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Discovery of a Novel HIV-1 Integrase/p75 Interacting Inhibitor by Docking Screening, Biochemical Assay, and in Vitro Studies.
Protein-protein interaction between lens epithelium-derived growth factor (LEDGF/p75) and HIV-1 integrase becomes an attractive target for anti-HIV drug development. The blockade of this interaction by small molecules could potentially inhibit HIV-1 replication. These small molecules are termed as LEDGINs; and several newly identified LEDGINs have been reported to significantly reduce HIV-1 replication. Through this project, we have finished the docking screening of the Maybridge database against the p75 binding site of HIV-1 integrase using both DOCK and Autodock Vina software. Finally, we have successfully identified a novel scaffold LEDGINs inhibitor DW-D-5. Its antiviral activities and anticatalytic activity of HIV-1 integrase are similar to other LEDGINs under development. We demonstrated that the combination of DW-D-5 and FDA approved anti-HIV drugs resulted in additive inhibitory effects on HIV-1 replication, indicating that DW-D-5 could be an important component of combination pills for clinic use in HIV treatment.
Silibinin, a novel chemokine receptor type 4 antagonist, inhibits chemokine ligand 12-induced migration in breast cancer cells.
PURPOSE: C-X-C chemokine receptor type 4 (CXCR4) signaling has been demonstrated to be involved in cancer invasion and migration; therefore, CXCR4 antagonist can serve as an anti-cancer drug by preventing tumor metastasis. This study aimed to identify the CXCR4 antagonists that could reduce and/or inhibit tumor metastasis from natural products. METHODS AND RESULTS: According to the molecular docking screening, we reported here silibinin as a novel CXCR4 antagonist. Biochemical characterization showed that silibinin blocked chemokine ligand 12 (CXCL12)-induced CXCR4 internalization by competitive binding to CXCR4, therefore inhibiting downstream intracellular signaling. In human breast cancer cells MDA-MB-231, which expresses high levels of CXCR4, inhibition of CXCL12-induced chemomigration can be found under silibinin treatment. Overexpression of CXCL12 sensitized MDA-MB-231 cells to the inhibition of silibinin, which was abolished by CXCR4 knockdown. The inhibition of silibinin was also observed in MCF-7/CXCR4 cells rather than MCF-7 cells that express low level of CXCR4. CONCLUSIONS: Our work demonstrated that silibinin is a novel CXCR4 antagonist that may have potential therapeutic use for prevention of tumor metastasis.
Pimozide, a novel fatty acid binding protein 4 inhibitor, promotes adipogenesis of 3T3-L1 cells by activating PPARγ.
Pimozide is a conventional antipsychotic of the diphenylbutylpiperidine class that has been clinically used for over 30 years. The obvious side effect of this drug is weight gain. However, the mechanism of pimozide-induced weight gain is still unknown. In the present study, we identified pimozide as a novel fatty acid binding protein 4 (FABP4) inhibitor using molecular docking simulation as well as biochemical characterizations. BMS309403, a well-known FABP4 inhibitor, elevated the basal protein levels of PPARγ, therefore stimulating adipogenesis in adipocytes. The present study showed that the inhibitory effect of pimozide on FABP4 promoted adipocyte differentiation with the potency proportional to their propensities for weight gain. These effects in adipogenesis by pimozide may help to explain the weight gain that is frequently observed in patients treated with pimozide.
Herbalog: A tool for target-based identification of herbal drug efficacy through molecular docking.
BACKGROUND: Traditionally, molecular docking is primarily employed to screen pure compounds; the top-ranking chemicals are subsequently selected for experimental validation. Unlike synthetic chemicals, most natural products are commercially unavailable. The isolation and purification of each natural product is extremely time-consuming, which has restricted the screening of lead compounds from natural products. PURPOSE: We developed a protocol, Herbalog, to facilitate the identification of bioactive phytochemicals through molecular docking. METHODS: We wrote a script using Python and Autodock Vina for docking; ligand displacement and adipolysis assays were used to determine the anti-fatty acid binding protein (FABP) 4 activity of bioactive extracts. An ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry system was applied for identifying major peaks of bioactive extracts. RESULTS: Herbalog, a natural product database, contains 5,112 phytochemicals from 197 common herbs and a script that counts the number of hits from docking in each herb and calculates the hit rate of herbs. Herbalog prioritizes herbs according to their hit rates, and top-ranking herb candidates contain a large repertoire of hits. We used Herbalog as a screening tool and identified labdane diterpenoids from Andrographis paniculata as leading candidates of FABP4 inhibitors. CONCLUSION: Herbalog facilitates the discovery of herbs that possess the highest number of inhibitors or activators against target proteins, which reduces the sample preparation time for preliminary validation.
