RESUMO
CONTEXT: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). OBJECTIVE: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. METHODS: A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis. RESULTS: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. CONCLUSION: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans.
Assuntos
Proliferação de Células , Receptores da Tireotropina , Neoplasias da Glândula Tireoide , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Camundongos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Proliferação de Células/efeitos dos fármacos , Receptores da Tireotropina/antagonistas & inibidores , Receptores da Tireotropina/metabolismo , Ligantes , Linhagem Celular Tumoral , Administração Oral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/administração & dosagem , Metástase Neoplásica , Simulação de Acoplamento Molecular , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , FemininoRESUMO
Lung cancer cells in the tumor microenvironment facilitate immune evasion that leads to failure of conventional chemotherapies, despite provisionally decided on the genetic diagnosis of patients in a clinical setup. The current study follows three lung cancer patients who underwent "personalized" chemotherapeutic intervention. Patient-derived xenografts (PDXs) were subjected to tumor microarray and treatment screening with chemotherapies, either individually or in combination with the peptide R11-NLS-pep8; this peptide targets both membrane-associated and nuclear PCNA. Ex vivo, employing PDX-derived explants, it was found that combination with R11-NLS-pep8 stimulated antineoplastic effect of chemotherapies that were, although predicted based on the patient's genetic mutation, inactive on their own. Furthermore, treatment in vivo of PDX-bearing mice showed an exactly similar trend in the result, corroborating the finding to be translated into clinical setup.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Humanos , Camundongos , Animais , Sistemas de Liberação de Medicamentos , Peptídeos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Microambiente Tumoral , Modelos Animais de DoençasRESUMO
Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells' functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38+CD138+ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.
Assuntos
Mieloma Múltiplo , Linhagem Celular Tumoral , Humanos , Células Matadoras Naturais , Mieloma Múltiplo/metabolismo , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: "S2A". Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.
Assuntos
Autoimunidade/efeitos dos fármacos , Interleucina-2/análogos & derivados , Receptor 2 Desencadeador da Citotoxicidade Natural/química , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Artrite Reumatoide/prevenção & controle , Preparações de Ação Retardada , Avaliação Pré-Clínica de Medicamentos , Glicosilação , Meia-Vida , Interleucina-2/administração & dosagem , Interleucina-2/farmacocinética , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A*0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A*0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Evasão da Resposta Imune , Ligantes , Polissacarídeos , Proteínas do Envelope Viral/genéticaRESUMO
Pseudomonas aeruginosa is a highly virulent bacterium, particularly associated with the spread of multidrug resistance. Here we show that carbon dots (C-dots), synthesized from aminoguanidine and citric acid precursors, can selectively stain and inhibit the growth of P. aeruginosa strains. The aminoguanidine-C-dots were shown both to target P. aeruginosa bacterial cells and also to inhibit biofilm formation by the bacteria. Mechanistic analysis points to interactions between aminoguanidine residues on the C-dots' surface and P. aeruginosa lipopolysaccharide moieties as the likely determinants for both antibacterial and labeling activities. Indeed, the application of biomimetic membrane assays reveals that LPS-promoted insertion and bilayer permeation constitute the primary factors in the anti- P. aeruginosa effect of the aminoguanidine-C-dots. The aminoguanidine C-dots are easy to prepare in large quantities and are inexpensive and biocompatible and thus may be employed as a useful vehicle for selective staining and antibacterial activity against P. aeruginosa.
Assuntos
Antibacterianos/farmacologia , Carbono/química , Nanoestruturas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Materiais Biocompatíveis/química , Biofilmes/efeitos dos fármacos , Carbono/farmacologia , Farmacorresistência Bacteriana Múltipla , Guanidinas/química , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/crescimento & desenvolvimento , Coloração e RotulagemRESUMO
Protein-ligand electrostatic interaction can be looked upon as ion receptor-ligand interaction, and the binding cavity of protein can be either an anion or cation receptor depending on the charge of the guest. Here we focus on the exploration of pH-modulated binding of a number of anionic ligands, specific to the subdomain IIA cavity of HSA, such as carmoisine, tartrazine, cochineal red, and warfarin. The logarithm of the binding constant is found to vary linearly with the square-root of ionic strength, indicating applicability of the Debye-Hückel limiting law to protein-ligand electrostatic binding equilibrium, and concludes that the subdomain IIA cavity is an anion receptor. The present approach is very unique that one can calculate the effective charge of the protein-based anion receptor pocket, and the calculated charge has been found to vary between +1 and +3 depending on the pH and ligand itself. The study also indicates that in such cases of specific ligand binding the pocket charge rather than the overall or surface charge of the macromolecule seems to have a paramount role in determining the strength of interaction. For the first time, it is demonstrated that the Debye-Hückel interionic interaction model can be successfully applied to understand the protein-based receptor-ligand electrostatic interaction in general.
Assuntos
Compostos Azo/metabolismo , Naftalenossulfonatos/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo , Tartrazina/metabolismo , Varfarina/metabolismo , Ânions/química , Ânions/metabolismo , Compostos Azo/química , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Modelos Moleculares , Estrutura Molecular , Naftalenossulfonatos/química , Estrutura Terciária de Proteína , Eletricidade Estática , Tartrazina/química , Varfarina/químicaRESUMO
Our recent report on the binding of Cochineal Red A, a food dye, with HSA and BSA at pH 7.4 has revealed that electrostatic forces is the principal cause of interaction. In that study issues relating to complications arising out of modulation of dye binding affinity of BSA with pH had not been explored. Here we have further explored the interaction of Cochineal Red A with BSA in pH range 4.8-7.8. Surprisingly, this system behaves differently in the texture of interaction pattern at two extremes of studied pH range, unlike HSA. Importantly, the charge on the amino acid side chains in the binding pocket is likely to play a significant role.
Assuntos
Compostos Azo/química , Dicroísmo Circular , Simulação de Acoplamento Molecular , Soroalbumina Bovina/química , Albumina Sérica/química , Animais , Sítios de Ligação , Bovinos , Aditivos Alimentares/química , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Naftalenossulfonatos , Espectrometria de Fluorescência , Eletricidade EstáticaRESUMO
Formation of ion pair between charged molecule and protein can lead to interesting biochemical phenomena. We report the evolution of thermodynamics of the binding of tartrazine, a negatively charged azo colorant, and serum albumins with salt. The dye binds predominantly electrostatically in low buffer strengths; however, on increasing salt concentration, affinity decreases considerably. The calculated thermodynamic parameters in high salt indicate manifestation of nonelectrostatic interactions, namely, van der Waals force and hydrogen bonding. Site-marker competitive binding studies and docking simulations indicate that the dye binds with HSA in the warfarin site and with BSA at the interface of warfarin and ibuprofen binding sites. The docked poses indicate nearby amino acid positive side chains, which are possibly responsible for electrostatic interaction. Using the Debye-Hückel interionic attraction theory for binding equilibria, it is shown that, for electrostatic binding the calculated free energy change increases linearly with square root of ionic strength. Also UV-vis, fluorescence, CD data indicate a decrease of interaction with salt concentration. This study quantitatively relates how ionic strength modulates the strength of the protein-ligand electrostatic interaction. The binding enthalpy and entropy have been found to compensate one another. The enthalpy-entropy compensation (EEC), general property of weak intermolecular interactions, has been discussed.
Assuntos
Albumina Sérica/química , Tartrazina/química , Termodinâmica , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Humanos , Sais/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Eletricidade EstáticaRESUMO
Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.