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1.
Eur J Med Chem ; 176: 393-409, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31125894

RESUMO

Novel substituted purine isosters, were designed and synthesized as potential inhibitors of the Epidermal Growth Factor Receptor (EGFR). The compounds were rationally designed through bioisosteric replacement of the central quinazoline core of lapatinib, an approved drug that inhibits both EGFR and HER2, another important member of this family of receptors. The new target molecules were evaluated as inhibitors of receptor phosphorylation at the cellular level, for their direct inhibitory action on the intracellular receptor kinase domain and for their cytotoxicity against the non-small cell lung cancer cell line A549 and breast cancer HCC1954, cell lines which are associated with overexpression of EGFR and HER2, respectively. The most potent derivatives were further studied for their cellular uptake levels and in vivo pharmacokinetic properties. One compound (23) displayed a noteworthy pharmacokinetic profile, and higher intracellular accumulation in comparison to lapatinib in the A549 cells, possibly due to its higher lipophilicity. This lead compound (23) was assessed for its efficacy in an EGFR positive xenograft model, where it successfully inhibited tumor growth, with a similar efficacy with that of lapatinib and with minimal phenotypic toxicity.


Assuntos
Antineoplásicos/uso terapêutico , Lapatinib/análogos & derivados , Lapatinib/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Domínio Catalítico , Linhagem Celular Tumoral , Feminino , Humanos , Lapatinib/síntese química , Lapatinib/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Purinas/síntese química , Purinas/química , Purinas/farmacocinética , Receptor ErbB-2/química
2.
Mol Immunol ; 109: 149-156, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30951934

RESUMO

Our aim was to construct a CD40×HER2 single chain diabody (ScDb) and determine its tumor-specific immune activation and anti-HER2 function. Overlap extension-polymerase chain reaction was applied in the construction of ScDb, and the protein was expressed with the pET28a (+)-Rosetta prokaryotic expression system. Soluble ScDb was purified by a nickel-nitrilotriacetic acid column. Dendritic cells (DC) was stimulated by ScDb and inhibited 4T1 cells proliferation in vitro. In 4T1 tumor mice model, lymphocyte infiltration was prominently detected in ScDb group, Caspase-3 expression was significantly upregulated. ScDb was labeled using quantum dots. Immunofluorescence assay indicated ScDb exhibited high affinity to HER2. T6-17 cells were inhibited by ScDb in vitro. The phosphorylation and expression levels of AKT, ERK were markedly decreased. In T6-17 tumor mice model. Compared to CD40 ScFv, HER2 ScFv and normal saline groups, tumor volume diminished significantly in ScDb group, and tumor cells showed extensive deformation, and pervasive karyopyknosis and karyorrhexis were found. In the present study, we successfully constructed a ScDb fragment and expressed it using a prokaryotic expression system. The in vivo and in vitro experimental results indicated that ScDb could inhibit the proliferation of tumor cells by stimulating the tumor-specific immunoreaction and blocking the HER2-related signaling pathway.


Assuntos
Neoplasias/imunologia , Neoplasias/patologia , Receptor ErbB-2/metabolismo , Anticorpos de Cadeia Única/farmacologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor ErbB-2/química , Linfócitos T/efeitos dos fármacos
3.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832287

RESUMO

The combination of hydrodynamic and electrophoretic experiments and computer simulations is a powerful approach to study the interaction between proteins. In this work, we present hydrodynamic and electrophoretic experiments in an aqueous solution along with molecular dynamics and hydrodynamic modeling to monitor and compute biophysical properties of the interactions between the extracellular domain of the HER2 protein (eHER2) and the monoclonal antibody trastuzumab (TZM). The importance of this system relies on the fact that the overexpression of HER2 protein is related with the poor prognosis breast cancers (HER2++ positives), while the TZM is a monoclonal antibody for the treatment of this cancer. We have found and characterized two different complexes between the TZM and eHER2 proteins (1:1 and 1:2 TZM:eHER2 complexes). The conformational features of these complexes regulate their hydrodynamic and electrostatic properties. Thus, the results indicate a high degree of molecular flexibility in the systems that ultimately leads to higher values of the intrinsic viscosity, as well as lower values of diffusion coefficient than those expected for simple globular proteins. A highly asymmetric charge distribution is detected for the monovalent complex (1:1 complex), which has strong implications in correlations between the experimental electrophoretic mobility and the modeled net charge. In order to understand the dynamics of these systems and the role of the specific domains involved, it is essential to find biophysical correlations between dynamics, macroscopic transport and electrostatic properties. The results should be of general interest for researchers working in this area.


