EP6 for future diagnosis and therapy of V. affinis venom allergy. Full article

In order to innovatively develop high-activity ACE inhibitory peptides from edible fungi, the conditions for a double-enzymatic hydrolysis preparation of ACE inhibitory peptides from Flammulina velutipes were optimized by response surface methodology. After purification by macroporous resin, gel chromatography, and RP-HPLC, a crude peptide fraction was obtained; its ACE inhibition rate was 85.73 ± 0.95% (IC₅₀ = 0.83 ± 0.09 mg/mL). Based on LC-MS/MS sequencing, the four novel peptides, namely, FAGGP, FDGY, FHPGY, and WADP, were screened by computer analysis and molecular docking technology. The four peptides exhibited a binding energy between −9.4 and −10.3 kcal/mol, and formed hydrogen bonds with Tyr523, Ala354, and Glu384 in the S1 pocket, Tyr520 and His353 in the S2 pocket, and His383 in the HEXXH zinc-coordinating motif of ACE, indicating their good affinity with the ACE active site. The IC₅₀ values of the four ACE inhibitory peptides were 29.17, 91.55, 14.79, and 41.27 μM, respectively, suggesting that these peptides could potentially contribute to the development of new antihypertensive products. Full article

Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments. Full article

Background/Objectives: PEGylated liposomes are widely recognized for their biocompatibility and capacity to extend systemic circulation via “stealth” properties. However, the PEG corona often limits tumor penetration and cellular internalization. Targeting matrix metalloproteinase-2 (MMP-2), frequently upregulated in breast cancer stroma, presents an opportunity to enhance tissue-specific drug delivery. In this study, we engineered MMP-2-responsive GPLGVRG peptide-modified cleavable PEGylated liposomes for targeted paclitaxel (PTX) delivery. Methods: Molecular docking simulations employed the MMP-2 crystal structure (PDB ID: 7XJO) to assess GPLGVRG peptide binding affinity. A cleavable, enzyme-sensitive peptide-PEG conjugate (Chol-PEG_2K-GPLGVRG-PEG_5K) was synthesized via small-molecule liquid-phase synthesis and characterized by ¹H NMR and MALDI-TOF MS. Liposomes incorporating this conjugate (S-Peps-PEG_5K) were formulated to evaluate whether MMP-2-mediated peptide degradation triggers detachment of long-chain PEG moieties, thereby enhancing internalization by 4T1 breast cancer cells. Additionally, the effects of tumor microenvironmental pH (~6.5) and MMP-2 concentration on drug release dynamics were investigated. Results: Molecular docking revealed robust GPLGVRG-MMP-2 interactions, yielding a binding energy of −7.1 kcal/mol. The peptide formed hydrogen bonds with MMP-2 residues Tyr A:23 and Arg A:53 (bond lengths: 2.4–2.5 Å) and engaged in hydrophobic contacts, confirming MMP-2 as the primary recognition site. Formulations containing 5 mol% Chol-PEG_2K-GPLGVRG-PEG_5K combined with 0.15 µg/mL MMP-2 (S-Peps-PEG_5K +MMP) exhibited superior internalization efficiency and significantly reduced clonogenic survival compared to controls. Notably, acidic pH (~6.5) induced MMP-2-mediated cleavage of the GPLGVRG peptide, accelerating S-Peps-PEG_5K dissociation and facilitating drug release. Conclusions: MMP-2-responsive, cleavable PEGylated liposomes markedly improve PTX accumulation and controlled release at tumor sites by dynamically modulating their stealth properties, offering a promising strategy to enhance chemotherapy efficacy in breast cancer. Full article

The development of orally bioavailable non-peptidomimetic glucagon-like peptide-1 receptor agonists (GLP-1RAs) offers a promising therapeutic avenue for the treatment of type 2 diabetes mellitus (T2DM) and obesity. An extensive in silico approach combining structure-based drug design and ligand-based strategies together with pharmacokinetic properties and drug-likeness predictions is implemented to identify novel non-peptidic GLP-1RAs from the COCONUT and Marine Natural Products (CMNPD) libraries. More than 700,000 compounds were screened by shape-based similarity filtering in combination with precision docking against the orthosteric site of the GLP-1 receptor (PDB ID: 6X1A). The docked candidates were further assessed with the molecular mechanics MM-GBSA tool to check the binding affinities; the final list of candidates was validated by running a 500 ns long MD simulation. Twenty final hits were identified, ten from each database. The hits contained compounds with reported antidiabetic effects but with no evidence of GLP-1 agonist activity, including hits 1, 6, 7, and 10. These findings proposed a novel mechanism for these hits through GLP-1 activity and positioned the other hits as potential promising scaffolds. Among the studied compounds—especially hits 1, 5, and 9—possessed strong and stable interactions with critical amino acid residues such as TRP-203, PHE-381, and GLN-221 at the active site of the 6X1A-substrate along with favorable pharmacokinetic profiles. Moreover, the RMSF and RMSD plots further suggested the possibility of stable interactions. Specifically, hit 9 possessed the best docking score with a ΔG_bind value of −102.78 kcal/mol, surpassing even the control compound in binding affinity. The ADMET profiling also showed desirable drug-likeness and pharmacokinetic characteristics for hit 9. The pipeline of computational integration underscores the potential of non-peptidic alternatives in natural product libraries to pursue GLP-1-mediated metabolic therapy into advanced preclinical validation. Full article

