Protein Affinity Research Articles

Essential oil from Fructus Alpiniae zerumbet ameliorates vascular endothelial cell senescence in diabetes by regulating PPAR-γ signalling: A 4D label-free quantitative proteomics and network pharmacology study

Ethnopharmacological relevanceVascular endothelial cell senescence is associated with cardiovascular complications in diabetes. Essential oil from Fructus Alpiniae zerumbet (Pers.) B.L.Burtt & R.M.Sm. (EOFAZ) has potentially beneficial and promising diabetes-related vascular endothelial cell senescence-mitigating effects; however, the underlying molecular mechanisms remain unclear. Aim of the studyTo investigate the molecular effects of EOFAZ on vascular endothelial cell senescence in diabetes. Materials and methodsA diabetes mouse model was developed using a high-fat and high-glucose diet (HFD) combined with intraperitoneal injection of low-dose streptozotocin (STZ, 30 mg/kg) and oral treatment with EOFAZ. 4D label-free quantitative proteomics, network pharmacology, and molecular docking techniques were employed to explore the molecular mechanisms via which EOFAZ alleviates diabetes-related vascular endothelial cell senescence. A human aortic endothelial cells (HAECs) senescence model was developed using high palmitic acid and high glucose (PA/HG) concentrations in vitro. Western blotting, immunofluorescence, SA-β-galactosidase staining, cell cycle, reactive oxygen species (ROS), cell migration, and enzyme linked immunosorbent assays were performed to determine the protective role of EOFAZ against vascular endothelial cell senescence in diabetes. Moreover, the PPAR-γ agonist rosiglitazone, inhibitor GW9662, and siRNA were used to verify the underlying mechanism by which EOFAZ combats vascular endothelial cell senescence in diabetes. ResultsEOFAZ treatment ameliorated abnormal lipid metabolism, vascular histopathological damage, and vascular endothelial aging in diabetic mice. Proteomics and network pharmacology analysis revealed that the differentially expressed proteins (DEPs) and drug-disease targets were associated with the peroxisome proliferator-activated receptor gamma (PPAR-γ) signalling pathway, a key player in vascular endothelial cell senescence. Molecular docking indicated that the small-molecule compounds in EOFAZ had a high affinity for the PPAR-γ protein. Western blotting and immunofluorescence analyses confirmed the significance of DEPs and the involvement of the PPAR-γ signalling pathway. In vitro, EOFAZ and rosiglitazone treatment reversed the effects of PA/HG on the number of senescent endothelial cells, expression of senescence-related proteins, the proportion of cells in the G0/G1 phase, ROS levels, cell migration rate, and expression of pro-inflammatory factors. The protective effects of EOFAZ against vascular endothelial cell senescence in diabetes were aborted following treatment with GW9662 or PPAR-γ siRNA. ConclusionsEOFAZ ameliorates vascular endothelial cell senescence in diabetes by activating PPAR-γ signalling. The results of the present study highlight the potential beneficial and promising therapeutic effects of EOFAZ and provide a basis for its clinical application in diabetes-related vascular endothelial cell senescence.