Assuntos
Antineoplásicos Imunológicos/química , Simulação de Acoplamento Molecular , Receptor ErbB-2/química , Trastuzumab/química , Antineoplásicos Imunológicos/farmacologia , Sítios de Ligação , Humanos , Hidrodinâmica , Ligação Proteica , Receptor ErbB-2/metabolismo , Eletricidade Estática , Trastuzumab/farmacologia
4.
Artif Cells Nanomed Biotechnol ; 47(1): 665-673, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30829072

RESUMO

The present study was aimed to develop an effective nanoliposomal vaccine delivery system with P435 HER2/neu-derived peptide conjugated to Maleimide-PEG2000-DSPE. The nanoliposome formulation composed of DSPC/DSPG/Chol/DOPE and monophosphoryl lipid A was used as an adjuvant. Liposomal formulations were prepared and their physical properties were characterized. Anti-tumoral efficacy of formulations was evaluated by immunization of tumor-bearing BALB/c mice and the generated immune response was studied by using ELISpot and flow cytometry analysis. The results of the study demonstrated Lip + DOPE + P535 formulation caused the lowest tumor size and the longest survival time in TUBO mice model and could make it a promising candidate in developing effective vaccines against HER2-positive breast cancers.


Assuntos
Vacinas Anticâncer , Neoplasias Mamárias Experimentais , Nanopartículas , Peptídeos , Receptor ErbB-2 , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Linhagem Celular Tumoral , Feminino , Lipossomos , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologia , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Receptor ErbB-2/uso terapêutico
5.
Proc Natl Acad Sci U S A ; 116(9): 3863-3872, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30733293

RESUMO

Although human epidermal growth factor receptor 2 (HER2)-targeted therapies have dramatically improved the clinical outcome of HER2-positive breast cancer patients, innate and acquired resistance remains an important clinical challenge. New therapeutic approaches and diagnostic tools for identification, stratification, and treatment of patients at higher risk of resistance and recurrence are therefore warranted. Here, we unveil a mechanism controlling the oncogenic activity of HER2: heteromerization with the cannabinoid receptor CB2R. We show that HER2 physically interacts with CB2R in breast cancer cells, and that the expression of these heteromers correlates with poor patient prognosis. The cannabinoid Δ9-tetrahydrocannabinol (THC) disrupts HER2-CB2R complexes by selectively binding to CB2R, which leads to (i) the inactivation of HER2 through disruption of HER2-HER2 homodimers, and (ii) the subsequent degradation of HER2 by the proteasome via the E3 ligase c-CBL. This in turn triggers antitumor responses in vitro and in vivo. Selective targeting of CB2R transmembrane region 5 mimicked THC effects. Together, these findings define HER2-CB2R heteromers as new potential targets for antitumor therapies and biomarkers with prognostic value in HER2-positive breast cancer.


Assuntos
Neoplasias da Mama/líquido cefalorraquidiano , Terapia de Alvo Molecular , Receptor CB2 de Canabinoide/genética , Receptor ErbB-2/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dronabinol/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-cbl/genética , Receptor CB2 de Canabinoide/química , Receptor ErbB-2/química , Transdução de Sinais
6.
Chem Commun (Camb) ; 55(12): 1730-1733, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30657476

RESUMO

It is significant to in situ monitor membrane proteins at the single-cell level for understanding the cell function. Herein, an integrated platform was designed and constructed for highly efficient cell capture and in situ sensitive SERS determination of HER2 activity in cells through rational spatial organization of multi-functional cyclic RGD nanopatterns at a disordered mesoporous gold film via electrochemical activation.


