RESUMO
Inhibitors of the interaction between Neuropilin-1 (NRP-1) and Vascular Endothelial Growth Factor-A165 (VEGF-A165) hold significant promise as therapeutic and diagnostic agents directed against cancers overexpressing NRP-1. In our efforts in this field, a few series of strong and fairly stable peptide-like inhibitors of the general formula Lys(Har)1-Xaa2-Xaa3-Arg4 have been previously discovered. In the current work, we focused on Lys(Har)-Dap/Dab-Pro-Arg sequence. The aim was to examine whether replacing C-terminal Arg with its homologs and mimetics would yield more stable yet still potent inhibitors. Upon considering the results of modelling and other factors, ten novel analogues with Xaa4 = homoarginine (Har), 2-amino-4-guanidino-butyric acid (Agb), 2-amino-3-guanidino-propionic acid (Agp), citrulline (Cit), 4-aminomethyl-phenylalanine [Phe(4-CH2-NH2)] were designed, synthesized and evaluated. Two of the proposed modifications resulted in inhibitors with activity slightly lower [e.g. IC50 = 14.3 µM for Lys(Har)-Dab-Pro-Har and IC50 = 19.8 µM for Lys(Har)-Dab-Pro-Phe(4-CH2-NH2)] than the parent compounds [e.g. IC50 = 4.7 µM for Lys(Har)-Dab-Pro-Arg]. What was a surprise to us, the proteolytic stability depended more on position two of the sequence than on position four. The Dab2-analogues exhibited half-life times beyond 60 h. Our results build up the knowledge on the structural requirements that effective VEGF-A165/NRP-1 inhibitors should fulfil.
Assuntos
Arginina , Neuropilina-1 , Peptidomiméticos , Fator A de Crescimento do Endotélio Vascular , Humanos , Arginina/química , Arginina/análogos & derivados , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/metabolismo , Peptidomiméticos/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Diabetic retinopathy (DR) is a leading cause of vision loss in working-age adults. Despite an established standard of care for advanced forms of DR, some patients continue to lose vision after treatment. This may be due to the development of diabetic macular ischemia (DMI), which has no approved treatment. Neuropilin-1 (Nrp-1) is a coreceptor with two ligand-binding domains, with semaphorin-3A (Sema3A) binding to the A-domain and vascular endothelial growth factor-A (VEGF-A) binding to the B-domain. Sema3A directs a subset of neuronal growth cones as well as blood vessel growth by repulsion; when bound to Nrp-1, VEGF-A mediates vascular permeability and angiogenesis. Modulating Nrp-1 could therefore address multiple complications arising from DR, such as diabetic macular edema (DME) and DMI. BI-Y is a monoclonal antibody that binds to the Nrp-1 A-domain, antagonizing the effects of the ligand Sema3A and inhibiting VEGF-A-induced vascular permeability. This series of in vitro and in vivo studies examined the binding kinetics of BI-Y to Nrp-1 with and without VEGF-A165, the effect of BI-Y on Sema3A-induced cytoskeletal collapse, the effect of BI-Y on VEGF- A165-induced angiogenesis, neovascularization, cell integrity loss and permeability, and retinal revascularization. The data show that BI-Y binds to Nrp-1 and inhibits Sema3A-induced cytoskeletal collapse in vitro, may enhance revascularization of ischemic areas in an oxygen-induced retinopathy mouse model, and prevents VEGF-A-induced retinal hyperpermeability in rats. However, BI-Y does not interfere with VEGF-A-dependent choroidal neovascularization. These results support further investigation of BI-Y as a potential treatment for DMI and DME. SIGNIFICANCE STATEMENT: Diabetic macular ischemia (DMI) is a complication of diabetic retinopathy (DR) with no approved pharmacological treatment. Diabetic macular edema (DME) commonly co-occurs with DMI in patients with DR. This series of preclinical studies in mouse and rat models shows that neuropilin-1 antagonist BI-Y may enhance the revascularization of ischemic areas and prevents vascular endothelial growth factor-A (VEGF-A)-induced retinal hyperpermeability without affecting VEGF-A-dependent choroidal neovascularization; thus, BI-Y may be of interest as a potential treatment for patients with DR.