Discovery of 4,5-Dihydro-1H-thieno[2',3':2,3]thiepino [4,5-c]pyrazole-3-carboxamide Derivatives as the Potential Epidermal Growth Factor Receptors for Tyrosine Kinase Inhibitors.
The epidermal growth factor receptors (EGFRs), in which overexpression (known as upregulation) or overactivity have been associated with a number of cancers, has become an attractive molecular target for the treatment of selective cancers. We report here the design and synthesis of a novel series of 4,5-dihydro-1H-thieno [2',3':2,3]thiepino[4,5-c]pyrazole-3-carboxamide derivatives and the screening for their inhibitory activity on the EGFR high-expressing human A549 cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). A Docking simulation was performed to fit compound 6g and gifitinib into the EGFR to determine the probable binding models, and the binding sites and modes conformation of 6g and gifitinib were exactly similar, the two compounds were stabilized by hydrogen bond interactions with MET769. Combining with the biological activity evaluation, compound 6g demonstrated the most potent inhibitory activity (IC50 = 9.68 ± 1.95 μmol·L⁻1 for A549). Conclusively, 4,5-dihydro-1H-thieno[2',3':2,3]thiepino[4,5-c]pyrazole-3-carboxamide derivatives as the EGFR tyrosine kinase inhibitors were discovered, and could be used as potential lead compounds against cancer cells.
Using molecular docking screening for identifying hyperoside as an inhibitor of fatty acid binding protein 4 from a natural product database
The inhibition of fatty acid binding protein 4 (FABP4) by using small molecules could potentially provide therapeutic opportunities for metabolic disorders treatment. According to the results of our in-house virtual screening on the herbal molecules database, this study reports flavonols as an ideal scaffold for FABP4 inhibitors development. Among the popular flavonols examined, we identified hyperoside as a promising FABP4 inhibitor. Identical to the well-known FABP4 inhibitor BMS309403, hyperoside induced lipid accumulation and upregulated peroxisome proliferator-activated receptor γ (PPARγ) protein expression during the adipocyte differentiation process. Furthermore, both PPARγ antagonist and FABP4 overexpression attenuated hyperoside-induced adipogenesis, indicating that hyperoside promoted adipogenesis in adipocytes via the FABP4/PPARγ pathway. We anticipate hyperoside to be a promising, novel FABP4 inhibitor for antidiabetic drug development.
The V2475F CPVT1 mutation yields distinct RyR2 channel populations that differ in their responses to cytosolic Ca2+ and Mg2.
Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/-)-RyR2 channels to both activating and inactivating levels of cytosolic Ca2+ , plus attenuation of Mg2+ inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/-)-RyR2 channels exhibited reduced Mg2+ inhibition and 23% were actually activated by Mg2+ . Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical domain 1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients. KEY POINTS: RyR2 mutations can cause type-1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a lethal, autosomal-dominant arrhythmic disease. However, the changes in RyR2 ion-channel function that result from the many different patient mutations are rarely investigated in detail and often only recombinant RyR2, homozygous for the mutation, is studied. As CPVT1 is a heterozygous disease and the tetrameric RyR2 channels expressed in the heart will contain varying numbers of mutated monomers, we have investigated the range of RyR2 single-channel abnormalities found in the hearts of mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2. Specific alterations to ligand regulation of V2475F(+/-)-RyR2 were observed. Multiple sub-populations of channels exhibited varying degrees of abnormality. In particular, an increased sensitivity to activating and inactivating cytosolic [Ca2+ ], and reduced sensitivity to Mg2+ inhibition were evident. Our results provide mechanistic insight into the changes to RyR2 gating that destabilise sarcoplasmic reticulum Ca2+ -release causing life-threatening arrhythmias in V2475F(+/-)-CPVT1 patients.
