RESUMEN
BACKGROUND: Chordoma is an extremely rare, locally aggressive malignant bone tumor originating from undifferentiated embryonic remnants. There are no effective therapeutic strategies for chordoma. Herein, we aimed to explore cellular interactions within the chordoma immune microenvironment and provide new therapeutic targets. METHODS: Spectrum flow cytometry and multiplex immunofluorescence (IF) staining were used to investigate the immune microenvironment of chordoma. Cell Counting Kit-8, Edu, clone formation, Transwell, and healing assays were used to validate tumor functions. Flow cytometry and Transwell assays were used to analyze macrophage phenotype and chemotaxis alterations. Immunohistochemistry, IF, western blot, PCR, and ELISA assays were used to analyze molecular expression. An organoid model and a xenograft mouse model were constructed to investigate the efficacy of maraviroc (MVC). RESULTS: The chordoma immune microenvironment landscape was characterized, and we observed that chordoma exhibits a typical immune exclusion phenotype. However, macrophages infiltrating the tumor zone were also noted. Through functional assays, we demonstrated that chordoma-secreted CCL5 significantly promoted malignancy progression, macrophage recruitment, and M2 polarization. In turn, M2 macrophages markedly enhanced the proliferation, invasion, and migration viability of chordoma. CCL5 knockdown and MVC (CCL5/CCR5 inhibitor) treatment both significantly inhibited chordoma malignant progression and M2 macrophage polarization. We established chordoma patient-derived organoids, wherein MVC exhibited antitumor effects, especially in patient 4, with robust killing effect. MVC inhibits chordoma growth and lung metastasis in vivo. CONCLUSIONS: Our study implicates that the CCL5-CCR5 axis plays an important role in the malignant progression of chordoma and the regulation of macrophages, and that the CCL5-CCR5 axis is a potential therapeutic target in chordoma.
Asunto(s)
Cordoma , Macrófagos Asociados a Tumores , Humanos , Animales , Ratones , Macrófagos Asociados a Tumores/metabolismo , Cordoma/tratamiento farmacológico , Cordoma/metabolismo , Macrófagos , Maraviroc/metabolismo , Modelos Animales de Enfermedad , Microambiente Tumoral , Quimiocina CCL5/metabolismoRESUMEN
Cancer-associated fibroblasts (CAF) can promote tumor growth, metastasis, and therapeutic resistance in esophageal squamous cell carcinoma (ESCC), but the mechanisms of action remain elusive. Our objective was to identify secreted factor(s) that mediate the communication between CAFs and ESCC tumor cells with the aim of identifying potential druggable targets. Through unbiased cytokine arrays, we have identified CC motif chemokine ligand 5 (CCL5) as a secreted factor that is increased upon co-culture of ESCC cells and CAFs, which we replicated in esophageal adenocarcinoma (EAC) with CAFs. Loss of tumor-cell-derived CCL5 reduces ESCC cell proliferation in vitro and in vivo and we propose this is mediated, in part, by a reduction in ERK1/2 signaling. Loss of tumor-derived CCL5 reduces the percentage of CAFs recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists, namely Maraviroc. Maraviroc treatment reduced tumor volume, CAF recruitment, and ERK1/2 signaling in vivo, thus, mimicking the effects observed with genetic loss of CCL5. High CCL5 or CCR5 expression is associated with worse prognosis in low-grade esophageal carcinomas. IMPLICATIONS: These data highlight the role of CCL5 in tumorigenesis and the therapeutic potential of targeting the CCL5-CCR5 axis in ESCC.
Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Proliferación Celular , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacología , Quimiocinas/metabolismo , Quimiocinas/farmacología , Quimiocinas/uso terapéutico , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Fibroblastos/metabolismo , Ligandos , Maraviroc/metabolismo , Maraviroc/farmacología , Maraviroc/uso terapéutico , AnimalesRESUMEN
OBJECTIVES: LN comprises various glomerular lesions, including endocapillary hypercellularity with macrophage infiltration. In this study, we aimed to clarify the involvement of macrophage-tropic chemokine receptors in the pathogenesis of these glomerular lesions. METHODS: MRL/lpr mouse-derived monoclonal IgG3 antibody-producing hybridomas, 2B11.3 and B1, were injected intraperitoneally into BALB/c mice [wild type (WT)] to induce endocapillary hypercellularity and wire-loop lesions, respectively. The expression of chemokine and chemokine receptors was analysed by quantitative real-time PCR and IF. The roles of chemokine receptors in these lesions were evaluated using chemokine receptor-deficient mice or a selective CCR5 antagonist, maraviroc. RESULTS: 2B11.3 caused glomerular endocapillary hypercellularity with a significant number of glomerular CD68-positive macrophages. Further, enhanced expression of CCL2, CCL3, CCR2, CCR5 and CX3CR1 was observed in the renal cortex, compared with B1 injection, which induced wire-loop lesions. In 2B11.3-induced glomerular lesions, CD68 -positive glomerular macrophages expressed CCL2, CCL3, CCR2, CCR5 and CX3CR1, while glomerular endothelial cells expressed CCL2, CCL3, CX3CL1 and CCR2. When 2B11.3 was injected, CCR2-/- and CCR5-/-, but not CX3CR1-/-, mice exhibited reduced endocapillary hypercellularity, attenuated glomerular macrophage infiltration and improved serum blood urea nitrogen levels. Only CCR2-/- mice developed wire-loop lesions. B1 injection caused wire-loop lesions in these chemokine receptor-deficient mice to a similar extent as WT. Maraviroc treatment reduced 2B11.3-induced endocapillary hypercellularity and improved serum blood urea nitrogen levels. CONCLUSION: CCR2 and CCR5 regulate glomerular macrophage infiltration and contribute to the development of glomerular endocapillary hypercellularity in LN. CCR5 inhibition can be a specific therapy for endocapillary hypercellularity without inducing wire-loop lesions.
Asunto(s)
Enfermedades Renales , Nefritis Lúpica , Receptores CCR2 , Receptores CCR5 , Animales , Anticuerpos Monoclonales , Células Endoteliales/metabolismo , Inmunoglobulina G/metabolismo , Enfermedades Renales/metabolismo , Nefritis Lúpica/patología , Macrófagos/metabolismo , Maraviroc/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismoRESUMEN
C-C chemokine receptor 5 (CCR5), which is part of the chemokine receptor family, is a member of the G protein-coupled receptor superfamily. The interactions of CCR5 with HIV-1 during viral entry position it as an effective therapeutic target for designing potent antiviral therapies. The small-molecule Maraviroc was approved by the FDA as a CCR5 drug in 2007, while clinical trials failure has characterised many of the other CCR5 inhibitors. Thus, the continual identification of potential CCR5 inhibitors is, therefore, warranted. In this study, a structure-based discovery approach has been utilised to screen and retrieved novel potential CCR5 inhibitors from the Asinex antiviral compound (â¼ 8,722) database. Explicit lipid-bilayer molecular dynamics simulation, in silico physicochemical and pharmacokinetic analyses, were further performed for the top compounds. A total of 23 structurally diverse compounds with binding scores higher than Maraviroc were selected. Subsequent molecular dynamics (MD) simulations analysis of the top four compounds LAS 51495192, BDB 26405401, BDB 26419079, and LAS 34154543, maintained stability at the CCR5 binding site. Furthermore, these compounds made pertinent interactions with CCR5 residues critical for the HIV-1 gp120-V3 loop binding such as Trp86, Tyr89, Phe109, Tyr108, Glu283 and Tyr251. Additionally, the predicted in silico physicochemical and pharmacokinetic descriptors of the selected compounds were within the acceptable range for drug-likeness. The results suggest positive indications that the identified molecules may represent promising CCR5 entry inhibitors. Further structural optimisations and biochemical testing of the proposed compounds may assist in the discovery of effective HIV-1 therapy.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Inhibidores de Fusión de VIH , Infecciones por VIH , VIH-1 , Humanos , Maraviroc/farmacología , Maraviroc/metabolismo , Maraviroc/uso terapéutico , Antagonistas de los Receptores CCR5/farmacología , Antagonistas de los Receptores CCR5/química , Antagonistas de los Receptores CCR5/uso terapéutico , Receptores de Quimiocina/metabolismo , Receptores de Quimiocina/uso terapéutico , Ciclohexanos/farmacología , Ciclohexanos/química , Triazoles/farmacología , Triazoles/química , Inhibidores de Fusión de VIH/farmacología , Inhibidores de Fusión de VIH/química , Inhibidores de Fusión de VIH/uso terapéutico , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores CCR5/uso terapéutico , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/tratamiento farmacológicoRESUMEN