Assuntos
Neovascularização de Coroide , Retinopatia Diabética , Edema Macular , Doenças Retinianas , Animais , Camundongos , Ratos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Ligantes , Edema Macular/tratamento farmacológico , Edema Macular/metabolismo , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/metabolismo , Roedores/metabolismo , Semaforina-3A , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mechanisms of infection are deciphered at the host-pathogen interface.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Endossomos , Neuropilina-1 , Receptores Virais , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Endossomos/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Neuropilina-1/antagonistas & inibidores , Receptores Virais/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Rede trans-Golgi/virologia , Internalização do Vírus/efeitos dos fármacos , HumanosRESUMO
PURPOSE: Targeted therapy in cancer researches is a promising approach that can resolve drawbacks of systematic therapeutics. Nanobodies are potent therapeutics due to their high specificity and affinity to the target. METHODS: In this study, we evaluated the effect of the combination of anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) and anti-neuropilin-1 (anti-NRP1) nanobodies both in vitro (MTT, and tube formation assay) and in vivo (chick chorioallantoic membrane (CAM), and Nude mice treatment assay). RESULTS: Our results showed that the combination of two nanobodies (anti-VEGFR2/NRP-1 nanobodies) significantly inhibited proliferation as well as tube formation of human endothelial cells effective than a single nanobody. In addition, the mixture of both nanobodies inhibited vascularization of chick chorioallantoic membrane ex ovo CAM assay as compared to a single nanobody. Moreover, the mixture of both nanobodies significantly inhibited tumor growth of the mice (tumor volume and weight) higher than individual nanobodies (P < 0.05). CONCLUSION: Our results offer a promising role of combination therapies in cancer therapy as well as angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização Patológica/terapia , Neuropilina-1/antagonistas & inibidores , Anticorpos de Domínio Único/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Membrana Corioalantoide , Células HCT116 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Anticorpos de Domínio Único/administração & dosagemRESUMO
Progression of multiple myeloma is regulated by factors intrinsic to the clonal plasma cells (PC) and by the immune effector cells in the tumor microenvironment. In this study, we investigated the interaction between CD304 expression on myeloid-derived suppressor cells (MDSC) and galectin-1 from malignant PCs in the context of autologous stem cell transplantation (ASCT) for multiple myeloma. Using high-throughput screening, CD304 expression on circulating monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/-) was compared before and after ASCT. There was a significantly higher M-MDSC expression of CD304 before ASCT and a clear correlation between circulating pre-ASCT M-MDSC frequency and serum galectin-1 concentration. Treatment of pre-ASCT M-MDSCs, but not post-ASCT M-MDSCs, with galectin-1 in vitro expanded the M-MDSC population and increased expression of CD304. High galectin-1 expression by malignant PCs was associated with poor clinical outcomes. M-MDSC development and expression of CD304 were differentially induced when healthy donor peripheral blood mononuclear cells were cultured with the human multiple myeloma cell lines RPMI-8226 and JJN3, which express high and low galectin-1, respectively. Inhibition of galectin-1 reduced M-MDSC proliferation induced by RPMI-8226 cells but not by JJN3 cells, and blockade of CD304 reduced M-MDSC migration induced by RPMI-8226 cells but not by JJN3 cells. In addition, blockade of CD304 reversed suppression of the in vitro cytotoxic effect of melphalan by pre-ASCT M-MDSCs. Our data demonstrate that multiple myeloma-derived galectin-1 could mediate the tumor-promoting effect of M-MDSCs through its interaction with CD304 on M-MDSCs and contribute to multiple myeloma progression after ASCT.See related Spotlight on p. 488.
Assuntos
Galectina 1/metabolismo , Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Células Supressoras Mieloides/imunologia , Neuropilina-1/imunologia , Antineoplásicos Alquilantes/uso terapêutico , Linhagem Celular Tumoral , Humanos , Melfalan/uso terapêutico , Monócitos/imunologia , Células Supressoras Mieloides/citologia , Neuropilina-1/antagonistas & inibidores , Transplante Autólogo , Microambiente TumoralRESUMO
Neuropilin-1 (NRP-1) regulates a range of physiological and pathological processes, including angiogenesis. Targeting of NRP1 is considered a significant approach in cancer therapy. In the present study, a novel antiNRP1 immunotoxin (αNRP1 IT) was developed by genetic fusion of a single domain (VHH) anti-NRP-1 antibody fragment to a truncated diphtheria toxin. The αNRP1 IT was expressed into bacterial cells as an inclusion body (IB). Expression of αNRP1 IT was confirmed by SDS-PAGE and western blotting. Recombinant αNRP1 IT was purified using nickel affinity chromatography. Toxicity and antiangiogenesis effect of αNRP1 IT was investigated both in vitro and in vivo. Results showed that αNRP1 IT significantly reduced the viability of human umbilical vein endothelial cell line (HUVEC) (p < .05). The αNRP1 IT significantly inhibited tube formation of HUVEC cells (p < .001). Furthermore, αNRP1 IT inhibited angiogenesis in Chick Chorioallantoic Membrane (CAM) Assay. These data suggest the potential of αNRP1 IT as a novel therapeutic in targeted cancer therapy.