Gonadotropin-Releasing Hormone (GnRH) is a crucial neuropeptide that regulates reproductive functions in vertebrates. The study identifies and characterizes (GnRH) in the brain of Tenualosa ilisha, an iconic and lucrative Clupeiform fish from River Ganga, India. The current study aimed to analyze the GnRH gene in T. ilisha using an in silico study. The GnRH gene of T. ilisha comprises a full-length nucleotide sequence of 605 base pairs with an open reading frame of 312 base pairs, which encodes 103 deduced amino acids (aa), respectively. It was found that leucine (L) is the most abundant amino acid in the GnRH protein. Additionally, the ligand interactions of the GnRH were analyzed using computational approaches. The structural validation showed an excellent stereochemical quality of the GnRH protein sequence, with over 88% of residues in Ramachandran plot-favored regions. The binding site prediction revealed 6 ligand-binding pockets, with the largest pocket containing 12 amino acids. After ADME screening, 16 drug-like compounds were docked to GnRH protein. Top five ligands N-Ac-(4-Cl-Phe)-Trp-Lys-AlaNH2, LHRH_LYS (6), Seabream_GnRH, Leuprolide, and LHRH_Des-tyr (5) had binding affinities ranging from −7.5 to −5.6 kcal/mol. The stable binding site was confirmed by 100 ns molecular dynamics simulations, with RMSD values below 10 Å and key residues retaining ligand contacts. The GnRH-protein resulted in the development of a suitable peptide sequence of T. ilisha, showing similarity with the similar anadromous American shad (Alosa sapidissima). This will certainly aid in future therapeutic and captive breeding advances, thereby fostering the culture and conservation of the wild species. Full article

Background/Objectives: Tetrastatin, the globular non collagenous (NC1) domain of the α4 chain of collagen IV, was previously demonstrated to inhibit melanoma progression. We identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin and demonstrated its anti-angiogenic activity mediated through αvβ3 and α5β1 binding. As QS-13 peptide was not fully soluble in aqueous solution, we designed new peptides with better water solubility. The present work aimed to investigate the interactions of ten QS-13-derived peptides, exhibiting improved hydro-solubility, with αvβ3 and α5β1 integrins. Methods: Using bioinformatics tools such as GROMACS, VMD, and the Autodock4 suite, we investigated the ability of the substituted peptides to bind αvβ3 and α5β1 integrins in silico. Results: We demonstrated in silico that all substituted peptides were able to bind both integrins at the RGD-binding site and determined their theoretical binding energy. Conclusions: The new soluble peptides should be able to compete with natural integrin ligands such as fibronectin, but also FGF1, FGF2, IGF1, and IGF2. Taken together, these findings suggest that the QS-13-derived peptides are reliable anti-angiogenic and anti-tumor agents. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

Recent bioassay studies have unexpectedly supported the high (computationally predicted) binding affinities of angiotensin receptor blockers (ARBs) at α-adrenergic receptors (αARs) in isolated smooth muscle. Computational predictions from ligand docking studies are consistent with very low concentrations of ARBs (e.g., sartans or bisartans) that partially reduce (20–50%) the contractile response to phenylephrine, suggesting that some ARBs may function as partial inverse agonists at αARs. Virtual ligand screening (docking) and molecular dynamics (MD) simulations were carried out to explore the binding affinities and stabilities of selected non-peptide ligands (e.g., ARBs and small-molecule opioids) for several G-protein coupled receptor (GPCR) types, including angiotensin II (AngII) type 1 receptor (AT₁R), α1AR, α2AR, and μ-(µOR) and ժ-opioid receptors (ժOR). Results: All ligands docked preferentially to the binding pocket on the cell surface domain of the GPCR types investigated. Drug binding was characterized by weak interactions (hydrophobic, hydrogen bonding, pi-pi) and stronger ionic and salt-bridge interactions (cation-pi and cation-anion interactions). Ligands specific to each GPCR category showed considerable cross-binding with alternative GPCRs, with small-molecule medications appearing less selective than their peptide or ARB functional equivalents. ARBs that exhibit higher affinities for AT₁R also demonstrate higher affinities for µORs and ժORs than opiate ligands, such as fentanyl and naltrexone. Moreover, ARBs had a higher affinity for αARs than either alpha agonists (epinephrine and phenylephrine) or inhibitors (prazosin and doxazosin). MD simulations of membrane-embedded ARB-GPCR complexes proved stable over nanosecond time scales and suggested that some ARBs may behave as agonists or antagonists depending on the GPCR type. Based on the results presented in this and related investigations, we propose that agonists bind to the resting A-site of GPCRs, while inverse agonists occupy the desensitizing D-site, which partial agonists like morphine and fentanyl share, contributing to addiction. ARBs block both AngII and alpha receptors, suggesting that they are more potent antihypertensive drugs than ACE inhibitors. ARBs have the potential to inhibit morphine tolerance and appear to disrupt receptor desensitization processes, potentially by competing at the D-site. Our results suggest the possible therapeutic potential of ARBs in treating methamphetamine and opiate addictions. Full article

