Innovative design of CXCR4 cyclic peptide via molecular docking for 99mTc-Labeled SPECT/CT imaging in melanoma.

The Potential of Peptide Chemistry: from Small Molecules up to Innovative Protein Engineering

The Chemokine Receptor CXCR4 and the Metalloproteinase MT1-MMP Are Mutually Required during Melanoma Metastasis to Lungs

Objective To investigate the role and possible mechanism of Chemokine receptor CXCR4 in the drug resistance of sorafenib in renal cell carcinoma. Methods 786-O cells were inoculated into the anterior sciatic region of nude mice subcutaneously, 5×106 cells per point. The mice were given normal saline and sorafenib intragastric (80 mg/kg, 1 time/day) when the transplanted tumor volume reached about 100 mm3. The tumor volume in the saline group was more than 1 500 mm3 at the 5th week, and the tumor was taken as the control tissue. Sorafenib group tumors started to grow accelerately at week 8, and the tumor volume was more than 1 500 mm3 at week 13. The 13th week tumors were used as resistant tissue. The expression of CXCR4 in control tissues and drug resistant tissues was detected by real-time quantitative PCR, western blotting and immunohistochemistry. The pcDNA3.1-CXCR4 plasmid was constructed and transfected into 786-O cells. The expression of CXCR4 was detected by real-time quantitative PCR and western blotting. The drug reactivity of the cells was measured by CCK-8 and monoclonal assay to compare the drug resistance of the control group, CXCR4 overexpression group and CXCR4 overexpression + CXCR4 inhibitor AMD3100 group. The phosphorylation of PKB, ERK and STAT3 in the control group, the sorafenib alone group, the overexpressing CXCR4+ sorafenib group and the overexpressing CXCR4+ sorafenib+ AMD3100 group were determined by Western blotting. Results Compared with the control tissues, the mRNA levels of CXCR4 in the drug-resistant tissues increased (3.22±0.23) times, and the levels of protein expression increased (2.33±0.47) according to western blotting, the differences were statistically significant (P<0.01). After overexpression of CXCR4, the mRNA expression of CXCR4 increased (78.3±5.3) times, and the protein expression level increased (2.80±0.95) times, and the differences were statistically significant (P<0.01), indicating that the expression model was established successfully. The drug response curves of the control group, CXCR4 overexpression group and CXCR4 overexpression+ AMD3100 group on sorafenib were measured by cck8 method, and the IC50 was (7.5±0.8) μmol/L, (10.3±0.7) μmol/L, (5.7±0.6) μmol/L, the differences were statistically significant (P<0.05); The numbers of clones formed in the above three groups were 26±5, 56±12 and 42±9, respectively. The differences were statistically significant (P<0.05). Sorafenib could reduce the phosphorylation of PKB, ERK and STAT3, and overexpression of CXCR4 could reverse the inhibition of phosphatidylation of PKB, ERK and STAT3 by sorafenib. After inhibition of chemokine receptor CXCR4 activity by AMD3100, PKB, ERK, STAT3 phosphorylation was re-suppressed. Conclusions CXCR4 can promote renal cell carcinoma sorafenib resistance. The expression of CXCR4 increased in secondary resistant tumor tissue increased; CXCR4 may promote drug resistance by activating the cell viable pathway. The inhibition of CXCR4 signaling pathway is expected to improve the therapeutic effect of sorafenib in renal cell carcinoma. Key words: Renal cell carcinoma; Chemokine receptor CXCR4; Sorafenib; Drug resistant

Preclinical evaluation of melanocortin-1 receptor (MC1-R) specific 68Ga- and 44Sc-labeled DOTA-NAPamide in melanoma imaging

Preclinical evaluation of MC1R targeting theranostic pair [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH

Recruitment of mononuclear leukocytes within atherosclerotic lesions is a critical step in atherogenesis. Mice lacking the chemokine receptor CCR2, highly expressed on macrophages but also on T lymphocytes, show a striking reduction of atherosclerotic lesion formation. The chemokine receptor CXCR3 is a marker of activated T helper type 1 lymphocytes, the principal T lymphocyte type detected within atheroma. We investigated whether the deletion of both of these 2 important receptors expressed on the principal inflammatory cells present in atheroma would further affect atherogenesis in vivo. We crossed ApoE(-/-) mice with either CCR2(-/-) or CXCR3- mice and crossed ApoE(-/-) CCR2(-/-) mice with the ApoE(-/-) CXCR3- mice to generate a triple knockout strain. Analysis of atherosclerosis development after 10 weeks of high-cholesterol diet revealed differential effects on early atherosclerotic lesions in the abdominal aorta and on advanced lesions in aortic roots. ApoE(-/-) CXCR3- mice, but not the triple knockout mice, displayed significantly reduced atherosclerotic lesion development within abdominal aortas compared with ApoE(-/-) CCR2(-/-) and ApoE(-/-) mice. This reduction of lesion formation correlated with an upregulation of antiinflammatory molecules such as interleukin-10, interleukin-18BP, and endothelial nitric oxide synthase and with an increased number of regulatory T lymphocytes within atherosclerotic lesions. In contrast, lesion size development within the aortic roots was more enhanced in ApoE(-/-) and ApoE(-/-) CXCR3- mice compared with ApoE(-/-) CCR2(-/-) and triple knockout mice. Blocking chemokine signaling in vivo through deletion of the chemokine receptors CCR2 and CXCR3 has differential effects during atherogenesis. In addition, our results point to an important role of regulatory T lymphocytes during early atherogenesis.