Assuntos
Toxina Diftérica/administração & dosagem , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Imunotoxinas/administração & dosagem , Neovascularização Patológica/prevenção & controle , Neuropilina-1/antagonistas & inibidores , Anticorpos de Domínio Único/administração & dosagem , Animais , Camelus , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Galinhas , Relação Dose-Resposta a Droga , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Células MCF-7 , Masculino , Neovascularização Patológica/imunologia , Neuropilina-1/imunologiaAssuntos
Antagonistas de Androgênios/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/química , Semaforina-3A/metabolismo , Macrófagos Associados a Tumor/imunologia , Apoptose , Proliferação de Células , Humanos , Masculino , Neuropilina-1/antagonistas & inibidores , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Semaforina-3A/genética , Células Tumorais CultivadasRESUMO
The COVID-19 pandemic continues without specific treatment. In this study it is proposed compounds that can be developed as adjuvant / complementary drugs against COVID-19. Through a search for molecular docking, for the development of a new drug using pharmacological compounds targeting the b1 region in neuropilin-1 (NRP1), which is important for the interaction with the S1 region of the S-Protein of SARS-CoV-2, to slow down the infection process of this virus. A molecular docking was performed using almost 500,000 compounds targeted to interact in the region between amino acids (Thr316, Asp320, Ser346, Thr349, and Tyr353) in NRP1 to determine compounds able to hinder the interaction with the S1 region in the S-Protein. In this study, ten compounds are proposed as potential inhibitors between S1 region in the S-Protein of SARS-CoV-2 with the b1 region in NRP1, to develop a new adjuvant / complementary drug against COVID-19, and to hinder the interaction between SARS-CoV-2 and human cells, with a high probability to be safe in humans, validated by web servers for prediction of ADME and toxicity (PreADMET).
Assuntos
Simulação de Acoplamento Molecular , Neuropilina-1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Reposicionamento de Medicamentos , Humanos , Neuropilina-1/metabolismo , SARS-CoV-2/isolamento & purificação , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: Despite the improvement of relapse-free survival mediated by anti-angiogenic drugs like sunitinib (Sutent®), or by combinations of anti-angiogenic drugs with immunotherapy, metastatic clear cell Renal Cell Carcinoma (mccRCC) remain incurable. Hence, new relevant treatments are urgently needed. The VEGFs coreceptors, Neuropilins 1, 2 (NRP1, 2) are expressed on several tumor cells including ccRCC. We analyzed the role of the VEGFs/NRPs signaling in ccRCC aggressiveness and evaluated the relevance to target this pathway. METHODS: We correlated the NRP1, 2 levels to patients' survival using online available data base. Human and mouse ccRCC cells were knocked-out for the NRP1 and NRP2 genes by a CRISPR/Cas9 method. The number of metabolically active cells was evaluated by XTT assays. Migration ability was determined by wound closure experiments and invasion ability by using Boyden chamber coated with collagen. Production of VEGFA and VEGFC was evaluated by ELISA. Experimental ccRCC were generated in immuno-competent/deficient mice. The effects of a competitive inhibitor of NRP1, 2, NRPa-308, was tested in vitro and in vivo with the above-mentioned tests and on experimental ccRCC. NRPa-308 docking was performed on both NRPs. RESULTS: Knock-out of the NRP1 and NRP2 genes inhibited cell metabolism and migration and stimulated the expression of VEGFA or VEGFC, respectively. NRPa-308 presented a higher affinity for NRP2 than for NRP1. It decreased cell metabolism and migration/invasion more efficiently than sunitinib and the commercially available NRP inhibitor EG00229. NRPa-308 presented a robust inhibition of experimental ccRCC growth in immunocompetent and immunodeficient mice. Such inhibition was associated with decreased expression of several pro-tumoral factors. Analysis of the TCGA database showed that the NRP2 pathway, more than the NRP1 pathway correlates with tumor aggressiveness only in metastatic patients. CONCLUSIONS: Our study strongly suggests that inhibiting NRPs is a relevant treatment for mccRCC patients in therapeutic impasses and NRPa-308 represents a relevant hit.