Abstract C081: Novel and selective inhibitors of KRASG12V

Abstract KRAS is one the most mutated proteins in cancers and are commonly identified in pancreatic adenocarcinoma, colorectal and non-small cell lung cancer. Owing to its role in a different human cancer, KRAS has long been considered as an important therapeutic target in precision oncology. Although KRAS was considered undruggable due to the protein’s structural complexity, recent allele specific inhibitors that covalently bind and trap KRAS in its inactive GDP bound state have demonstrated promising approach for generating anti RAS therapies and is validated by the accelerated approval of Sotorasib and Adagrasib by the FDA for patients with lung cancer. Success of targeting mutant KRASG12C has opened opportunities to tackle other oncogenic KRAS variants such as KRASG12V. KRASG12V mutations occur frequently in multiple tumour histotypes such as adenocarcinoma of Pancreas (~32%), colorectal adenocarcinoma (~20%) and Lung (~19%) and represents a significant unmet medical need. Here we describe preclinical profile of a potent and selective KRASG12V inhibitor VRTX171. VRTX171 is a tool compound that was potent across a KRASG12V mutated cancer cell line and found to be several fold less active in non-KRASG12V mutant cell lines. Using a de novo drug design approach the compound was optimized to offer desired potency with high selectivity over WT KRAS and other KRAS mutant isoforms. Affinity for target protein, was assessed by HTRF based target engagement assay and inhibition of Raf/Mitogen-activated protein kinase (MAPK) pathway was determined using Western blot analysis in multiple cancer cell lines harbouring KRASG12V mutation. In-vivo studies of VRTX171 in Xenograft models harbouring KRASG12V mutations, as single agent as well as in combination with other targeted therapies are under consideration. Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi. Novel and selective inhibitors of KRASG12V [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C081.

A Hybrid Design for the Functional Assay of LvLRRm (Protein Containing LRR Domain) of the White Leg Shrimp, Litopenaeus vannamei

In the crustacean immune system, leucine-rich repeat (LRR) is one of the major structures for recognizing pathogen-associated molecular patterns (PAMPs). LRR domain-containing proteins belong to the LRR family, which is a large group of proteins with more than 6000 genes in the database. They are involved in very diverse physiological functions, mainly by interacting with other proteins. In a previous study, the LvLRRm, a transmembrane protein containing only LRR domain, was identified in the white leg shrimp, Litopenaeus vannamei. Its versatile role in performing multiple immunomodulation activities has been reported. However, there is still a lack of research on its efficient function at the protein level. To investigate its interactions with other proteins, we applied a convenient method called the ‘Hybrid LRR technique’ to produce a recombinant LvLRRm. The LvLRRm and hagfish’s variable lymphocyte receptors (VLRs) fragments were fused to the conserved LxxLxLxxN motif while retaining the β-strand. In addition, we established interactions between hybrid proteins and the flagellin of Salmonella typhimurium by performing surface plasmon resonance (SPR) analysis. The results of the SPR analysis demonstrated notable affinity for both LvLRRm and hybrid proteins towards Salmonella flagellin. The designed LvLRRm hybrid proteins bring insight for universal applications without losing protein functions.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p < 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p < 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p < 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.

A First-in-Human, Open-Label, Phase I Dose Escalation Trial of Daily Oral Nmt Inhibitor Zelenirstat in Patients with Relapsed/Refractory B-Cell Lymphomas and Advanced Solid Tumors