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

Background: LASP-1 (LIM and SH3 protein-1) mediates cell migration, proliferation and survival in several breast cancer cell lines. LASP-1 is a scaffold protein that plays a critical role in cytoskeletal organization and cell motility. Silencing of LASP-1 in breast cancer cells inhibits cell migration and proliferation by 40%. We discovered that LASP-1 associates with chemokine receptors that are involved in migration of tumor cells or leukocytes in the tumor microenvironment or to the metastatic sites. LASP-1 directly binds to the chemokine receptors CXCR1, CXCR2, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated migration and possibly cell survival. This established LASP-1 as a key component of the “CXC chemokine receptor chemosynapse”. This is highly significant as chemokine receptors CXCR2, CXCR4 and CXCR3 play a paramount role in primary and metastatic breast cancer. CXCR1 is involved in self-renewal of breast cancer stem cells that contribute to metastasis and drug resistance. The increase in expression level of LASP-1 with an increasing invasiveness of the tumor cells implicates a role for LASP-1 in tumor progression and lymph node metastases. Nuclear localization of LASP-1 (44% of LASP-1 positive specimens) was observed in tissue sections of aggressive and invasive breast cancer that positively correlated with poor survival rate. This is suggestive of a key role for nuclear LASP-1 in breast cancer progression. Methodology/Results: We investigated the chemokine-mediated nuclear translocation of LASP-1 in breast cancer and endothelial cell lines. Initially, cell surface expression of CXCR4 was profiled in breast cancer cell lines by FACS analysis. Medium to high CXCR4 expressers were stimulated with CXCL12 and the subcellular status of LASP-1 was examined by confocal microscopy. LASP-1 accumulated in the nucleus of the CXCL12-stimulated cells. Surprisingly, in MDA-MB-231 cells that were isolated from the mouse bone metastatic lesions had LASP-1 in the nucleus even in the absence of any stimulation by CXCL12. Additionally, the nuclear translocation of LASP-1 was observed in EGF- or heregulin-stimulated cells. Ligand activated CXCR2 in human endothelial cell line was also able to drive the nuclear accumulation of LASP-1. In order ascertain the role played by nuclear LASP-1 it was stably knocked down (KD). The control silenced (NS) and LASP-1 KD cells were grown in 3D-matrigel. Morphology of the 3D-clusters and the profile of the secreted cytokines were assessed by light microscopy and cytokine antibody microarray respectively along with additional biochemical methods and gene expression analysis by oligo microarray. Conclusions/Significance: We demonstrate here for the first time that LASP-1 translocates to the nucleus through activation of i) GPCRs -chemokine receptors CXCR2, CXCR4 and ii) Receptor tyrosine kinases - EGF receptor and HER2. Shuttling of LASP1 to the nucleus through the activation of a variety of receptors present in the tumor cells, macrophages, neutrophils and endothelial cells of the tumor microenvironment is of high significance. Silencing of LASP-1 in breast cancer cells revealed an altered profile of the 3D-clusters and the secreted cytokines. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-01.