Assuntos
Células Neoplásicas Circulantes/metabolismo , Peptídeos Cíclicos/química , Receptor ErbB-2/química , Linhagem Celular Tumoral , Ouro/química , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Células Neoplásicas Circulantes/química , Peptídeos Cíclicos/metabolismo , Porosidade , Receptor ErbB-2/metabolismo , Análise Espectral Raman
7.
Anal Bioanal Chem ; 410(28): 7489-7498, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30232524

RESUMO

A high serum HER-2 extracellular domain (sHER-2 ECD) level has a reverse association with tumor behaviors. In this study, a portable platform for the disease biomarker sHER-2 ECD detection has been established using a pressure-based bioassay. The pressure bioassay consists of a monoclonal antibody immobilized on an eight-well strip, the analyte HER-2, and another monoclonal antibody labeled with the Pt nanoparticles (PtNPs), which have the catalytic ability to decompose H2O2 into H2O and O2(g). The increased pressure due to O2(g) generation is measured by a hand-held pressure meter. A total of 34 serum samples were collected to validate the performance of the pressure bioassay. The results showed that the pressure bioassay platform of HER-2 had a dynamic range from 2 to 50 ng/mL with a limit of detection (LOD) of 2 ng/mL, which was consistent with the ELISA result. In the real serum samples, there was a significant correlation between sHER-2 ECD level and several clinicopathological parameters, especially tissue HER-2 status. Furthermore, the sHER-2 ECD level was found to decrease after targeted therapy in a patient with tHER-2 positive. Overall, this bioassay can facilitate breast cancer diagnosis and prognosis in clinical scenarios and resource-limited areas.


Assuntos
Neoplasias da Mama/sangue , Receptor ErbB-2/sangue , Anticorpos Monoclonais , Bioensaio/métodos , Biomarcadores Tumorais/sangue , Feminino , Humanos , Pressão , Receptor ErbB-2/química
8.
Cell Physiol Biochem ; 49(1): 271-281, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30138940

RESUMO

BACKGROUND/AIMS: Smart molecular probes are required in the application of Magnetic resonance imaging (MRI) for biochemical and clinical research. This study aims to investigate the diagnostic values of estrogen receptor (ER), progesterone receptor (PR), folate receptor (FR) and human epidermal growth factor receptor 2 (HER-2)-targeted molecular probes in the MRI diagnosis of breast cancer. METHODS: Initially, a total of 508 female breast cancer patients were selected for breast cancer subtype classification by immunohistochemistry. Subsequently, the tumor size, lymph node metastasis, and histological grade of different breast cancer subtypes were compared. Molecular probes of Ab-ER-USPIO, Ab-PR-USPIO, Ab-FR-USPIO and Ab-HER-2-USPIO were constructed and screened. The specific binding of molecular probes to breast cancer cells was detected both in vitro and in vivo by Prussian blue staining and MRI using T1 and T2 weighted images. Finally, in vivo toxicity of Ab-HER-2-USPIO was analyzed using hematoxylin and eosin staining. RESULTS: We identified the following subtypes of breast cancer: Luminal A (ER-positive, FR-positive, HER-2-negative), Luminal B (ER-positive, FR-positive, HER-2-positive), HER-2 overexpression (ER-negative, FR-negative, HER-2-positive), and triple-negative breast cancer (ER-negative, FR-negative, HER-2-negative). Featuring favorable in vitro biocompatibility and low in vivo toxicity, Ab-HER-2-USPIO can specifically bind to breast cancer cells BT47 and SKBR3, thus enhancing the quality of T1 weighted MRI images. CONCLUSION: The results indicate that HER-2-targeted MRI molecular probes may be used in the clinical diagnosis of breast cancer and facilitate the development of promising strategies for breast cancer treatments.


Assuntos
Neoplasias da Mama/diagnóstico , Meios de Contraste/química , Receptores de Folato com Âncoras de GPI/metabolismo , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Animais , Anticorpos/química , Anticorpos/imunologia , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dextranos/química , Feminino , Receptores de Folato com Âncoras de GPI/química , Humanos , Imuno-Histoquímica , Metástase Linfática , Imagem por Ressonância Magnética , Nanopartículas de Magnetita/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Receptores Estrogênicos/química , Receptores de Progesterona/química
9.
Chem Commun (Camb) ; 54(53): 7314-7317, 2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29904764

RESUMO

We developed highly bright and stable far-red emissive AIEdots by using a new kind of click-functional PEG grafted amphiphilic polymer to coat hydrophobic AIE-active polymers (PDFDP). Furthermore, an anti-HER2 recombinant fully human antibody was produced and conjugated on the AIEdots via metal-free click chemistry to fabricate in vivo tumor-targeting nanoprobes.