Background Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, regulates multiple membrane-bound signal transduction pathways driving cancer cell biology. This modification is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2. Aberrant NMT expression has been identified in cancer cells and inhibition of myristoylation represents a novel anticancer treatment strategy. Zelenirstat (PCLX-001) is an oral, highly bioavailable, small molecule NMT inhibitor with strong affinity for both NMT1 and NMT2 proteins (IC 50 of 5nM and 8nM, respectively). In vitro hematologic cancer cell lines were exquisitely sensitive to zelenirstat. Zelenirstat regressed subcutaneous tumors in xenograft models derived from lymphoma cell lines, as well as in a refractory DLBCL patient (pt) derived xenograft model. Zelenirstat also demonstrated anticancer activity in multiple solid tumor xenograft models. Based on pre-clinical data, we conducted a phase I dose escalation trial to determine safety, tolerability, maximum tolerated dose (MTD), and pharmacokinetics (PK) of zelenirstat. Methods Pts with relapsed/refractory (R/R) B-cell lymphomas and advanced solid tumors were enrolled in a multicenter, open label, standard phase I dose escalation trial of once daily (OD) oral zelenirstat, administered in 28-day cycles until progressive disease or unacceptable toxicity. Seven dose levels were studied: 20 mg, 40 mg, 70 mg, 100 mg, 140 mg, 210 mg, and 280 mg. Dose limiting toxicity (DLT), attributable to zelenirstat during cycle 1, was defined as: grade (Gr) 4 thrombocytopenia, Gr 3 thrombocytopenia with bleeding, Gr 4 neutropenia ≥ 7 days, febrile neutropenia, clinically significant > Gr 3 non-hematologic toxicity, or any significant toxicity warranting a hold of zelenirstat. Plasma PK were obtained on C1D1, C1D15, and trough concentrations C1D2, C1D16 and prior to each subsequent cycle. Response assessments occurred every 2 cycles to determine best overall response. Results Between Sept 2021 and Jul 2023, 21 DLT-evaluable pts [4 R/R B-cell lymphoma (3 DLBCL, 1 FL grade 3b); 17 advanced solid tumor (7 colorectal, 2 pancreas, 2 ovarian, 1 each of sarcoma, NSCLC, pleural mesothelioma, melanoma, CUP, bladder)] received zelenirstat at doses ranging from 20 mg OD to 280 mg OD. Dose escalation occurred without DLT until the 280 mg cohort where two DLTs were reported: a bladder carcinoma pt who developed nausea, vomiting, and drug-induced fever necessitating a hold of zelenirstat and a colorectal cancer (CRC) pt who had Gr 3 dehydration from nausea, vomiting, diarrhea, and oral mucositis. The maximal administered dose was 280 mg OD and the presumptive MTD was 210 mg OD. Drug compliance was high, and of 3 pts within the 210 mg cohort, none required dose reduction. Common treatment-emergent all-grade adverse events (AEs) at doses up to the 210 mg cohort were gastrointestinal and reported in 29% of pts. These were primarily diarrhea, nausea, or vomiting. Gr 2 thrombocytopenia occurred in 17% of patients. No other AEs were seen at a frequency of > 5%. There were no AEs of neuropathy, alopecia, or QT interval prolongation. Noncompartmental PK analyses demonstrated rapid oral absorption, a terminal half-life of approximately 10 hrs, rapid achievement of steady state, and no induction of metabolism. Zelenirstat trough plasma concentrations at 100 mg, 140 mg, and 210 mg doses markedly exceeded the EC 90 required to inhibit cultured cancer cells. Stable disease as best response was seen in five patients (pancreas at 40 mg; CRC at 140 mg; and two ovarian and one CRC at 210 mg). At the 210 mg dose, one of the ovarian cancer pts had a corresponding reduction of CA-125 (20%) while the CRC pt had a reduction in CEA (50%). Conclusions Zelenirstat is safe and well tolerated up to the 210 mg OD dose, which we have established as the presumptive MTD. Additional pts, including a phase IIA study in pts with R/R B-cell NHL, are accruing at this dose for further safety and activity exploration. The absence of severe toxicities, attainment of plasma concentrations highly active in preclinical models, and early evidence of antitumor activity support the ongoing development of zelenirstat for pts with R/R B-cell lymphomas and advanced solid tumors.

Nucleolin binds to and regulates transcription of hepatitis B virus covalently closed circular DNA minichromosome.

Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.

Acetylcholine-Binding Protein Affinity Profiling of Neurotoxins in Snake Venoms with Parallel Toxin Identification

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins.

Designing of fragment based inhibitors with improved activity against E. coli AmpC β-lactamase compared to the conventional antibiotics

