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1.
BMC Immunol ; 25(1): 29, 2024 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-38730320

RESUMEN

BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.


Asunto(s)
Reacciones Cruzadas , Inmunoterapia , Receptor de Muerte Celular Programada 1 , Animales , Humanos , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Ratones , Reacciones Cruzadas/inmunología , Inmunoterapia/métodos , Concentración de Iones de Hidrógeno , Neoplasias/inmunología , Neoplasias/terapia , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Epítopos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Ratones Endogámicos C57BL , Femenino
2.
Mol Psychiatry ; 27(11): 4770-4780, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35948663

RESUMEN

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder, which is characterized by cognitive deficit due to synaptic loss and neuronal death. Extracellular amyloid ß plaques are one of the pathological hallmarks of AD. The autophagic lysosomal pathway is the essential mechanism to maintain cellular homeostasis by driving clearance of protein aggregates and is dysfunctional in AD. Here, we showed that inhibiting MEK/ERK signaling using a clinically available MEK1/2 inhibitor, trametinib (GSK1120212, SNR1611), induces the protection of neurons through autophagic lysosomal activation mediated by transcription factor EB (TFEB) in a model of AD. Orally administered trametinib recovered impaired neural structures, cognitive functions, and hippocampal long-term potentiation (LTP) in 5XFAD mice. Trametinib also reduced Aß deposition via induction of autophagic lysosomal activation. RNA-sequencing analysis revealed upregulation of autophagic lysosomal genes by trametinib administration. In addition, trametinib inhibited TFEB phosphorylation at Ser142 and promoted its nuclear translocation, which in turn induced autophagic lysosomal related genes, indicating that trametinib activates the autophagic lysosomal process through TFEB activation. From these observations, we concluded that MEK inhibition provides neuronal protection from the Aß burden by increasing autophagic lysosomal activity. Thus, MEK inhibition may be an effective therapeutic strategy for AD.


Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Lisosomas/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Placa Amiloide/metabolismo , Autofagia
3.
Biochem Biophys Res Commun ; 482(2): 215-220, 2017 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-27840051

RESUMEN

Protein phosphatase 5 (PP5) is a serine/threonine phosphatase that belongs to the PPP family phosphatases. PP5 and the other phosphatases of the PPP family share significantly similar catalytic domain structure. Due to this structural similarity, natural competitive inhibitors such as okadaic acid and cantharidin exhibit broad specificity over the PPP family phosphatases. In this study, we report the identification of three PP5 inhibitors, Ro 90-7501, aurothioglucose, and N-oleoyldopamine, along with a novel inhibitory mechanism of Ro 90-7501. Unlike other inhibitors binding to the phosphatase domain, Ro 90-7501 inhibited PP5 in a TPR-dependent manner. This TPR-dependent PP5 inhibition shown by Ro 90-7501 is a unique and novel inhibitory mechanism, which might be a useful tool for studies of PP5 on both regulatory mechanism and drug discovery.


Asunto(s)
Aminas/química , Bencimidazoles/química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Sitios de Unión , Activación Enzimática , Unión Proteica , Dominios Proteicos
4.
Biochem Biophys Res Commun ; 478(4): 1647-52, 2016 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-27591899

RESUMEN

NOD1 is an intracellular sensor of innate immunity which is related to a number of inflammatory diseases. NOD1 is known to be difficult to express and purify for structural and biochemical studies. Based on the fact that Hsp90 and its cochaperone SGT1 are necessary for the stabilization and activation of NOD1 in mammals, SGT1 was chosen as a fusion partner of the leucine-rich repeat (LRR) domain of NOD1 for its soluble expression in Escherichia coli. Fusion of human SGT1 (hSGT1) to NOD1 LRR significantly enhanced the solubility, and the fusion protein was stabilized by coexpression of mouse Hsp90α. The expression level of hSGT1-NOD1 LRR was further enhanced by supplementation of rare codon tRNAs and exchange of antibiotic marker genes.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas de Ciclo Celular/genética , Escherichia coli/genética , Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Humanos , Immunoblotting , Ratones , Proteína Adaptadora de Señalización NOD1/genética , Reacción en Cadena de la Polimerasa , Estabilidad Proteica , Secuencias Repetitivas de Aminoácido/genética , Solubilidad
5.
J Biol Chem ; 288(1): 215-22, 2013 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-23184943

RESUMEN

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Proteínas del Citoesqueleto/química , Glicoproteínas/química , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Musculares/química , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Calcio/química , Calcio/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Nucleótidos/química , Proteínas de Unión a Fosfato , Unión Proteica , Mapeo de Interacción de Proteínas/métodos
6.
Bioorg Med Chem Lett ; 21(6): 1593-7, 2011 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-21353544

RESUMEN

Novel 2,4-diaminoquinazoline derivatives originating from a virtual screening approach were designed, synthesized and their biological activities as heat shock protein 90 (Hsp90) inhibitors were evaluated. The prepared compounds exhibited significant anti-proliferative activities against DU-145, HT-29, HCT-116, A375P and MCF-7 cancer cell lines. The selected compounds were tested against Her2, a client protein of Hsp90, and showed significant reduction in Her2 protein expression. Compound 6b was found the most potent, reduced Her2 protein expression levels and induced Hsp70 protein expression levels significantly.


Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Quinazolinas/síntesis química , Quinazolinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Humanos , Quinazolinas/química
7.
Bioorg Med Chem ; 19(5): 1714-20, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21306907

RESUMEN

Structure based drug design (SBDD) was used to discover heat shock protein 90 (HSP90) inhibitors useful in the treatment of cancer. By using the crystal structure of HSP90-ligand complex (1uyi), a docking model was prepared and was validated by external dataset containing known HSP90 inhibitors. This validated model was then used to virtually screen commercial databases, selected hits of which were bought and sent for real biological evaluation. Further as an alternative method, pharmacophores were generated using crystal structure conformations of ligands in HSP90 complexes (1uyi and 2bz5) and where used for virtual screening. Both cases yielded several hits containing novel scaffolds, particularly compound KHSP8 showed an IC(50) value of 0.902 µM in case of colon cancer (HT29), which is comparable to doxorubicin (0.828 µM). These compounds were being now used as leads for constructing small molecular libraries to get compounds with favourable pharmacokinetics and drug like properties.


Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Diseño de Fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Modelos Moleculares , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Humanos , Concentración 50 Inhibidora , Estructura Molecular
8.
Bioorg Med Chem Lett ; 19(16): 4839-42, 2009 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-19560353

RESUMEN

Structure-based virtual screening identified pyrimidine-2,4,6-trione and 4H-1,2,4-triazole-3-thiol as novel scaffolds of Hsp90 ATPase inhibitors. Their binding modes in the ATP-binding pocket of Hsp90 were analyzed using AutoDoc program combined with molecular dynamics (MD) simulations.


Asunto(s)
Antineoplásicos/química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Pirimidinas/química , Triazoles/química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Técnicas Químicas Combinatorias , Simulación por Computador , Bases de Datos Factuales , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Receptor ErbB-2/metabolismo
9.
FEBS Lett ; 586(16): 2535-41, 2012 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-22750442

RESUMEN

Pendrin is a transmembrane chloride/anion exchanger highly expressed in thyroid, kidney, and inner ear. Endoplasmic reticulum (ER)-retention of improperly folded Pendrin mutants is considered as the major cause for Pendred syndrome. However, the folding and degradation mechanisms of Pendrin are poorly understood. Here, we report that treatment of 17-AAG, an Hsp90 inhibitor, facilitates the folding of Pendrin through heat shock transcription factor 1 (Hsf1)-dependent induction of molecular chaperones. Furthermore, we demonstrate that Rma1, an E3 ubiquitin ligase localized in the ER membrane, is involved in Pendrin degradation.


Asunto(s)
Benzoquinonas/farmacología , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Lactamas Macrocíclicas/farmacología , Proteínas de Transporte de Membrana/química , Chaperonas Moleculares/química , Ubiquitina-Proteína Ligasas/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Factores de Transcripción del Choque Térmico , Humanos , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Transportadores de Sulfato , Factores de Transcripción/metabolismo
10.
Biosens Bioelectron ; 25(6): 1307-12, 2010 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-19914055

RESUMEN

A rapid and efficient strategy to detect novel heat shock protein 90 (Hsp90) inhibitors as anti-cancer agents was developed with field-effect transistor (FET) sensor based on carboxylated polypyrrole nanotubes (CPNTs). First of all, the CPNTs were successfully fabricated by using cylindrical micelle templates in a water-in-oil emulsion system, and the functional carboxyl groups were effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer. A liquid-ion gated FET-type sensor was readily constructed on the basis of CPNTs as the conductive channel. The CPNTs were covalently immobilized onto the microelectrode substrate to maintain stable electrical contact between the CPNTs and the microelectrodes in the liquid phase. Subsequently, Hsp90 was attached to the CPNTs surface through condensation reactions between the terminal amino groups in Hsp90 amino acid residues and the carboxyl groups on the CPNTs. The Hsp90-conjugated CPNT FET sensor provided a convenient and sensitive method to observe the affinity between Hsp90 to Hsp90 inhibitors in real-time. This result suggests that the FET sensor will open up the potential application for new anti-cancer drug discovery (Hsp90 inhibitors) after a judicious optimization.


Asunto(s)
Antineoplásicos/análisis , Benzoquinonas/análisis , Técnicas Biosensibles/instrumentación , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/análisis , Nanotubos/química , Polímeros/química , Pirroles/química , Transistores Electrónicos , Diseño de Equipo , Análisis de Falla de Equipo , Nanotubos/ultraestructura , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
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