Assuntos
Anticorpos/química , Corantes Fluorescentes/química , Nanopartículas/química , Imagem Óptica , Polímeros/química , Receptor ErbB-2/química , Tensoativos/química , Linhagem Celular Tumoral , Química Click , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/química
10.
J Biosci ; 43(2): 295-306, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29872018

RESUMO

The ErbB signalling pathway has been studied extensively owing to its role in normal physiology and its dysregulation in cancer. Reverse engineering by mathematical models use the reductionist approach to characterize the network components. For an emergent, system-level view of the network, we propose a data analytics pipeline that can learn from the data generated by reverse engineering and use it to re-engineer the system with an agent-based approach. Data from a kinetic model that estimates the parameters by fitting to experiments on cell lines, were encoded into rules, for the interactions of the molecular species (agents) involved in biochemical reactions. The agent model, a digital representation of the cell line system, tracks the activation of ErbB1-3 receptors on binding with ligands, resulting in their dimerization, phosphorylation, trafficking and stimulation of downstream signalling through P13-Akt and Erk pathways. The analytics pipeline has been used to mechanistically link HER expression profile to receptor dimerization and activation of downstream signalling pathways. When applied to drug studies, the efficacy of a drug can be investigated in silico. The anti-tumour activity of Pertuzumab, a monoclonal antibody that inhibits HER2 dimerization, was simulated by blocking 80% of the cellular HER2 available, to observe the effect on signal activation.


Assuntos
Receptores ErbB/genética , Modelos Teóricos , Neoplasias/genética , Receptor ErbB-2/genética , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Simulação por Computador , Receptores ErbB/química , Humanos , Cinética , Neoplasias/tratamento farmacológico , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Receptor ErbB-2/química , Transdução de Sinais/genética
11.
Ann Anat ; 218: 141-155, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29680777

RESUMO

Stromal cells/telocytes (SCs/TCs) were recently described in the human adult trigeminal ganglion (TG). As some markers are equally expressed in SCs/TCs and endothelial cells, we hypothesized that a subset of the TG SCs/TCs is in fact represented by endothelial progenitor cells of a myelomonocytic origin. This study aimed to evaluate whether the interstitial cells of the human adult TG correlate with the myelomonocytic lineage. We used primary antibodies for c-erbB2/HER-2, CD31, nestin, CD10, CD117/c-kit, von Willebrand factor (vWF), CD34, Stro-1, CD146, α-smooth muscle actin (α-SMA), CD68, VEGFR-2 and cytokeratin 7 (CK7). The TG pial mesothelium and subpial vascular microstroma expressed c-erbB2/HER-2, CK7 and VEGFR-2. SCs/TCs neighbouring the neuronoglial units (NGUs) also expressed HER-2, which suggests a pial origin. These cells were also positive for CD10, CD31, CD34, CD68 and nestin. Endothelial cells expressed CD10, CD31, CD34, CD146, nestin and vWF. We also found vasculogenic networks with spindle-shaped and stellate endothelial progenitors expressing CD10, CD31, CD34, CD68, CD146 and VEGFR-2. Isolated mesenchymal stromal cells expressed Stro-1, CD146, CK7, c-kit and nestin. Pericytes expressed α-SMA and CD146. Using transmission electron microscopy (TEM), we found endothelial-specific Weibel-Palade bodies in spindle-shaped stromal progenitors. Our study supports the hypothesis that an intrinsic vasculogenic niche potentially involved in microvascular maintenance and repair might be present in the human adult trigeminal ganglion and that it might be supplied by either the pial mesothelium or the bone marrow niche.