One of the most common primary resistance mechanism of multi-drug resistant (MDR) Gram negative pathogenic bacteria to combat β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems is the generation of β- lactamases. The uropathogenic E. coli is mostly getting multi-drug resistance due to the synthesis of AmpC β-lactamases and therefore new antibiotics and inhibitors are needed to treat the evolving infections. The current study was designed for targetting AmpC β-lactamase of E. coli using molecular docking based virtual screening, linking fragments for designing novel compounds and binding mode analysis using molecular dynamic simulation with target protein. The FCH group all-purpose fragment library consisting of 9388 fragments has been screened against AmpC β-lactamase protein of E. coli and the antibiotics and anti-infectives used in treatment of Urinary tract Infections (UTIs) were also screened with AmpC β-lactamase protein. Among the 9388 fragments, 339 fragment candidates were selected and linked with cefepime antibiotic having maximum binding affinity for AmpC target protein. Computational analysis of interactions as well as molecular dynamics (MD) simulations were also conducted for identifying the most promising ligand-pocket complexes from docking investigations to comprehend their thermodynamic properties and verify the docking outcomes as well. Overall, the linked complexes (LCs) showed good binding interactions with AmpC β-lactamase. Interestingly, our fragment-based LCs remained relatively stable in comparison with cefepime antibiotic. Moreover, S12 fragment linked complex remained the most stable during 50 ns with remarkable number of interactions indicating it as promising candidate in novel lead discovery against MDR E. coli infections.

Enantioselective metabolism of fenpropathrin enantiomers by carboxyl/choline esterase 6 in Tetranychus cinnabarinus.

Tetranychus cinnabarinus is a polyphagous pest mite commonly found in agriculture. As an excellent acaricide, fenpropathrin (FEN) is frequently used to control T. cinnabarinus in agriculture. However, commercial FEN is a racemate with two enantiomers, R-FEN and S-FEN. Considering that investigations on the metabolism of FEN by T. cinnabarinus are based on racemate FEN, it is important to investigate the enantioselective metabolism of FEN in T. cinnabarinus. S-FEN was more toxic to T. cinnabarinus than R-FEN by more than 68.8-fold. Moreover, the synergist bioassay revealed that carboxylesterase and cytochrome P450 were the primary enzymes engaged in the detoxification of FEN in T. cinnabarinus, with carboxylesterase playing a leading role. Seven genes were substantially different after the induction of S-FEN and R-FEN. TcCCE06 was screened and selected as a key gene that related to FEN metabolism in T. cinnabarinus. The metabolic results showed that the recombinant TcCCE06 effectively metabolized 32.1% of the R-FEN and 13.8% of the S-FEN within 4 h of incubation. Moreover, R-FEN was demonstrated to have a higher affinity for the TcCCE06 protein than S-FEN based on molecular docking. Our results indicated that TcCCE06 mediates the enantioselective metabolism of FEN in T. cinnabarinus. Our findings will contribute to a more comprehensive understanding of the mechanisms underlying the differential toxicity of the FEN enantiomers against T. cinnabarinus. Furthermore, they also provide a new perspective for the development of enantiomer-enriched acaricides with higher activity and lower pesticide dosage and pollution risks. © 2023 Society of Chemical Industry.

Insights into the structural, electronic, quantum chemical properties and molecular docking studies on novel NAMPT inhibitor molecule

This research paper presents a comprehensive study on a novel NAMPT (Nicotinamide phosphoribosyl transferase) inhibitor molecule, N- [4- [(4R)-1,4-dimethyl-6-oxidanylidene-4,5-dihydropyridazin-3-yl] phenyl]-5,7-dihydropyrrolo [3,4-b] pyridine-6-carboxamide], with the potential applications in cancer therapy. We performed quantum chemical calculations with DFT and topological investigations on the molecule to understand its electronic properties and chemical behaviour. The study covers basic molecular properties, molecular planarity and various electronic properties, including the HOMO (Highest Occupied Molecular Orbital), LUMO (Lowest Unoccupied Molecular Orbital) energy levels, the energy gap of 4.09 eV, ionization potential, electron affinity, electronegativity, chemical potential, chemical hardness, chemical softness, electrophilicity index, Mulliken atomic charges, Laplacian bond order, and ESP (Electrostatic Potential) properties. Moreover, we studied the nature of bonding and interactions within the molecule using ELF (Electron Localization Function), LOL (Localized Orbital Locator), RDG (Reduced Density Gradient), and NCI (Non-covalent Interaction). An analysis of the title molecule's molecular thermodynamic properties demonstrates its intrinsic stability under thermal and quantum effects. Molecular docking studies were also performed to reveal how the molecule interacts with its target protein, exhibiting a strong binding affinity for NAMPT target proteins (7PPF and 5WI0), which could help develop future cancer drugs. This multifaceted investigation provides a detailed understanding of the molecular structure and interactions of the molecule with NAMPT protein, potentially paving the way for its potential use as a cancer therapy.