Defective iron homeostasis and hematological abnormalities in Niemann-Pick disease type C1
Background: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium. The lysosomal system regulates key aspects of iron homeostasis, which prompted us to investigate whether there are hematological abnormalities and iron metabolism defects in NPC1. Methods: Iron-related hematological parameters, systemic and tissue metal ion and relevant hormonal and proteins levels, expression of specific pro-inflammatory mediators and erythrophagocytosis were evaluated in an authentic mouse model and in a large cohort of NPC patients. Results: Significant changes in mean corpuscular volume and corpuscular hemoglobin were detected in Npc1-/- mice from an early age. Hematocrit, red cell distribution width and hemoglobin changes were observed in late-stage disease animals. Systemic iron deficiency, increased circulating hepcidin, decreased ferritin and abnormal pro-inflammatory cytokine levels were also found. Furthermore, there is evidence of defective erythrophagocytosis in Npc1-/- mice and in an in vitro NPC1 cellular model. Comparable hematological changes, including low normal serum iron and transferrin saturation and low cerebrospinal fluid ferritin were confirmed in NPC1 patients. Conclusions: These data suggest loss of iron homeostasis and hematological abnormalities in NPC1 may contribute to the pathophysiology of this disease.
Metabolomics detects clinically silent neuroinflammatory lesions earlier than neurofilament-light chain in a focal multiple sclerosis animal model.
BACKGROUND: Despite widespread searches, there are currently no validated biofluid markers for the detection of subclinical neuroinflammation in multiple sclerosis (MS). The dynamic nature of human metabolism in response to changes in homeostasis, as measured by metabolomics, may allow early identification of clinically silent neuroinflammation. Using the delayed-type hypersensitivity (DTH) MS rat model, we investigated the serum and cerebrospinal fluid (CSF) metabolomics profiles and neurofilament-light chain (NfL) levels, as a putative marker of neuroaxonal damage, arising from focal, clinically silent neuroinflammatory brain lesions and their discriminatory abilities to distinguish DTH animals from controls. METHODS: 1H nuclear magnetic resonance (NMR) spectroscopy metabolomics and NfL measurements were performed on serum and CSF at days 12, 28 and 60 after DTH lesion initiation. Supervised multivariate analyses were used to determine metabolomics differences between DTH animals and controls. Immunohistochemistry was used to assess the extent of neuroinflammation and tissue damage. RESULTS: Serum and CSF metabolomics perturbations were detectable in DTH animals (vs. controls) at all time points, with the greatest change occurring at the earliest time point (day 12) when the neuroinflammatory response was most intense (mean predictive accuracy [SD]-serum: 80.6 [10.7]%, p
Distribution and efficacy of ofatumumab and ocrelizumab in humanized CD20 mice following subcutaneous or intravenous administration.
Approval of B-cell-depleting therapies signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the administration route of anti-CD20 monoclonal antibodies (mAbs) alters tissue distribution patterns and subsequent downstream effects. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT)/CT 72 h after SC/IV administration of ofatumumab or SC/IV administration of ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC/IV ofatumumab or SC/IV ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, albeit with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, where no differences between the route of administration or mAb were observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was mostly achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain.
The ryanodine receptor mutational characteristics and its indication for cancer prognosis.
Ca2+ signaling is altered substantially in many cancers. The ryanodine receptors (RYRs) are among the key ion channels in Ca2+ signaling. This study aimed to establish the mutational profile of RYR in cancers and investigate the correlation between RYR alterations and cancer phenotypes. The somatic mutation and clinical data of 11,000 cancer patients across 33 cancer types was downloaded from The Cancer Genome Atlas (TCGA) database. Subsequent data processing was performed with corresponding packages of the R software. Mutational profile was analyzed and its correlation with tumor mutational burden (TMB), patient prognosis, age and smoking status was analyzed and compared. All three RYR isoforms exhibited random mutational distribution without hotspot mutations when all cancers were analyzed together. The number of mutations in RYR2 (2388 mutations) far overweight that of RYR1 (1439 mutations) and RYR3 (1573 mutations). Linear correlation was observed between cumulative TMB and cumulative number of mutations for all RYR isoforms. Patients with RYR mutations exhibited significantly higher TMB than those without RYR mutations for most cancer types. Strong correlation was also revealed in the average number of mutations per person between pairs of RYR isoforms. No stratification of patient overall survival (OS) by mutational status was found for all three RYR isoforms when all cancers were analyzed together, however, significant stratification of OS by RYR mutations was revealed in several individual cancers, most strikingly in LUAD (P = 0.0067, RYR1), BLCA (P = 0.00071, RYR2), LUSC (P = 0.036, RYR2) and KIRC (P = 0.0042, RYR3). Furthermore, RYR mutations were correlated with higher age, higher smoking history grading and higher number of pack years. Characteristic mutation profile of RYRs in cancers has been revealed for the first time. RYR mutations were correlated with TMB, age, smoking status and capable of stratifying the prognosis of patients in several cancer types.
Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria.
Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease.
Statin-activation of RyR1 is a class effect but separable from HMG-CoA reductase inhibition.
BACKGROUND AND PURPOSE: Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. We therefore investigated if RyR1 activation is a standard property of commonly-prescribed statins. Using a range of structurally-diverse statin analogues we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. EXPERIMENTAL APPROACH: Compounds were screened for RyR1 activity utilising [3 H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. KEY RESULTS: All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca2+ . We synthesised the one essential structural moiety that all statins must possess for HMG-CoA reductase inhibition, the R-3,5-dihydroxypentanoic acid unit, and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four analogues that lacked the ability to modulate RyR1. CONCLUSION AND IMPLICATIONS: That cervistatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, thus paving the way for the development of effective cardioprotective statins with improved patient tolerance.
Neutrophils incite and macrophages avert electrical storm after myocardial infarction
AbstractSudden cardiac death, arising from abnormal electrical conduction, occurs frequently in patients with coronary heart disease. Myocardial ischemia simultaneously induces arrhythmia and massive myocardial leukocyte changes. In this study, we optimized a mouse model in which hypokalemia combined with myocardial infarction triggered spontaneous ventricular tachycardia in ambulatory mice, and we showed that major leukocyte subsets have opposing effects on cardiac conduction. Neutrophils increased ventricular tachycardia via lipocalin-2 in mice, whereas neutrophilia associated with ventricular tachycardia in patients. In contrast, macrophages protected against arrhythmia. Depleting recruited macrophages in Ccr2−/− mice or all macrophage subsets with Csf1 receptor inhibition increased both ventricular tachycardia and fibrillation. Higher arrhythmia burden and mortality in Cd36−/− and Mertk−/− mice, viewed together with reduced mitochondrial integrity and accelerated cardiomyocyte death in the absence of macrophages, indicated that receptor-mediated phagocytosis protects against lethal electrical storm. Thus, modulation of leukocyte function provides a potential therapeutic pathway for reducing the risk of sudden cardiac death.
Dysregulation of Tweak and Fn14 in skeletal muscle of spinal muscular atrophy mice.
BACKGROUND: Spinal muscular atrophy (SMA) is a childhood neuromuscular disorder caused by depletion of the survival motor neuron (SMN) protein. SMA is characterized by the selective death of spinal cord motor neurons, leading to progressive muscle wasting. Loss of skeletal muscle in SMA is a combination of denervation-induced muscle atrophy and intrinsic muscle pathologies. Elucidation of the pathways involved is essential to identify the key molecules that contribute to and sustain muscle pathology. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNF receptor superfamily member fibroblast growth factor-inducible 14 (Fn14) pathway has been shown to play a critical role in the regulation of denervation-induced muscle atrophy as well as muscle proliferation, differentiation, and metabolism in adults. However, it is not clear whether this pathway would be important in highly dynamic and developing muscle. METHODS: We thus investigated the potential role of the TWEAK/Fn14 pathway in SMA muscle pathology, using the severe Taiwanese Smn-/-; SMN2 and the less severe Smn2B/- SMA mice, which undergo a progressive neuromuscular decline in the first three post-natal weeks. We also used experimental models of denervation and muscle injury in pre-weaned wild-type (WT) animals and siRNA-mediated knockdown in C2C12 muscle cells to conduct additional mechanistic investigations. RESULTS: Here, we report significantly dysregulated expression of Tweak, Fn14, and previously proposed downstream effectors during disease progression in skeletal muscle of the two SMA mouse models. In addition, siRNA-mediated Smn knockdown in C2C12 myoblasts suggests a genetic interaction between Smn and the TWEAK/Fn14 pathway. Further analyses of SMA, Tweak-/-, and Fn14-/- mice revealed dysregulated myopathy, myogenesis, and glucose metabolism pathways as a common skeletal muscle feature, providing further evidence in support of a relationship between the TWEAK/Fn14 pathway and Smn. Finally, administration of the TWEAK/Fn14 agonist Fc-TWEAK improved disease phenotypes in the two SMA mouse models. CONCLUSIONS: Our study provides mechanistic insights into potential molecular players that contribute to muscle pathology in SMA and into likely differential responses of the TWEAK/Fn14 pathway in developing muscle.