Assuntos
Células Endoteliais/ultraestrutura , Células-Tronco/ultraestrutura , Células Estromais/ultraestrutura , Telócitos/ultraestrutura , Gânglio Trigeminal/ultraestrutura , Biomarcadores/análise , Células Endoteliais/química , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Receptor ErbB-2/química , Células-Tronco/química , Células Estromais/química , Telócitos/química , Gânglio Trigeminal/anatomia & histologia , Gânglio Trigeminal/química , Nervo Trigêmeo/química , Nervo Trigêmeo/ultraestrutura , Corpos de Weibel-Palade/química , Corpos de Weibel-Palade/ultraestrutura
12.
Appl Biochem Biotechnol ; 186(1): 85-108, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29508211

RESUMO

Targeting ErbB family of receptors is an important therapeutic option, because of its essential role in the broad spectrum of human cancers, including non-small cell lung cancer (NSCLC). Therefore, in the present work, considerable effort has been made to develop an inhibitor against HER family proteins, by combining the use of pharmacophore modelling, docking scoring functions, and ADME property analysis. Initially, a five-point pharmacophore model was developed using known HER family inhibitors. The generated model was then used as a query to screen a total of 468,880 compounds of three databases namely ZINC, ASINEX, and DrugBank. Subsequently, docking analysis was carried out to obtain hit molecules that could inhibit the HER receptors. Further, analysis of GLIDE scores and ADME properties resulted in one hit namely BAS01025917 with higher glide scores, increased CNS involvement, and good pharmaceutically relevant properties than reference ligand, afatinib. Furthermore, the inhibitory activity of the lead compounds was validated by performing molecular dynamic simulations. Of note, BAS01025917 was found to possess scaffolds with a broad spectrum of antitumor activity. We believe that this novel hit molecule can be further exploited for the development of a pan-HER inhibitor with low toxicity and greater potential.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos/química , Cristalografia por Raios X , Desenho de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/química
13.
Nature ; 554(7691): 189-194, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29420467

RESUMO

Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.


Assuntos
Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação de Sentido Incorreto , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/efeitos adversos , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-3/química , Receptor ErbB-3/genética , Resultado do Tratamento
14.
PLoS One ; 13(2): e0190942, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29389942

RESUMO

HER-2 belongs to the human epidermal growth factor receptor (HER) family. Via different signal transduction pathways, HER-2 regulates normal cell proliferation, survival, and differentiation. Recently, it was reported that MCF10A, BT474, and MDA-MB-231 cells bearing the HER2 K753E mutation were resistant to lapatinib. Present study revealed that HER-2 mutant K753E showed some contrasting behaviour as compared to wild, L768S and V773L HER-2 in complex with lapatinib while similar to previously known lapatinib resistant L755S HER-2 mutant. Lapatinib showed stable but reverse orientation in binding site of K753E and the highest binding energy among studied HER2-lapatinib complexes but slightly lesser than L755S mutant. Results indicate that K753E has similar profile as L755S mutant for lapatinib. The interacting residues were also found different from other three studied forms as revealed by free energy decomposition and ligplot analysis.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Mutação , Inibidores de Proteínas Quinases/metabolismo , Quinazolinas/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Humanos , Lapatinib , Simulação de Dinâmica Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Quinazolinas/química , Receptor ErbB-2/genética , Transdução de Sinais
15.
Int J Biol Macromol ; 111: 569-586, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29329808

RESUMO

Activation of EGFR starts by ligand binding at the extracellular domain which results in homo and heterodimerization, leading to phosphorylation, activation of downstream signaling pathways which upregulate expression of genes, proliferation and angiogenesis. Abnormalities in the expression of EGFR play a critical role in the development of different types of cancer. HER2 is the preferred heterodimerization partner for EGFR; this biological characteristic together with the high percentage of structural homology has been exploited in the design of dual synthetic inhibitors against EGFR/HER2. Herein we combined structural data and molecular dynamics (MD) simulations coupled to an MMGBSA approach to provide insight into the binding mechanism between two dual synthetics (lapatinib and TAK-285) and one dual natural inhibitor (EGCG) which target EGFR/HER2. In addition, we proposed some EGCG derivatives which were filtered through in silico screening. Structural analysis demonstrated that the coupling of synthetic, natural or newly designed compounds impacts the conformational space of EGFR and HER2 differently. Energetic analysis points out that lapatinib and TAK-285 have better affinity for inactive EGFR than the active EGFR state or HER2, whereas some EGCG derivatives seem to form binding affinities similar to those observed for lapatinib or TAK-285.