Cu (II)-catalyzed: synthesis of imidazole derivatives and evaluating their larvicidal, antimicrobial activities with DFT and molecular docking studies

This paper deals with the evaluation of novel imidazole molecules for their antimicrobial and larvicidal activities. A series of imidazole derivatives 1(a–f) and 2(a–e) were prepared by the Mannich base technique using a Cu(II) catalyst. The Cu(phen)Cl2 catalyst was found to be more effective than other methods. FTIR, elemental analyses, mass spectrometry, 1H NMR, and 13C NMR spectroscopy were performed to elucidate the structures of the synthesised compounds. Antimicrobial and larvicidal activities were investigated for all compounds. The antibacterial activity of compounds (2d) and (2a) were highly active in S.aureus (MIC: 0.25 μg/mL) and K.pneumoniae (MIC: 0.25 μg/mL) compared to ciprofloxacin. Compound (1c) was significantly more effective than clotrimazole in C.albicans (MIC: 0.25 μg/mL). Molecular docking studies of compound 2d showed a higher binding affinity for the 1BDD protein (− 3.4 kcal/mol) than ciprofloxacin (− 4.4 kcal/mol). Compound 1c had a higher binding affinity (− 6.0 kcal/mol) than clotrimazole (− 3.1 kcal/mol) with greater frontier molecular orbital energy and reactivity properties of compound 1c (∆E gap = 0.13 eV). The activity of compound 1a (LD50: 34.9 μg/mL) was more effective in the Culex quinquefasciatus than permethrin (LD50: 35.4 μg/mL) and its molecular docking binding affinity for 3OGN protein (− 6.1 kcal/mol). These newly synthesised compounds can act as lead molecules for the development of larvicides and antibiotic agents.

Design and synthesis of novel hederagonic acid analogs as potent anti-inflammatory compounds capable of protecting against LPS-induced acute lung injury

Acute lung injury (ALI) presents a significant clinical challenge due to its high mortality rates and the lack of effective treatment strategies. The most effective approaches to treating ALI include disrupting inflammatory cascades and associated inflammatory damage within the lung. Hederagenin was utilized as a core skeleton to design and synthesize 33 hederagonic acid derivatives. Among these derivatives, compound 29 demonstrated potent anti-inflammatory activity without inducing cytotoxicity, inhibiting nitric oxide (NO) release by 78–86 %. Detailed structure-activity relationship studies and the reverse virtual screening of ALI-related targets revealed that compound 29 exhibits a high affinity for the STING protein. Mechanistic studies revealed that compound 29 suppresses macrophage activation, inhibits the nuclear translocation of IRF3 and p65, and disrupts the STING/IRF3/NF-κB signaling pathway, thereby attenuating the inflammatory response. The in vivo administration of compound 29 was sufficient to protect against lipopolysaccharide (LPS)-induced ALI by suppressing the production of inflammatory mediators, including IL-6, TNF-α, and IFN-β, thereby preserving lung tissue integrity. These results substantiate the anti-inflammatory efficacy of compound 29, both in vitro and in vivo, indicating its potential as a promising lead compound in ALI treatment strategies.

Lupenone, a wonder chemical obtained from Euphorbia segetalis to boost affinity for the transcriptional factor escalating drought-tolerance in Solanum Lycopersicum: A cutting-edge computational biology approach.