Small-molecule glucagon-like peptide-1 receptor agonists (GLP-1RAs) represent an innovative advancement in oral therapeutics, addressing key limitations associated with injectable peptide-based incretin therapies. These nonpeptidic agents exert their actions primarily through non-canonical binding orthosteric sites within the GLP-1 receptor transmembrane domain, enabling selective G protein (Gs)-biased signaling with reduced β-arrestin-mediated adverse effects. Orforglipron has notably advanced through Phase 3 clinical development, demonstrating significant reductions in hemoglobin A1c and body weight (up to 7.9%) with favorable tolerability. Conversely, promising candidates such as danuglipron and lotiglipron were discontinued due to hepatotoxicity, underscoring critical safety concerns intrinsic to small-molecule GLP-1RA development. Current clinical candidates, including GSBR-1290, CT-996, and ECC5004, continue to offer substantial potential due to their oral bioavailability, simplified dosing regimens, and favorable gastrointestinal tolerability. Nevertheless, challenges persist regarding hepatic safety, pharmacodynamic variability, and limited long-term outcome data. This review integrates current structural, pharmacological, and clinical evidence, highlights key mechanistic innovations—including biased agonism, covalent binding strategies, and allosteric modulation—and discusses future directions for this rapidly evolving therapeutic class in metabolic disease management. Full article

Background: The well-studied 18-residue-long proline-rich antimicrobial designer peptide Api137 utilizes at least two lethal intracellular mechanisms that target the bacterial 70S ribosome. First, Api137 stalls the ribosome by binding to the peptidyl-transferase center, trapping the release factor, and inhibiting protein expression. Second, Api137 disrupts the assembly of the large 50S subunit of the ribosome, resulting in partially assembled pre-50S dead-end particles that are unable to form the functional 70S ribosome. Methods: All six proline residues in Api137 were substituted with 4S- and 4R-fluoro-l-proline (Fpr), which promote the cis- and trans-conformer ratio of the preceding Xaa-Pro-bond, respectively. The effect on the antibacterial activity was studied using Escherichia coli. The underlying mechanisms were investigated by studying 70S ribosome binding, inhibition of in vitro translation, and ribosome profile analysis. Results: Interestingly, the analogs were equipotent to Api137, except for the 4S-Fpr11 and 4S-Fpr16 analogs, which were four times more or less active, respectively. The most active 4S-Fpr11 analog competed the least with Api137 for its ribosome binding site, suggesting a shifted binding site. Both Fpr14 and the 4S-Fpr16 analogs disturbed 50S subunit assembly less than Api137 or not at all. The strongest effect was observed with the 4R-Fpr16 analog resulting in the lowest 70S ribosome content and the highest pre-50S particle content. This peptide also showed the strongest competition with Api137 for its binding site. However, its antibacterial activity was similar to that of Api137, possibly due to its slower cellular uptake. Conclusions: Api137 inhibits protein translation and disrupts 50S assembly, which can be adjusted by substituting specific proline residues with fluoroproline. 4R-Fpr16 potently inhibits ribosome assembly and offers a novel, unexploited clinical mechanism for future antibiotic development. Full article