Assuntos
Receptores ErbB/química , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Hidroxibutiratos/farmacologia , Lapatinib , Simulação de Dinâmica Molecular , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Conformação Proteica/efeitos dos fármacos , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos
16.
Phys Chem Chem Phys ; 20(5): 3438-3444, 2018 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-29328338

RESUMO

Nanobodies offer a viable alternative to antibodies for engineering high affinity binders. Their small size has an additional advantage: it allows exploiting computational protocols for optimizing their biophysical features, such as the binding affinity. The efficient prediction of this quantity is still considered a daunting task especially for modelled complexes. We show how molecular dynamics can successfully assist in the binding affinity prediction of modelled nanobody-protein complexes. The approximate initial configurations obtained by in silico design must undergo large rearrangements before achieving a stable conformation, in which the binding affinity can be meaningfully estimated. The scoring functions developed for the affinity evaluation of crystal structures will provide accurate estimates for modelled binding complexes if the scores are averaged over long finite temperature molecular dynamics simulations.


Assuntos
Complexo Antígeno-Anticorpo/química , Simulação de Dinâmica Molecular , Proteínas/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/metabolismo , Humanos , Muramidase/química , Muramidase/imunologia , Estrutura Terciária de Proteína , Proteínas/química , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Alinhamento de Sequência , Temperatura Ambiente
17.
Hum Antibodies ; 26(2): 103-111, 2018 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-29036807

RESUMO

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for detection and treatment of different types of cancers such as breast, ovarian, stomach cancer. In this study, we developed a monoclonal antibody against the extracellular domain (ECD) of HER2 biomarker of breast cancer. For this purpose, the ECD-HER2 gene was amplified and cloned into an expression vector. Gene was generated in Escherichia coli BL21 (DE3) strain for expression of recombinant protein. The expressed protein was separated by SDS-PAGE and detected by anti-his monoclonal antibody in immunoblotting. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells injected with recombinant ECD-HER2 and screened by ELISA for the production of monoclonal antibody. The results indicate that out of three candidate hybridoma cells one clone (1E7) was producing the highest titer and antibody specificity was envisioned in ELISA results. In vivo scaling up culture of hybridoma cells in BALB/C mice lead to significant increase in the monoclonal antibody concentration up to 16 mg/ml. Immunochemical methods demonstrated the specificity of developed antibody against ECD-HER2 protein.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Antineoplásicos Imunológicos/isolamento & purificação , Biomarcadores Tumorais/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Antineoplásicos Imunológicos/metabolismo , Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Fusão Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mieloma Múltiplo/imunologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Baço/citologia , Baço/imunologia
18.
Methods Mol Biol ; 1674: 229-237, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28921442

RESUMO

Drug delivery is of paramount importance, since the drug needs to be delivered to a specific site, in adequate concentration, avoiding degradation in order to provide therapeutic efficacy. Different nanocarriers have been used over the years for this purpose and liposomes are well-established systems due to the high biocompatibility and the possibility to vehiculate both hydrophilic and lipophilic drugs. In order to circumvent the rapid clearance by the reticuloendothelial system and to avoid the healthy cells exposure to the drug, long circulating liposomes containing polyethyleneglycol (PEG) and functionalized liposomes for targeted delivery have been developed. Immunoliposomes consist of liposomes containing antibodies or antibody fragments attached at the membrane surface. This attachment can be performed using PEG lipids, containing a reactive terminal group such as maleimide and thiolated antibodies. Additionaly, the use of PEG chains as spacers increases antibody-antigen affinity, since the antibody is not shielded by the steric hindrance of PEG and also due to the correct orientation of antibodies for interaction with receptors on cell surface. In this chapter, we describe and discuss in details the protocol to prepare anti-epidermal growth factor receptor (anti-EGFR) and anti-human epidermal growth factor receptor 2 (anti-HER2) liposomes using cetuximab and trastuzumab as antibodies. We present the direct coupling method based on the maleimide thioether reaction for these immunoliposomes preparation and present some characterization steps and in vitro studies in cell culture which can be used for better understanding these nanocarriers.


