RESUMEN

α-Synuclein (α-syn), a disordered cytoplasmatic protein, plays a fundamental role in the pathogenesis of Parkinson's disease (PD). Here, we have shown, using photophysical measurements, that addition of FKBP12 to α-syn solutions, dramatically accelerates protein aggregation, leading to an explosion of dendritic structures revealed by fluorescence and phase-contrast microscopy. We have further demonstrated that this aberrant α-syn aggregation can be blocked using a recently discovered non-immunosuppressive synthetic inhibitor of FKBP12, ElteN378. The role of FKBP12 and of ElteN378 in the α-syn aggregation mechanism has been elucidated using molecular dynamics simulations based on an effective coarse-grained model. The reported data not only reveal a new potent synthetic drug as a candidate for early stage treatment of α-syn dependent neurodegenerations but also pave the way to a deeper understanding of the mechanism of action of FKBP12 on α-syn oligomeric aggregation, a topic which is still controversial.

Asunto(s)

Piperidinas/farmacología , Agregado de Proteínas/efectos de los fármacos , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , alfa-Sinucleína/química , Dendrímeros/química , Cinética , Simulación de Dinámica Molecular , Piperidinas/química , Unión Proteica , Conformación Proteica , Transducción de Señal , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/metabolismo

RESUMEN

Progression of chronic kidney disease associated with progressive fibrosis and impaired tubular epithelial regeneration is still an unmet biomedical challenge because, once chronic lesions have manifested, no effective therapies are available as of yet for clinical use. Prompted by various studies across multiple organs demonstrating that preconditioning regimens to induce endogenous regenerative mechanisms protect various organs from later incurring acute injuries, we here aimed to gain insights into the molecular mechanisms underlying successful protection and to explore whether such pathways could be utilized to inhibit progression of chronic organ injury. We identified a protective mechanism controlled by the transcription factor ARNT that effectively inhibits progression of chronic kidney injury by transcriptional induction of ALK3, the principal mediator of antifibrotic and proregenerative bone morphogenetic protein-signaling (BMP-signaling) responses. We further report that ARNT expression itself is controlled by the FKBP12/YY1 transcriptional repressor complex and that disruption of such FKBP12/YY1 complexes by picomolar FK506 at subimmunosuppressive doses increases ARNT expression, subsequently leading to homodimeric ARNT-induced ALK3 transcription. Direct targeting of FKBP12/YY1 with in vivo morpholino approaches or small molecule inhibitors, including GPI-1046, was equally effective for inducing ARNT expression, with subsequent activation of ALK3-dependent canonical BMP-signaling responses and attenuated chronic organ failure in models of chronic kidney disease, and also cardiac and liver injuries. In summary, we report an organ-protective mechanism that can be pharmacologically modulated by immunophilin ligands FK506 and GPI-1046 or therapeutically targeted by in vivo morpholino approaches.

Asunto(s)

Translocador Nuclear del Receptor de Aril Hidrocarburo/biosíntesis , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Línea Celular , Progresión de la Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Fallo Renal Crónico/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pirrolidinas/farmacología , Transducción de Señal/efectos de los fármacos , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/metabolismo , Factor de Transcripción YY1/antagonistas & inhibidores , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismo

RESUMEN

Stroke is a disease of the leading causes of mortality and disability across the world, but the benefits of drugs curative effects look less compelling, intracellular calcium overload is considered to be a key pathologic factor for ischemic stroke. Gualou Guizhi decoction (GLGZD), a classical Chinese medicine compound prescription, it has been used to human clinical therapy of sequela of cerebral ischemia stroke for 10 years. This work investigated the GLGZD improved prescription against intracellular calcium overload could decreased the concentration of [Ca2+]i in cortex and striatum neurone of MCAO rats. GLGZD contains Trichosanthin and various small molecular that they are the potential active ingredients directed against NR2A, NR2B, FKBP12 and Calnodulin target proteins/enzyme have been screened by computer simulation. "Multicomponent systems" is capable to create pharmacological superposition effects. The Chinese medicine compound prescriptions could be considered as promising sources of candidates for discovery new agents.