Special attention is required when pharmacological treatment is indicated for a pregnant woman. P-glycoprotein (MDR1) is a well-known transporter localized in the maternal blood-facing apical membrane of placental syncytiotrophoblast and is considered to play an important role in protecting the developing fetus. Maraviroc, a MDR1 substrate that is registered for treatment of HIV infection, shows a low toxicity profile, suggesting favorable tolerability also if administered to pregnant women. Nevertheless, there is only poor understanding to date regarding the extent to which it permeates across the placental barrier and what are the transport mechanisms involved. Endeavoring to clarify the passage of maraviroc across placenta, we used in this study the method of closed-circuit perfusion of maraviroc across human placental cotyledon. The data obtained confirmed slight involvement of MDR1, but they also suggest possible interaction with other transport system(s) working in the opposite direction from that of MDR1. Complementary in vitro studies, including cellular experiments on choriocarcinoma BeWo cells as well as transporter-overexpressing MDCKII and A431 cell lines and accumulation in placental fresh villous fragments, revealed maraviroc transport by MRP1, OATP1A2, and OATP1B3 transporters. Based on mRNA expression data in the placental tissue, isolated trophoblasts, and fetal endothelial cells, especially MRP1 and OATP1A2 seem to play a crucial role in cooperatively driving maraviroc into placental tissue. By the example of maraviroc, we show here the important interplay of transporters in placental drug handling and its possibility to overcome the MDR1-mediated efflux.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Maraviroc/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico/metabolismo , Placenta/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Acridinas/farmacología , Animales , Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/farmacología , Línea Celular Tumoral , Perros , Interacciones Farmacológicas , Femenino , Regulación de la Expresión Génica , Humanos , Células de Riñón Canino Madin Darby , Maraviroc/sangre , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/genética , Perfusión , Placenta/efectos de los fármacos , Circulación Placentaria , Embarazo , Ritonavir/farmacología , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/antagonistas & inhibidores , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/genética , Tetrahidroisoquinolinas/farmacologíaRESUMEN
Due to the lack of efficient therapeutic options and clinical trial limitations, the FDA-approved drugs can be a good choice to handle Coronavirus disease (COVID-19). Many reports have enough evidence for the use of FDA-approved drugs which have inhibitory potential against target proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we utilized a structure-based drug design approach to find possible drug candidates from the existing pool of FDA-approved drugs and checked their effectiveness against the SARS-CoV-2. We performed virtual screening of the FDA-approved drugs against the main protease (Mpro) of SARS-CoV-2, an essential enzyme, and a potential drug target. Using well-defined computational methods, we identified Glecaprevir and Maraviroc (MVC) as the best inhibitors of SARS-CoV-2 Mpro. Both drugs bind to the substrate-binding pocket of SARS-CoV-2 Mpro and form a significant number of non-covalent interactions. Glecaprevir and MVC bind to the conserved residues of substrate-binding pocket of SARS-CoV-2 Mpro. This work provides sufficient evidence for the use of Glecaprevir and MVC for the therapeutic management of COVID-19 after experimental validation and clinical manifestations.
Asunto(s)
Betacoronavirus/enzimología , Maraviroc/farmacología , Inhibidores de Proteasas/farmacología , Quinoxalinas/farmacología , Sulfonamidas/farmacología , Ácidos Aminoisobutíricos , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Simulación por Computador , Ciclopropanos , Evaluación Preclínica de Medicamentos/métodos , Lactamas Macrocíclicas , Leucina/análogos & derivados , Maraviroc/química , Maraviroc/metabolismo , Estructura Molecular , Prolina/análogos & derivados , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , SARS-CoV-2 , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMEN
The aim of the present study was to quantitatively evaluate the drug-drug interactions (DDIs) of maraviroc (MVC) with various perpetrator drugs, including telaprevir (TVR), using an in vitro data-informed physiologically based pharmacokinetic (PBPK) model. MVC showed significant active uptake and biliary excretion in sandwich-cultured human hepatocytes, and biphasic organic anion transporting polypeptide (OATP)1B1-mediated uptake kinetics in transfected cells (high-affinity K m â¼5 µM). No measureable active uptake was noted in OATP1B3- and OATP2B1-transfceted cells. TVR inhibited OATP1B1-mediated MVC transport in vitro, and also exhibited CYP3A time-dependent inhibition in human hepatocytes (inactivation constant, K I = 2.24 µM, and maximum inactivation rate constant, k inact = 0.0112 minute-1). The inactivation efficiency (k inact/K I) was approximately 34-fold lower in human hepatocytes compared with liver microsomes. A PBPK model accounting for interactions involving CYP3A, P-glycoprotein (P-gp), and OATP1B1 was developed based on in vitro mechanistic data. MVC DDIs with ketoconazole (inhibition of CYP3A and P-gp), ritonavir (inhibition of CYP3A and P-gp), efavirenz (induction of CYP3A), rifampicin (induction of CYP3A and P-gp; inhibition of OATP1B1), and TVR (inhibition of CYP3A, P-gp, and OATP1B1) were well described by the PBPK model with optimized transporter K i values implying that OATP1B1-mediated uptake along with CYP3A metabolism determines the hepatic clearance of MVC, and P-gp-mediated efflux limits its intestinal absorption. In summary, MVC disposition involves intestinal P-gp/CYP3A and hepatic OATP1B1/CYP3A interplay, and TVR simultaneously inhibits these multiple mechanisms leading to a strong DDI-about 9.5-fold increase in MVC oral exposure.