Drought is the single greatest abiotic factor influencing crop yield worldwide. Plants remain in one area for extended periods, making them vulnerable to natural and man-made influences. Understanding plant drought responses will help us develop strategies for breeding drought-resistant crops. Large proteome analysis revealed that leaf and root tissue proteins respond to drought differently depending on the plant's genotype. Commonly known as tomatoes, Solanum Lycopersicum is a globally important vegetable crop. However, drought stress is one of the most significant obstacles to tomato production, making the development of cultivars adapted to dry conditions an essential goal of agricultural biotechnology. Breeders have put quite a lot of time and effort into the tomato to increase its productivity, adaptability, and resistance to biotic and abiotic challenges. However, conventional tomato breeding has only improved drought resistance due to the complexity of drought traits. The resilience of tomatoes under drought stress has been the subject of extensive study. Using contemporary sequencing approaches like genomics, transcriptomics, proteomics, and metabolomics has dramatically aided in discovering drought-responsive genes. One of the most prominent families of plant transcription factors, WRKY genes, plays a crucial role in plant growth and development in response to natural and abiotic stimuli. To develop plants that can withstand both biotic and abiotic stress, understanding the relationships between WRKY-proteins (transcription factors) and other proteins and ligands in plant cells is essential. This is despite the fact that tomatoes have a long history of domestication. This research aims to utilize Lupenone, a hormone produced in plant roots in response to stress, to increase drought resistance in plants. Lupenone exhibits a strong affinity for the WRKY protein at -9.64 kcal/mol. Molecular docking and modeling studies show that these polyphenols have a significant role in making Solanum Lycopersicum drought-resistant and improving the quality of its fruit. As a result of climate change, droughts are occurring more frequently and persisting for more extended periods, making it necessary to breed crops resistant to drought. While considerable variability for tolerance exists in wild cousins, little is known about the processes and essential genes influencing drought tolerance in cultivated tomato species.

New Insights Into Pyridoxal Kinase Inhibitors and Their Antileukemic Effects.

Pyridoxal kinase (PDXK) plays a pivotal role as an essential enzyme in cellular processes. It catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine to generate pyridoxal 5'-phosphate (PLP), the bioactive form of vitamin B6. An intriguing link has emerged between elevated expression levels of PDXK and PLP and various types of carcinomas, including leukemia. Leukemic cells have an increased need for vitamin B6 to sustain their survival and rapid growth, highlighting the potential of targeting PDXK-PLP as a promising therapeutic target in cancer treatment. To discover a novel and promising PDXK inhibitor, we conducted a comprehensive screening of compounds derived from both natural sources and drug-like databases. Our approach involved employing structure-based virtual screening and molecular docking techniques to attenuate the phosphorylation of PLP. Among the top six compounds, ZINC095099376 (referred to as C03) emerged as the most potent inhibitor of PDXK, primarily due to its exceptional binding affinity and remarkable specificity for the target protein. Furthermore, our investigation revealed that compound C03 establishes crucial interactions with key residues within the substrate binding site, indicating that it binds at the same site as the co-crystallized ligand. Remarkably, compound C03 inhibited the endogenous PDXK expression, showed anti-proliferative activity, and triggered an intrinsic pathway for apoptosis via the activation of key apoptotic factors in leukemic cells. In summary, these findings strongly indicate that compound C03 holds promise as a novel inhibitor of PDXK, offering the potential for the development of effective treatments for leukemia.

Multifunctional Nanoclay-Based Hemostatic Materials for Wound Healing: A Review.