Assuntos
Anticorpos Monoclonais/química , Lipossomos/química , Sulfetos/química , Cetuximab/química , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/química , Humanos , Maleimidas/química , Polietilenoglicóis/química , Receptor ErbB-2/química , Trastuzumab/química , Células Tumorais Cultivadas
19.
J Photochem Photobiol B ; 176: 69-80, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28964888

RESUMO

In our endeavor towards the development of potent molecules for cancer diseases, we have designed and synthesized a series of 2,4,5-trisubstituted imidazole derivatives (B1-B24) and characterized by using various spectroscopic techniques. All these compounds are further evaluated for their in vitro anti-cancer, anti-oxidant activities and molecular docking studies against EGFR, HER2 protein receptors. The in vitro anti-cancer activity analysis reveals that compounds B11 and B16 were found to be effective scaffolds against the tested human cancer cell lines IMR-32, A549 and HeLa. Particularly, B16 and B11 showed effective cytotoxicity against A549 and IMR-32 with IC50 values of 09.521±0.54µM and 10.294±0.43µM, respectively. Moreover, compounds B17, B18 and B23 showed potent activity towards the anti-oxidant screening with IC50 values of 5.87±1.73µM, 6.29±1.27µM and 4.95±1.81µM, respectively compared to standard ascorbic acid. Molecular docking was performed against the EGFR, HER2 protein receptors to provide more insight into their mechanism of interaction by comparing with standard EGFR, HER2 inhibitors like Gefitinib (EFGR), Lapatanib (EGFR), Afitinib (HER2) and Canertinib (HER2). Compounds B15, B16, B11 and B10 were exhibiting their minimum binding energies. Out of the aforementioned docked molecules, B15 and B16 showed the best binding energies of -11.15kcalmol-1, -10.70kcalmol-1 and -10.49kcalmol-1, -10.12kcalmol-1 against EGFR and HER2 protein receptors, respectively. The molecular docking results are well corroborated with the in vitro anti-cancer activity finding.


Assuntos
Antioxidantes/metabolismo , Receptores ErbB/metabolismo , Imidazóis/metabolismo , Receptor ErbB-2/metabolismo , Células A549 , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Antioxidantes/química , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Drogas , Receptores ErbB/química , Gefitinibe , Células HEK293 , Células HeLa , Humanos , Ligações de Hidrogênio , Imidazóis/química , Simulação de Acoplamento Molecular , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/toxicidade , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/toxicidade , Estrutura Terciária de Proteína , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/toxicidade , Receptor ErbB-2/química , Relação Estrutura-Atividade , Termodinâmica
20.
J Immunol Methods ; 451: 28-36, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28827189

RESUMO

Next generation sequencing (NGS) is widely applied in immunological research, but has yet to become common in antibody epitope mapping. A method utilizing a 12-mer random peptide library expressed in bacteria coupled with magnetic-based cell sorting and NGS correctly identified >75% of epitope residues on the antigens of two monoclonal antibodies (trastuzumab and bevacizumab). PepSurf, a web-based computational method designed for structural epitope mapping was utilized to compare peptides in libraries enriched for monoclonal antibody (mAb) binders to antigen surfaces (HER2 and VEGF-A). Compared to mimotopes recovered from Sanger sequencing of plated colonies from the same sorting protocol, motifs derived from sets of the NGS data improved epitope prediction as defined by sensitivity and precision, from 18% to 82% and 0.27 to 0.51 for trastuzumab and 47% to 76% and 0.19 to 0.27 for bevacizumab. Specificity was similar for Sanger and NGS, 99% and 97% for trastuzumab and 66% and 67% for bevacizumab. These results indicate that combining peptide library screening with NGS yields epitope motifs that can improve prediction of structural epitopes.


Assuntos
Anticorpos Monoclonais/metabolismo , Antineoplásicos Imunológicos/metabolismo , Bevacizumab/metabolismo , Mapeamento de Epitopos/métodos , Epitopos , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca de Peptídeos , Receptor ErbB-2/genética , Trastuzumab/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Algoritmos , Motivos de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antineoplásicos Imunológicos/imunologia , Bevacizumab/imunologia , Sítios de Ligação de Anticorpos , Biologia Computacional , Bases de Dados Genéticas , Separação Imunomagnética , Modelos Químicos , Ligação Proteica , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , Trastuzumab/imunologia , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
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