Assuntos
Carcinoma de Células Renais/terapia , Neoplasias Renais/terapia , Animais , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , Modelos Moleculares , Metástase Neoplásica , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/genética , Neuropilina-2/antagonistas & inibidores , Neuropilina-2/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.
Assuntos
Betacoronavirus/fisiologia , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , COVID-19 , Células CACO-2 , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Furina/metabolismo , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/química , Neuropilina-1/genética , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Neuropilin-1 (NRP-1), non-tyrosine kinase receptor, was initially identified as axonal protein and later recognized as co-receptor for vascular endothelial growth factor (VEGF). Neuropilins (NRPs) are involved in vascular development and tumor angiogenesis. Over the last years, many studies have been accomplished to inhibit angiogenesis. In this study, the nanobody library was panned against immobilized NRP-1 antigen. High affinity and specificity nanobodies were selected through monoclonal ELISA. The selected nanobodies inhibited proliferation and tube formation of HUVEC and MCF-7 cells in vitro and ex vivo. The results highlight potential of anti-NRP1 nanobodies in inhibition of angiogenesis both in vitro and ex vivo and promises development of novel therapeutics against pathologic angiogenesis.
Assuntos
Neovascularização Patológica/imunologia , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/imunologia , Anticorpos de Domínio Único/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Although bevacizumab (Avastin®) has been approved as an antiangiogenic agent against some cancers, the efficacy is transient and unsatisfactory in other cancers most likely owing to the presence of alternative proangiogenic factors. Therefore, simultaneous blocking of several proangiogenic factors may be a promising strategy for antiangiogenic cancer therapeutics. Accordingly, neuropilin-1 (NRP1) is an attractive target because it serves as a multifunctional receptor for the vascular endothelial growth factor (VEGF) family. Here, we aimed to generate and test an anti-VEGFA and anti-NRP1 dual-targeting bispecific antibody (named as IDB0076) by genetic fusion of an NRP1-targeting peptide to the C-terminus of the bevacizumab heavy chain. Similar to the parental antibody (bevacizumab), IDB0076 suppressed VEGFA-induced migration of human endothelial cells. In contrast, IDB0076 inhibited endothelial-cell migration induced by other angiogenesis growth factors and manifested a more potent antitumor activity than that of bevacizumab in a murine tumor xenograft model. When toxicity was preliminarily evaluated in cynomolgus monkeys, IDB0076 showed no substantial adverse effects, e.g., the absence of noticeable nephrotoxicity, which has previously been documented for the combination therapy of bevacizumab and an anti-NRP1 antibody. Thus, VEGFA-and-NRP1 dual-targeting bispecific antibody IDB0076 may be a potent and safe anticancer agent worthy of further preclinical and clinical studies.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Neuropilina-1/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/química , Antineoplásicos/química , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuropilina-1/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The aims of this study were to find a treatment for acute kidney injury in sepsis and study the role of miR-128-3p in this process. We generated a model of septic acute kidney injury through cecal ligation and puncture (CLP) induction and screened differentially expressed microRNAs through microarray. The mechanism used by miR-128-3p in inflammatory response to septic acute kidney injury was investigated using cell transfection assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, enzyme-linked immunosorbent assay, western blot, and Dual-Luciferase Reporter Assay. miRNA microarray screening revealed that miR-128-3p was significantly upregulated in the kidneys of mice with CLP-induced septic acute kidney injury. The level of inflammatory factors TNF-α, IL-1 ß, and IL-6 decreased. In contrast, cell viability increased and apoptosis decreased with the addition of miR-128-3p inhibitors in TCMK-1 cells treated with lipopolysaccharide (LPS). Using bioinformatics and luciferin reporter gene experiments, we found that NRP1 is a miR-128-3p target gene. Overexpression of NRP1 in LPS-treated TCMK-1 cells decreased the expression of TNF-α, IL-6, and IL-1ß; increased cell viability; and decreased apoptosis. The survival period of mice pretreated with miR-128-3p inhibitors was prolonged, infiltration of inflammatory cells into kidney tissue decreased, permeability of kidneys enhanced, and expression of inflammatory factors and renal apoptosis decreased. miR-128-3p targets NRP1 for cell degradation, promotes inflammatory cell infiltration, increases expression of inflammatory factors, decreases renal cell viability, and increases apoptosis in LPS-induced septic acute renal injury.