Asunto(s)

Isquemia Encefálica/tratamiento farmacológico , Calcio/metabolismo , Medicamentos Herbarios Chinos/farmacología , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/química , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/metabolismo , Tricosantina/administración & dosificación , Tricosantina/química , Tricosantina/farmacología

RESUMEN

NMR and X-ray crystallography are the two most widely used methods for determining protein structures. Our previous study examining NMR versus X-Ray sources of protein conformations showed improved performance with NMR structures when used in our Multiple Protein Structures (MPS) method for receptor-based pharmacophores (Damm, Carlson, J Am Chem Soc 129:8225-8235, 2007). However, that work was based on a single test case, HIV-1 protease, because of the rich data available for that system. New data for more systems are available now, which calls for further examination of the effect of different sources of protein conformations. The MPS technique was applied to Growth factor receptor bound protein 2 (Grb2), Src SH2 homology domain (Src-SH2), FK506-binding protein 1A (FKBP12), and Peroxisome proliferator-activated receptor-γ (PPAR-γ). Pharmacophore models from both crystal and NMR ensembles were able to discriminate between high-affinity, low-affinity, and decoy molecules. As we found in our original study, NMR models showed optimal performance when all elements were used. The crystal models had more pharmacophore elements compared to their NMR counterparts. The crystal-based models exhibited optimum performance only when pharmacophore elements were dropped. This supports our assertion that the higher flexibility in NMR ensembles helps focus the models on the most essential interactions with the protein. Our studies suggest that the "extra" pharmacophore elements seen at the periphery in X-ray models arise as a result of decreased protein flexibility and make very little contribution to model performance.

Asunto(s)

Proteína Adaptadora GRB2/química , Modelos Moleculares , PPAR gamma/química , Proteína 1A de Unión a Tacrolimus/química , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos Factuales , Diseño de Fármacos , Proteína Adaptadora GRB2/agonistas , Proteína Adaptadora GRB2/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética , PPAR gamma/agonistas , PPAR gamma/antagonistas & inhibidores , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Dominios Homologos src

RESUMEN

The recently proposed fast switching double annihilation (FS-DAM) [Cardelli et al., J. Chem. Theory Comput., 2015, 11, 423] is aimed at computing the absolute standard dissociation free energies for the chemical equilibrium RL ⇌ R + L occurring in solution through molecular dynamics (MD) simulations at the atomistic level. The technique is based on the production of fast nonequilibrium annihilation trajectories of one of the species (the ligand) in the solvated RL complex and in the bulk solvent. As detailed in the companion theoretical paper, the free energies of these two nonequilibrium annihilation processes are recovered by using an unbiased unidirectional estimate derived from the Crooks theorem exploiting the inherent Gaussian nature of the annihilation work. The FS-DAM technique was successfully applied to the evaluation of the dissociation free energy of the complexes of Zn(ii) cations with an inhibitor of the Tumor Necrosis Factor α converting enzyme. Here we apply the technique to a real drug-receptor system, by satisfactorily reproducing the experimental dissociation free energies of FK506-related bulky ligands towards the native FKBP12 enzyme and by predicting the dissociation constants for the same ligands towards the mutant I56D. The effect of such mutations on the binding affinity of FK506-related ligands is relevant for assessing the thermodynamic forces regulating molecular recognition in FKBP12 inhibition.

Asunto(s)

Inhibidores de la Calcineurina/metabolismo , Inmunosupresores/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , Tacrolimus/metabolismo , Termodinámica , Inhibidores de la Calcineurina/farmacología , Descubrimiento de Drogas , Humanos , Inmunosupresores/farmacología , Ligandos , Simulación de Dinámica Molecular , Mutación Puntual , Unión Proteica , Conformación Proteica , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/genética

RESUMEN

BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation.