Bleeding to death accounts for around 30-40% of all trauma-related fatalities. Current hemostatic materials are mainly mono-functional or have insufficient hemostatic capacity. Nanoclay has been recently shown to accelerate hemostasis, improve wound healing, and provide the resulting multifunctional hemostatic materials antibacterial, anti-inflammatory, and healing-promoting due to its distinctive morphological structure and physicochemical properties. Herein, the chemical design and action mechanism of nanoclay-based hemostatic, antibacterial, and pro-wound healing materials in the context of wound healing are discussed. The physiological processes of hemostasis and wound healing to elucidate the significance of nanoclay for functional wound hemostatic dressing design are outlined. A summary of the features of various nanoclay and product types used in wound hemostatic dressings is provided. Nanoclay can be antimicrobial due to the slow release of metal ions and has an abundant surface charge allowing for high affinity for proteins and cells, which can activate the coagulation reaction or facilitate tissue repair. Nanoclay with a microporous structure can be used as drug carriers to create composites critical for inhibiting bacterial growth on wounds or promoting the regeneration of vascular, muscle, and skin tissues. Directions for further research and innovation of nanoclay-based multifunctional materials for hemostasis and tissue regeneration are explored.

In silico designing of multiepitope-based-peptide (MBP) vaccine against MAPK protein express for Alzheimer's disease in Zebrafish

Understanding the role of the mitogen-activated protein kinases (MAPKs) signalling pathway is essential in advancing treatments for neurodegenerative disorders like Alzheimer's. In this study, we investigate in-silico techniques involving computer-based methods to extract the MAPK1 sequence. Our applied methods enable us to analyze the protein's structure, evaluate its properties, establish its evolutionary relationships, and assess its prevalence in populations. We also predict epitopes, assess their ability to trigger immune responses, and check for allergenicity using advanced computational tools to understand their immunological properties comprehensively. We apply virtual screening, docking, and structure modelling to identify promising drug candidates, analyze their interactions, and enhance drug design processes. We identified a total of 30 cell-targeting molecules against the MAPK1 protein, where we selected top 10 CTL epitopes (PAGGGPNPG, GGGPNPGSG, SAPAGGGPN, AVSAPAGGG, AGGGPNPGS, ATAAVSAPA, TAAVSAPAG, ENIIGINDI, INDIIRTPT, and NDIIRTPTI) for further evaluation to determine their potential efficacy, safety, and suitability for vaccine design based on strong binding potential. The potential to cover a large portion of the world's population with these vaccines is substantial—88.5 % for one type and 99.99 % for another. In exploring the molecular docking analyses, we examined a library of compounds from the ZINC database. Among them, we identified twelve compounds with the lowest binding energy. Critical residues in the MAPK1 protein, such as VAL48, LYS63, CYS175, ASP176, LYS160, ALA61, LEU165, TYR45, SER162, ARG33, PRO365, PHE363, ILE40, ASN163, and GLU42, are pivotal for interactions with these compounds. Our result suggests that these compounds could influence the protein's behaviour. Moreover, our docking analyses revealed that the predicted peptides have a strong affinity for the MAPK1 protein. These peptides form stable complexes, indicating their potential as potent inhibitors. This study contributes to the identification of new drug compounds and the screening of their desired properties. These compounds could potentially help reduce the excessive activity of MAPK1, which is linked to Alzheimer's disease.

New oxindole carboxamides as inhibitors of DENV NS5 RdRp: Design, synthesis, docking and Biochemical characterization

Dengue is a mosquito-borne disease caused by the dengue virus belonging to family flaviviridae and has grown to be a major global public health issue. Despite decades of effort, the global comeback of dengue is evidence of the inadequacy of present management techniques. Due to the loss of healthy lives and the depletion of scarce medical resources, dengue has a significant negative economic impact in underdeveloped countries. In recent years, research for tackling the incidences of dengue infection has increased. The structure of the viral genome has been deciphered with the non-structural protein, known as NS5 serving as a potential target. NS5 consisting of an MTase domain involved in RNA capping and an RdRp domain involved in viral replication. In the presented work, a series of new Oxindoline Carboxamide derivatives were designed and synthesized for inhibiting the viral RNA dependent RNA-polymerase (RdRp) activity of DENV. The novel compounds were put through tests including molecular docking and surface plasmon resonance (SPR) binding analysis to evaluate their affinity for the viral protein and their potential as novel inhibitors of the virus. From a total of 12 derivative compounds, four compounds OCA-10c, OCA-10f, OCA-10j &OCA-10i, were found to exhibit high affinity for NS5 RdRp, the KD values being 1.376 μM, 1.63 μM, 7.08 μM & 9.32 μM respectively. Overall, we report novel inhibitors of DENV RdRp activity with potential to be utilized against DENV for treating humans after further optimization.