The human coagulation pathway orchestrates a complex series of events vital for maintaining vascular integrity, in which the intrinsic pathway plays a pivotal role in amplifying and propagating the coagulation response. Dysregulation of this pathway can lead to various bleeding disorders and thrombotic complications, posing significant health risks. In this pathway, the activation of Factor (F) X zymogen is catalyzed by the FVIIIa-FIXa binary complex, but knowledge about this is still incomplete. Understanding the structural and functional intricacies of the FVIIIa-FIXa-FX (zymogen) complex is imperative for unraveling the molecular mechanisms underlying coagulation regulation and guiding the development of targeted therapeutic interventions. In this study, utilizing Alphafold-Multimer and molecular dynamics (MD) simulations, we provide insights into factor interactions within the ternary complex and propose novel functional mechanisms contributing to the functional defects inflicted by their cross-reactive material (CRM) positive mutations. The amino acid residue replacement impairs the coagulation function by interfering with structure elements, including the following: (1) a knot-like structure between Arg-562 of FVIIIa’s 558-Loop (residue 555–571) and the 333-Loop of FIXa (residue 333–346) contributes to FVIIIa-FIXa binding; (2) the a2 region of FVIIIa (residue 716–740) opens the lid of active site (FIXa’s 266-Loop, residue 256–270) and facilitates substrate binding; (3) the activation peptide (AP) of FX zymogen (residue 143–194) not only assists in the activation of itself but also adheres the interface of the three factors like a double-sided tape. Our work provides novel insights for the pathogenesis of a number of reported clinical CRM-positive mutations and may lay the groundwork for the structure-based development of therapeutic interventions. Full article

The proteolytic processing of the SARS-CoV-2 spike glycoprotein by host cell membrane-associated proteases is a key step in both the entry of the invading virus into the cell and the release of the newly generated viral particles from the infected cell. Because of the critical importance of this step for the viral infectivity cycle, it has been a target of extensive efforts aimed at identifying highly specific protease inhibitors as potential antiviral agents. An alternative strategy to disrupt the pre-fusioviden processing of the SARS-CoV-2 S glycoprotein aims to protect the substrate rather than directly inhibit the proteases. In this work, we focused on furin, a serine protease located primarily in the Golgi apparatus, but also present on the cell membrane. Its cleavage site within the S glycoprotein is located within the stalk region of the latter and comprises an arginine-rich segment (SPRRARS), which fits the definition of the Cardin–Weintraub glycosaminoglycan recognition motif. Native mass spectrometry (MS) measurements confirmed the binding of a hexadecameric peptide representing the loop region at the S1/S2 interface and incorporating the furin cleavage site (FCS) to heparin fragments of various lengths, as well as unfractionated heparin (UFH), although at the physiological ionic strength, only UFH remains tightly bound to the FCS. The direct LC/MS monitoring of FCS digestion with furin revealed a significant impact of both heparin fragments and UFH on the proteolysis kinetics, although only the latter had IC50 values that could be considered physiologically relevant (0.6 ± 0.1 mg/mL). The results of this work highlight the importance of the long-range and relatively non-specific electrostatic interactions in modulating physiological and pathological processes and emphasize the multi-faceted role played by heparin in managing coronavirus infections. Full article

Background/Objectives: The increasing threat of antibiotic resistance and the declining efficiency of traditional drug discovery pipelines highlight the urgent need for novel drug targets and effective enzyme inhibitors against infectious diseases. Oligopeptidase B (OPB), a serine protease with trypsin-like specificity that processes low-molecular-weight peptides and oligopeptides, is present in bacteria and certain parasites but absent in mammals. This unique distribution makes OPB an attractive and selective target for antimicrobial drug development. Methods: Three-dimensional models of OPB from Serratia marcescens and Stenotrophomonas maltophilia, previously identified by our research group, were constructed via homology modeling using the best available OPB template from the RCSB Protein Data Bank. The S. marcescens OPB model was subjected to high-throughput virtual screening (HTVS) against the Natural Products Atlas (npatlas) database. Top-ranking compounds were further evaluated using Glide standard precision (SP) and extra precision (XP) docking protocols. Binding affinities were refined using molecular mechanics with generalized born and surface area (MM–GBSA) calculations. Molecular dynamics (MD) simulations assessed binding stability, while absorption distribution metabolism excretion and toxicity (ADMET) profiling evaluated drug-likeness and pharmacokinetic properties. Results: Ten natural product compounds demonstrated stronger binding affinities than antipain, a well-known oligopeptide-based protease inhibitor, as indicated by their more favorable MM–GBSA scores of −60.90 kcal/mol (S. marcescens) and −27.07 kcal/mol (S. maltophilia). Among these, dichrysobactin and validamycin E consistently exhibited favorable binding profiles across both OPB models. MD simulations confirmed the stability of their interactions with OPB active sites, maintaining favorable binding conformations throughout the simulation period. ADMET analysis suggested that while both compounds show promise, lead optimization is required to enhance their drug-like characteristics. Conclusions: This study identifies dichrysobactin and validamycin E as promising OPB inhibitors with potential antimicrobial activity. These findings support their further development as selective and potent agents against bacterial pathogens, including resistant strains, and underscore the need for experimental validation to confirm their efficacy and safety. Full article