Asunto(s)

Virus BK/fisiología , Células Epiteliales/virología , Túbulos Renales/virología , Sirolimus/farmacología , Proteína 1A de Unión a Tacrolimus/metabolismo , Tacrolimus/farmacología , Replicación Viral/efectos de los fármacos , Western Blotting , Células Cultivadas , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunosupresores/uso terapéutico , Lactante , Túbulos Renales/metabolismo , Infecciones por Polyomavirus/tratamiento farmacológico , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/genética , Infecciones Tumorales por Virus/tratamiento farmacológico , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología

RESUMEN

In drug discovery, small molecules must often discriminate between healthy and diseased cells. This feat is usually accomplished by binding to a protein that is preferentially expressed in the target cell or on its surface. However, in many cases, the expression of an individual protein may not generate sufficient cyto-selectivity. Here, we demonstrate that bispecific molecules can better discriminate between similar cell types by exploiting their simultaneous affinity for two proteins. Inspired by the natural product FK506, we designed molecules that have affinity for both FKBP12 and HIV protease. Using cell-based reporters and live virus assays, we observed that these compounds preferentially accumulated in cells that express both targets, mimicking an infected lymphocyte. Treatment with FKBP12 inhibitors reversed this partitioning, while overexpression of FKBP12 protein further promoted it. The partitioning into the target cell type could be tuned by controlling the properties of the linker and the affinities for the two proteins. These results show that bispecific molecules create significantly better potential for cyto-selectivity, which might be especially important in the development of safe and effective antivirals and anticancer compounds.

Asunto(s)

Anticuerpos Biespecíficos/química , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Regulación de la Expresión Génica , Proteasa del VIH/genética , Proteína 1A de Unión a Tacrolimus/genética , Citometría de Flujo , Proteasa del VIH/metabolismo , Humanos , Estructura Molecular , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/metabolismo

RESUMEN

MOTIVATION: Using molecular similarity to discover bioactive small molecules with novel chemical scaffolds can be computationally demanding. We describe Ultra-fast Shape Recognition with Atom Types (UFSRAT), an efficient algorithm that considers both the 3D distribution (shape) and electrostatics of atoms to score and retrieve molecules capable of making similar interactions to those of the supplied query. RESULTS: Computational optimization and pre-calculation of molecular descriptors enables a query molecule to be run against a database containing 3.8 million molecules and results returned in under 10 seconds on modest hardware. UFSRAT has been used in pipelines to identify bioactive molecules for two clinically relevant drug targets; FK506-Binding Protein 12 and 11ß-hydroxysteroid dehydrogenase type 1. In the case of FK506-Binding Protein 12, UFSRAT was used as the first step in a structure-based virtual screening pipeline, yielding many actives, of which the most active shows a KD, app of 281 µM and contains a substructure present in the query compound. Success was also achieved running solely the UFSRAT technique to identify new actives for 11ß-hydroxysteroid dehydrogenase type 1, for which the most active displays an IC50 of 67 nM in a cell based assay and contains a substructure radically different to the query. This demonstrates the valuable ability of the UFSRAT algorithm to perform scaffold hops. AVAILABILITY AND IMPLEMENTATION: A web-based implementation of the algorithm is freely available at http://opus.bch.ed.ac.uk/ufsrat/.

Asunto(s)

11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1 , Algoritmos , Simulación por Computador , Inhibidores Enzimáticos , Simulación del Acoplamiento Molecular , Proteína 1A de Unión a Tacrolimus , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/metabolismo

RESUMEN

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site.