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas/fisiología , Hepatocitos/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Hígado/metabolismo , Maraviroc/metabolismo , Transporte Biológico/fisiología , Línea Celular , Células HEK293 , Humanos , Cinética , Proteínas de Transporte de Membrana/metabolismo , Microsomas Hepáticos/metabolismoRESUMEN
In this work, we combined accelerated molecular dynamics (aMD) and conventional molecular dynamics (cMD) simulations coupled with the potential of mean force (PMF), correlation analysis, principal component analysis (PCA), and protein structure network (PSN) to study the effects of dimerization and the mutations of I52V and V150A on the CCR5 homodimer, in order to elucidate the mechanism regarding cooperativity of the ligand binding between two protomers and to address the controversy about the mutation-induced dimer-separation. The results reveal that the dimer with interface involved in TM1, TM2, TM3, and TM4 is stable for the CCR5 homodimer. The dimerization induces an asymmetric impact on the overall structure and the ligand-binding pocket. As a result, the two protomers exhibit an asymmetric binding to the maraviroc (one anti-HIV drug). The binding of one protomer to the drug is enhanced while the other is weakened. The PSN result further reveals the allosteric pathway of the ligand-binding pocket between the two protomers. Six important residues in the pathway were identified, including two residues unreported. The results from PMF, PCA, and the correlation analysis clearly indicate that the two mutations induce strong anticorrelation motions in the interface, finally leading to its separation. The observations from the work could advance our understanding of the structure of the G protein-coupled receptor dimers and implications for their functions.
Asunto(s)
Simulación de Dinámica Molecular , Multimerización de Proteína , Receptores CCR5/química , Receptores CCR5/metabolismo , Regulación Alostérica , Ligandos , Maraviroc/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Receptores CCR5/genética , TermodinámicaRESUMEN
HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.
Asunto(s)
Antígenos CD4/química , Antígenos CD4/ultraestructura , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/ultraestructura , Receptores CCR5/química , Receptores CCR5/ultraestructura , Receptores del VIH/química , Receptores del VIH/ultraestructura , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Sitios de Unión , Antígenos CD4/aislamiento & purificación , Antígenos CD4/metabolismo , Línea Celular , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Proteína gp120 de Envoltorio del VIH/aislamiento & purificación , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/ultraestructura , Humanos , Ligandos , Maraviroc/química , Maraviroc/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores CCR5/aislamiento & purificación , Receptores CCR5/metabolismo , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/metabolismoRESUMEN
Biophysical methods and x-ray crystallography have revealed that class A G protein-coupled receptors (GPCRs) can form homodimers. We combined computational approaches with receptor cross-linking, energy transfer, and a newly developed functional export assay to characterize the residues involved in the dimerization interfaces of the chemokine receptor CCR5, the major co-receptor for HIV-1 entry into cells. We provide evidence of three distinct CCR5 dimeric organizations, involving residues of transmembrane helix 5. Two dimeric states corresponded to unliganded receptors, whereas the binding of the inverse agonist maraviroc stabilized a third state. We found that CCR5 dimerization was required for targeting the receptor to the plasma membrane. These data suggest that dimerization contributes to the conformational diversity of inactive class A GPCRs and may provide new opportunities to investigate the cellular entry of HIV-1 and mechanisms for its inhibition.
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Membrana Celular/metabolismo , VIH-1/fisiología , Maraviroc/metabolismo , Multimerización de Proteína , Receptores CCR5/química , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Antagonistas de los Receptores CCR5/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica , Receptores CCR5/genéticaRESUMEN
We performed an in vitro evaluation of the effect of maraviroc or dolutegravir on bilirubin to albumin binding. At typical treatment and low albumin concentrations, maraviroc had no impact, while dolutegravir affected bilirubin to albumin binding to an equivalent extent as sulfisoxazole. However in vivo, neither is likely to significantly impact bilirubin to albumin binding because of their low concentrations relative to albumin.