Anti-COVID-19, Anti-Inflammatory, and Anti-Osteoarthritis Activities of Sesamin from Sesamum indicum L.

During the COVID-19 (coronavirus disease 2019) outbreak, many people were infected, and the symptoms may persist for several weeks or months for recovering patients. This is also known as "long COVID" and includes symptoms such as fatigue, joint pain, muscle pain, et cetera. The COVID-19 virus may trigger hyper-inflammation associated with cytokine levels in the body. COVID-19 can trigger inflammation in the joints, which can lead to osteoarthritis (OA), while long-term COVID-19 symptoms may lead to joint damage and other inflammation problems. According to several studies, sesame has potent anti-inflammatory properties due to its major constituent, sesamin. This study examined sesamin's anti-inflammatory, anti-osteoarthritis, and anti-COVID-19 effects. Moreover, in vivo and in vitro assays were used to determine sesamin's anti-inflammatory activity against the RAW264.7 and SW1353 cell lines. Sesamin had a dose-dependent effect (20 mg/kg) in a monoiodoacetic acid (MIA)-induced osteoarthritis rat model. Sesamin reduced paw swelling and joint discomfort. In addition, the findings indicated that sesamin suppressed the expression of iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) in the RAW264.7 cell line within the concentration range of 6.25-50 μM. Furthermore, sesamin also had a suppressive effect on MMP (matrix metalloproteinase) expression in chondrocytes and the SW1353 cell line within the same concentration range of 6.25-50 μM. To examine the anti-viral activity, an in silico analysis was performed to evaluate sesamin's binding affinity with SARS-CoV-2 RdRp (severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase) and human ACE2 (angiotensin-converting enzyme 2). Compared to the controls, sesamin exhibited strong binding affinities towards SARS-CoV-2 RdRp and human ACE2. Furthermore, sesamin had a higher binding affinity for the ACE2 target protein. This study suggests that sesamin shows potential anti-SARS-CoV-2 activity for drug development.

Characterizing and predicting ccRCC-causing missense mutations in Von Hippel-Lindau disease.

Mutations within the Von Hippel-Lindau (VHL) tumor suppressor gene are known to cause VHL disease, which is characterized by the formation of cysts and tumors in multiple organs of the body, particularly clear cell renal cell carcinoma (ccRCC). A major challenge in clinical practice is determining tumor risk from a given mutation in the VHL gene. Previous efforts have been hindered by limited available clinical data and technological constraints. To overcome this, we initially manually curated the largest set of clinically validated VHL mutations to date, enabling a robust assessment of existing predictive tools on an independent test set. Additionally, we comprehensively characterized the effects of mutations within VHL using in silico biophysical tools describing changes in protein stability, dynamics and affinity to binding partners to provide insights into the structure-phenotype relationship. These descriptive properties were used as molecular features for the construction of a machine learning model, designed to predict the risk of ccRCC development as a result of a VHL missense mutation. Analysis of our model showed an accuracy of 0.81 in the identification of ccRCC-causing missense mutations, and a Matthew's Correlation Coefficient of 0.44 on a non-redundant blind test, a significant improvement in comparison to the previous available approaches. This work highlights the power of using protein 3D structure to fully explore the range of molecular and functional consequences of genomic variants. We believe this optimized model will better enable its clinical implementation and assist guiding patient risk stratification and management.

High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media.

Affinity-purification mass spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid-phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly nonspecific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from a dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing nonspecific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.