Asunto(s)

Inhibidores Enzimáticos/química , Rodio/química , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/metabolismo , Estructura Terciaria de Proteína , Rodio/farmacología , Proteína 1A de Unión a Tacrolimus/metabolismo

RESUMEN

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

Asunto(s)

Receptores de Activinas Tipo I/química , Miositis Osificante/enzimología , Receptores de Activinas Tipo I/antagonistas & inhibidores , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Secuencias de Aminoácidos , Animales , Proteína Morfogenética Ósea 4/fisiología , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Ratones , Modelos Moleculares , Mutación Missense , Miositis Osificante/genética , Unión Proteica , Pirazoles/química , Pirimidinas/química , Transducción de Señal , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/metabolismo

RESUMEN

Dengue is one of the most infectious viral diseases prevalent mainly in tropical countries. The virus is transmitted by Aedes species of mosquito, primarily Aedes aegypti. Dengue remains a challenging drug target for years as the virus eludes the immune responses. Currently, no vaccines or antiviral drugs are available for dengue prevention. Previous studies suggested that the immunosuppressive drug FK506 shows antimalarial activity, and its molecular target, FK506-binding protein (FKBP), was identified in the Plasmodium parasite. Likewise, a FKBP family protein has been identified in A. aegypti (AaFKBP12) in which AaFKBP12 is assumed to play a similar role in its life cycle. FKBPs belong to a highly conserved class of proteins and are considered as an attractive pharmacological target. Herein, we present a high-resolution crystal structure of AaFKBP12 at 1.3 Å resolution and discuss its structural features throwing light in facilitating the design of potential antagonists against the dengue-transmitting mosquito.

Asunto(s)

Aedes/química , Dengue/transmisión , Insectos Vectores/química , Proteína 1A de Unión a Tacrolimus/química , Aedes/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Dengue/prevención & control , Diseño de Fármacos , Humanos , Insectos Vectores/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores

RESUMEN

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias.

Asunto(s)

Activación del Canal Iónico , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Masculino , Modelos Biológicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosfolípidos/metabolismo , Ratas , Ratas Wistar , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Ovinos , Factores de Tiempo

RESUMEN

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.

Asunto(s)

Regulación hacia Abajo , Receptores ErbB/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , Anticuerpos Fosfo-Específicos , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Reactivos de Enlaces Cruzados , Dimerización , Regulación hacia Abajo/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Silenciador del Gen , Células HeLa , Humanos , Cinética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/genética

RESUMEN

Mammalian target of rapamycin (mTOR), a large multidomain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein-drug interactions at the domain level, while NMR gave insights into the protein-drug interactions at the residue level. The use of the FKBP12-FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.

Asunto(s)

Rastreo Diferencial de Calorimetría/métodos , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética/métodos , Proteínas Recombinantes de Fusión/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , Inhibidores Enzimáticos/química , Humanos , Ligandos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/química , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/química

RESUMEN

This article presents a mathematical model on the design of peptide inhibitors for proteins. This model is a combination of the two rules on protein-ligand interaction, Miyazawa-Jernigan (M-J) matrix and hidden Markov model (HMM). The model is applied to predict peptide inhibitors for the protein cyclophilin A (CypA) and FKBP12, and then validated by the highest occupied molecular orbital calculation, dock process between protein and inhibitor, and biological experiments. The results are encouraging and suggest that we have taken a step forward towards building a mathematical theory on the design of peptide inhibitors for proteins. The mathematical model is rough at present, but if it represents a correct direction of the theoretical trends of biology as we believe, then this theory can be further developed and become more and more precise.

Asunto(s)

Péptidos/química , Péptidos/farmacología , Proteínas/antagonistas & inhibidores , Ciclofilina A/antagonistas & inhibidores , Ciclofilina A/metabolismo , Ligandos , Cadenas de Markov , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Proteínas/metabolismo , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/metabolismo

RESUMEN

Recent years have observed significant advances in our understanding of how the serine/threonine kinase target of rapamycin (TOR) controls key cellular processes such as cell survival, growth and proliferation. Consistent with its role in cell proliferation, the mTOR pathway is frequently hyperactivated in a number of human malignancies and is thus considered to be an attractive target for anti-cancer therapy. Rapamycin and its analogs (rapalogs) function as allosteric inhibitors of mTORC1 and are currently used in the treatment of advanced renal cell carcinoma. Rapamycin and its derivatives bind to the small immunophilin FKBP12 to inhibit mTORC1 signalling through a poorly understood mechanism. Rapamycin/FKBP12 efficiently inhibit some, but not all, functions of mTOR and hence much interest has been placed in the development of drugs that target the kinase activity of mTOR directly. Several novel active-site inhibitors of mTOR, which inhibit both mTORC1 and mTORC2, were developed in the last year. In this manuscript, we provide a brief outline of our current understanding of the mTOR signalling pathway and review the molecular underpinnings of the action of rapamycin and novel active-site mTOR inhibitors as well as potential advantages and caveats associated with the use of these drugs in the treatment of cancer.