Assuntos
Injúria Renal Aguda/metabolismo , Mediadores da Inflamação/metabolismo , MicroRNAs/biossíntese , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/biossíntese , Sepse/metabolismo , Injúria Renal Aguda/etiologia , Animais , Expressão Gênica , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sepse/complicaçõesRESUMO
BACKGROUND: Neuropilin1 (NRP1) participates in cancer cell proliferation, migration, and metastasis as a multifunctional co-receptor by interacting with multiple signal pathways, but few studies have addressed the precise function of NRP1 in pancreatic cancer (PACA) cells. We aimed to study whether NRP1 gene silencing involved in the proliferation and migration of PACA cells in vitro. METHODS: A lentiviral vector expressing NRP1 shRNA was constructed and transfected into human PACA cells (CFPAC-1 and PANC-1). The expression of NRP1 protein and mRNA was detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) assay, respectively. CCK-8 assay, wound healing assay, and transwell assay were conducted to examine the effect of NRP1 silencing on cells proliferation and migration capability. RESULTS: Results of qRT-PCR and Western blot showed successfully established, stably transfected shRNA-NRP1 cells in PACA cells. The proliferation capacity of PACA cells in NRP1 shRNA group was lower significantly than that in the negative control (NC) group (P < .05). The invasion and migration capability of PACA cells in NRP1 shRNA group was lower significantly than that in the NC group (P < .01). CONCLUSIONS: NRP1-shRNA lentiviral interference vectors can effectively decrease NRP1 gene expression in PACA cells, thereby inhibiting cells proliferation and migration, which provides a basis for finding a valuable therapeutic target for PACA therapy.
Assuntos
Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/genética , Neuropilina-1/metabolismo , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Vetores Genéticos/administração & dosagem , Humanos , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Células Tumorais CultivadasRESUMO
Multifaceted roles of vascular endothelial growth factor (VEGF)-neuropilin-1 (NRP1) interaction have been implicated in cancer, but reports on small-molecule inhibitors of VEGF-NRP1 interaction are scarce. Herein, we describe the identification of 1, a novel nonpeptide small-molecule NRP1 antagonist with moderate activity via structure-based virtual screening. Ensemble docking and molecular dynamics (MD) simulations of 1 were carried out and an interesting binding model was obtained. We found that the "aromatic box" enclosed by Tyr297, Trp301 and Tyr353 of NRP1 is critical for NRP1-1 binding. Further structure modification of 1 based on the binding model derived from MD simulations resulted in the identification of 12a with significantly improved activity.
Assuntos
Descoberta de Drogas , Simulação de Dinâmica Molecular , Neuropilina-1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Neuropilina-1/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Regulatory T cells (Treg) are targeted for cancer immunotherapy because they suppress antitumor immunity. Although the importance of neuropilin-1 (NRP1) in the stability and function of intratumoral Tregs is well-documented, targeting of NRP1+ Tregs for anticancer immunotherapy has not been well explored. Here, we found that an NRP1 antagonist [Fc(AAG)-TPP11], generated by fusion of the NRP1-specific binding peptide TPP11 with the C-terminus of an effector function-deficient immunoglobulin Fc(AAG) variant, inhibits intratumoral NRP1+ Treg function and stability. Fc(AAG)-TPP11 triggered the internalization of NRP1, reducing its surface expression on Tregs and thereby inhibiting the suppressive function of Tregs. In two murine syngeneic tumor models, Fc(AAG)-TPP11 retarded tumor growth, comparable with a Treg-depleting anti-CTLA-4 antibody, without noticeable toxicity. Fc(AAG)-TPP11 inhibited NRP1-dependent Treg function, inducing unstable intratumoral Tregs, with reduced expression of Foxp3 and enhanced production of IFNγ, which subsequently increased the functionality and frequency of intratumoral CD8+ T cells. We also observed selective expression of NRP1 on Tregs isolated from human tumors, but not from the blood of healthy donors and patients with cancer, as well as ex vivo inhibition of intratumoral NRP1+ Treg function by Fc(AAG)-TPP11. Our results suggest that the NRP1 antagonist Fc(AAG)-TPP11 has therapeutic potential for the inhibition of intratumoral NRP1+ Tregs with limited unfavorable effects on peripheral Tregs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Fragmentos Fc das Imunoglobulinas/química , Melanoma/imunologia , Neuropilina-1/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Feminino , Terapia de Imunossupressão , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neuropilina-1/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