The SPRY-domain of the SOCS box protein 2 (SPSB2) plays an important role in the proteasomal degradation of inducible nitric oxide synthase (iNOS). SPSB2 knockout mice show prolonged expression of iNOS and enhanced killing of persistent pathogens such as Mycobacterium tuberculosis and Leishmania major, suggesting that inhibitors of the SPSB2-iNOS interaction represent a potential novel class of anti-infectives. In this study, attempts to discover small molecule inhibitors of SPSB2-iNOS interaction were performed using in silico guided fragment-based drug design (FBDD) approach. The best fragment hit STK441224, however, was found to bind promiscuously to SPSB2 by saturation transfer difference spectroscopy (STD), Carr-Purcell-Meiboom-Gill (CPMG), ¹⁹ F and [¹ H,¹⁵N]-HSQC NMR experiments, with an estimated KD of 1.8 mM by surface plasmon resonance (SPR). Further predictions by SiteMap and FTMap revealed that the iNOS binding site of SPSB2 is less druggable, explaining the poor outcome from the current FBDD campaign. Thus, other approaches to discover potent and specific inhibitors of the SPSB2-iNOS interaction were explored. Utilising an in silico structure-based drug design approach, a disulphide-bridged cyclic peptide Ac-c[CVDINNNC]-NH2 was designed and synthesised. It was found to bind to the iNOS binding site on SPSB2 with a KD of 4.4 nM, as shown by SPR, [¹H,¹⁵N]-HSQC and ¹⁹F NMR experiments, with approximately 70-fold improvement in affinity, compared to the linear peptide DINNN (KD ≈ 318 nM). An in vitro assay on macrophage cell lysates further showed a complete inhibition of SPSB2-iNOS interactions by the cyclic peptide. In addition, the solution structure of the cyclic peptide was found to closely match that of the crystal structure of SPSB2-bound linear peptide DINNN with a backbone RMSD of 1.21 Å. The designed peptide was also found to be stable against pepsin, trypsin and α-chymotrypsin, and in human plasma. The disulphide-bridged cyclic peptide, however, is reductively labile. To generate redox-stable inhibitors of SPSB2-iNOS interaction that would retain activity in the cell cytoplasm, two cyclic peptide analogues, one containing a thioether bridge (CP1) and the other a lactam bridge (CP2), as well as four cyclic peptidomimetics (M1-M4), incorporating organic moieties as cyclisation linkers, were generated. All analogues were able to bind to the iNOS binding site of SPSB2, with five of the six analogues binding with stronger affinities (3-15 fold) than the linear peptide DINNN (CP1, KD 31 nM; CP2, KD 21 nM; M1, KD 29 nM; M2, KD 99 nM; M3, KD 54 nM; M4, KD 465 nM), as determined by 19F NMR and SPR, respectively. All analogues were able to compete with full-length iNOS for binding to SPSB2 in macrophage cell lysates. As CP2 is the most potent redox-stable analogue, with more sites for derivatisation and is easier to synthesise compared to other analogues, CP2 is being used as the template to generate analogues for macrophage-targeted delivery studies. Binding studies by SPR and 19F NMR revealed an approximately 5-fold improvement in binding affinity of rhodamine B isothiocyanate (RBITC)-conjugated cyclic peptide analogue CP4 to the iNOS binding site of SPSB2 (KD ≈ 4 nM), while the oligohistidine-conjugated analogues CP5 and CP6 showed a modest 1-2 fold drop in their binding affinities to SPSB2 compared to CP2 (KD ≈ 33-51 nM). Imaging studies of mannose or GalNAc glycopolymer-conjugated analogues of CP4 (i.e. CP7 and CP8, respectively) by confocal laser scanning microscopy revealed that both analogues were taken up by bone marrow-derived macrophages but not HEK 293 cells. On the other hand, oligohistidine-conjugated analogue CP6 showed signs of endosomal escape after 9 h of incubation, although coincident signs of cell necrosis or apoptosis were observed in some of these macrophages. In summary, several potent and stable cyclic peptide and peptidomimetic inhibitors of SPSB2-iNOS interactions were identified in this study. One of these analogues was able to be derivatised for macrophage-targeted delivery studies without negatively affecting their binding to SPSB2. This study also showed that both mannose and GalNAc glycopolymers are viable choices for macrophage-targeted delivery although more work to improve the endosomal escape of this new class of cyclic peptide inhibitors of SPSB2-iNOS interaction to the cytoplasm of macrophages without causing cell injury is needed.

In this COVID-19 pandemic situation, an appropriate drug is urgent to fight against this infectious disease to save lives and prevent mortality. Repurposed drugs and vaccines are the immediate solutions for this medical emergency until discover a new drug to treat this disease. As of now, no specific drug is available to cure this disease completely. Several drug targets were identified in SARS-CoV-2, in which RdRp protein is one of the potential targets to inhibit this virus infection. In-Silico studies plays a vital role to understand the binding nature of the drugs at the atomic level against the disease targets. The present study explores the binding mechanism of reported 53 nucleoside and non-nucleoside RdRp inhibitors and Ivermectin which are in clinical trials. These molecules were screened by molecular docking simulation; in which, the molecules are showing high binding affinity and forming interactions with the key amino acids of active site of RdRp protein are chosen for molecular dynamics simulation (MD) and binding free energy analysis. The results of molecular docking and MD simulation studies reveal that IDX184 is a stable molecule and forms strong interactions with the key amino acids and shows high binding affinity towards RdRp. Hence, IDX184 may also be considered as a potential inhibitor of RdRp after clinical study. Communicated by Ramaswamy H. Sarma