The therapeutic targeting of oncogenic KRAS mutant-harboring (KRASMUT) non-small cell lung cancer (NSCLC) is an urgent unmet need in cancer therapy. Although NSCLC is often driven by epidermal growth factor receptor (EGFR) overexpression and/or mutations, no EGFR-targeted therapy is clinically available for KRASMUT NSCLC. In this study, we show that integrin ß3 expression is associated with the intrinsic resistance of KRASMUT NSCLCs to the anti-EGFR antibody cetuximab. Further analyses identified an integrin ß3-mediated ternary complex comprising NRP1-integrin ß3-KRASMUT and its downstream signaling of PI3K-Akt and RalB-TBK1 as a primary resistance mechanism of KRASMUT NSCLC to cetuximab treatment. Importantly, we demonstrate that the EGFR/NRP1 dual-targeting bispecific antibody, Ctx-TPP11, attenuates the downstream signaling driven by the ternary complex via the cellular co-internalization and degradation of the NRP1-coupled complex, resulting in the alleviation of cetuximab resistance in KRASMUT NSCLCs in vitro and in vivo, including patient-derived xenograft mouse models. Our study shows that the dual-targeting of EGFR and NRP1 with a bispecific antibody might be an effective therapeutic strategy for KRASMUT NSCLC.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neuropilina-1/metabolismo , Animais , Anticorpos Biespecíficos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Humanos , Integrina beta3/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Mutação , Neuropilina-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Complexo Ternário/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
miRNAs such as miR-148b play crucial regulatory role in tumor metastasis, but their applications are limited because they are easy to degrade in serum conditions and lack targeting ability. Herein, CC9-PEG-SSBPEI was synthesized and used as nano-carrier for miR-148b. DLS and gel retardation analyses indicated that CC9-PEG-SSBPEI could combine with miR-148b by charge interaction and formed into nanoparticles with the size changed from 811.6 nm to 146.4 nm. CC9-PEG-SSBPEI could protect miR-148b from RNase A degradation and showed a reduction sensitive release of miR-148b. FACS analysis and CLSM images displayed that the conjugated CC9 peptide improved the accumulation and penetration of the nanoparticles in HuH-7 liver cancer cells through binding with the target of miR-148b neuropilin-1(NRP-1) on the cell surface. The raised level of miR-148b in turn inhibited the expression of NRP-1 and suppressed the migration of HuH-7 liver cancer cells. Moreover, hemolysis and cytotoxicity assay demonstrated that the nanoparticles had good hemo- and cyto- compatibility. Hence, CC9-PEG-SSBPEI/miR-148b nanoparticles had the potential for targeting delivery of miR-148b and anti-metastasis of hepatocellular carcinoma (HCC) cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Marcação de Genes/métodos , Técnicas de Transferência de Genes , MicroRNAs/genética , Nanopartículas/química , Neuropilina-1/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Eritrócitos/citologia , Hemólise , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oxirredução , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoimina/química , Polietilenoimina/metabolismo , Ribonuclease Pancreático/químicaRESUMO
NRP-1 is an important co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). Many reports suggested that NRP-1 might also serve as a separate receptor for VEGF-A165 causing stimulation of tumour growth and metastasis. Therefore, compounds interfering with VEGF-A165/NRP-1 complex triggered interest in the design of new molecules, including peptides, as anti-angiogenic and anti-tumour drugs. Here, we report the synthesis, affinity and stability evaluation of the urea-peptide hybrids, based on general Lys(hArg)-AA2-AA3-Arg sequence, where hArg residue was substituted by Arg urea unit. Such substitution does not substantially affected affinity of compounds for NRP-1 but significantly increased their proteolytic stability in plasma.
Assuntos
Antineoplásicos/química , Neuropilina-1/antagonistas & inibidores , Peptidomiméticos/química , Ureia/química , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Amidas/química , Antineoplásicos/metabolismo , Desenho de Fármacos , Meia-Vida , Humanos , Neuropilina-1/metabolismo , Peptidomiméticos/metabolismo , Ligação Proteica , Ureia/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 µM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 µM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure-activity relationships.