This study investigates the binding affinity and interaction mechanism of chlorhexidine (CHX), a commonly used antimicrobial agent, with lysozyme (LZM), a crucial salivary enzyme in the oral cavity. Chicken egg white lysozyme (CEWLZM) was used as a model LZM. Tri‐N‐acetylchitotriose (NAG3) was applied in redocking analysis to determine the exact binding sites of the CEWLZM. The study elucidates how CHX interacts with LZM at the molecular level, employing surface plasmon resonance (SPR), molecular docking, and molecular dynamics (MD) simulations. The SPR analysis revealed that CHX as an exogenous substance showed significant affinity for CEWLZM, with an equilibrium dissociation constant of 43.39 ± 2.07 µM. Molecular docking and MD simulations further demonstrated that CHX primarily binds at the active site of CEWLZM, stabilizing the complex via hydrophobic interactions and hydrogen bonding. These findings suggest that CHX's presence in the oral cavity could impact lysozyme's natural antimicrobial functions, potentially influencing oral health. This research provides insight into optimizing CHX‐containing products, balancing antimicrobial efficacy with minimal interference with host defense enzymes, which may enhance the safety and effectiveness of CHX in oral care applications.

Lysozyme (LZM) is an important enzyme in medicine and industry. Tannic acid (TA) is used in brewing, wine industry, and as a food flavor enhancer. In nutritional and food science, LZM interacts with TA, notably in wine and saliva. This study aimed to investigate the binding interaction between LZM and TA using surface plasmon resonance, molecular docking, and molecular dynamics simulation. Chicken egg white lysozyme (CEWLZM) was applied as a model protein. Tri-N-acetylchitotriose (NAG3), the known inhibitor of CEWLZM, was used in the redocking experiments to determine the precise binding location within the complex. The average binding energies obtained from docking NAG3 and tannic acid to the target structure of CEWLZM were found to be -6.46 ± 0.05 kcal/mol and -7.52 ± 0.39 kcal/mol, respectively. The binding free energy of the CEWLZM-TA complex was then calculated as -27.61 kcal/mol by MMPBSA based on MD simulation trajectories. The observed interactions between the ligands and the lysozyme structure were mainly driven by hydrophobic, van der Waals, and H-bond interactions formed by the active site residues. MD simulations showed consistent and satisfactory binding distances between CEWLZM and TA throughout the analysis. SPR analysis was performed using 1X PBS buffer (pH 7.4) as coupling and running buffers, 30 μL/min as flow rate, and 2.5 mg/mL CEWLZM. Serial concentrations of TA (20-150 μM) were injected through immobilized CEWLZM, and the K D value of CEWLZM-TA binding was obtained as 4.17 × 10-5 M. This study could enhance existing literature and pave the way for future research in food science and oral biology.

BackgroundFactor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C-type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. Methods and resultsThe FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. ConclusionsProtein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating them for 72 h. These optimized conditions are well-suited for upscaling the production of recombinant FC fragments using a bioreactor.

Liquiritigenin suppresses osteoclastogenesis via multi-target mechanisms involving NF-κB/PI3K-AKT signaling pathways and metabolic reprogramming.

Normal unstimulated mouse lymph node lymphocytes (LNLs) migrated into filters in a gradient of normal mouse serum (NMS), heat-inactivated mouse serum (HI-MS), or zymosan-activated mouse serum (ZAS). Blind well chemotaxis chambers with 5-micrometer pore size cellulose nitrate membranes were used. Migration was assessed both by the leading front technique and the mean aggregate number. A concentration of 2.5 x 10(6) LNLs/ml or greater was needed to detect migration. Migration of LNLs to 1% NMS was time dependent and was inhibited by cytochalasin B. Comparison of the migration patterns of LNLs, neutrophils, and macrophages revealed that all cell types were responsive to NMS. LNLs responded as well to HI-MS as they did to NMS, neutrophils responded less well to HI-MS than to MMS, and macrophages did not respond to HI-MS. The LNL response to ZAS was significantly greater than the response to NMS to HI-MS and neutrophils and macrophages also responded strongly to ZAS. The migration of LNLs from various mouse strains to NMS revealed that the LNLs from different mouse strains possess varying degree of motility. The factor in mouse serum which induced migration was not strain specific. The LNLs from peripheral (inguinal, axillary, and brachial) nodes demonstrated greater motility in response to NMS than mesenteric LNLs. Using the checkerboard assay to discriminate chemotaxis from chemokinesis, mouse serum appeared to be solely chemokinetic when the leading front technique was used. However, using the mean aggregate number technique, mouse serum was determined to be both chemokinetic acid chemotactic for LNLs. The results indicate that the method can be reliably used to study those factors which influence the motility of normal or altered populations of lymphocytes.