Asunto(s)

Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/enzimología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo

RESUMEN

Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) to solid organ transplant (SOT) recipients has been shown safe and effective for the treatment of EBV-associated posttransplantation lymphoproliferative disorders (PTLDs). SOT recipients, however, require the continuous administration of immunosuppressive drugs to prevent graft rejection, and these agents may significantly limit the long-term persistence of transferred EBV-CTLs, precluding their use as prophylaxis. Tacrolimus (FK506) is one of the most widely used immunosuppressive agents in SOT recipients, and its immunosuppressive effects are largely dependent on its interaction with the 12-kDa FK506-binding protein (FKBP12). We have knocked down the expression of FKBP12 in EBV-CTLs using a specific small interfering RNA (siRNA) stably expressed from a retroviral vector and found that FKBP12-silenced EBV-CTLs are FK506 resistant. These cells continue to expand in the presence of the drug without measurable impairment of their antigen specificity or cytotoxic activity. We confirmed their FK506 resistance and anti-PTLD activity in vivo using a xenogenic mouse model, suggesting that the proposed strategy may be of value to enhance EBV-specific immune surveillance in patients at high risk of PTLD after transplantation.

Asunto(s)

Técnicas de Silenciamiento del Gen , Vectores Genéticos/farmacología , Herpesvirus Humano 4/inmunología , Inmunosupresores/uso terapéutico , ARN Interferente Pequeño/fisiología , Linfocitos T Citotóxicos/efectos de los fármacos , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Tacrolimus/farmacología , Traslado Adoptivo , Animales , Antígenos Virales/inmunología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Células Cultivadas/trasplante , Citotoxicidad Inmunológica , Resistencia a Medicamentos/genética , Humanos , Linfoma no Hodgkin/terapia , Linfoma no Hodgkin/virología , Ratones , Ratones SCID , Procesamiento Proteico-Postraduccional , ARN Mensajero/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Retroviridae/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/trasplante , Proteína 1A de Unión a Tacrolimus/efectos de los fármacos , Proteína 1A de Unión a Tacrolimus/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

BACKGROUND: The mammalian target of rapamycin protein (mTOR) is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues. As a critical downstream mediator of PI3K signaling, mTOR is important for lymphocyte development and function of mature T and B-cells. Most studies of mTOR in immune responses have relied on the use of pharmacological inhibitors, such as rapamycin. Rapamycin-FKBP12 complex exerts its immunosuppressive and anti-proliferative effect by binding outside the kinase domain of mTOR, and subsequently inhibiting downstream mTOR signaling. RESULTS: To determine the requirement for mTOR kinase activity in the immune system function, we generated knock-in mice carrying a mutation (D2338) in the catalytic domain of mTOR. While homozygous mTOR kd/kd embryos died before embryonic day 6.5, heterozygous mTOR+/kd mice appeared entirely normal and are fertile. mTOR +/kd mice exhibited normal T and B cell development and unaltered proliferative responses of splenocytes to IL-2 and TCR/CD28. In addition, heterozygousity for the mTOR kinase-dead allele did not sensitize T cells to rapamycin in a CD3-mediated proliferation assay. Unexpectedly, mTOR kinase activity towards its substrate 4E-BP1 was not decreased in hearts and livers from heterozygous animals. CONCLUSION: Altogether, our findings indicate that mTOR kinase activity is indispensable for the early development of mouse embryos. Moreover, a single wild type mTOR allele is sufficient to maintain normal postnatal growth and lymphocyte development and proliferation.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Desarrollo Embrionario/inmunología , Sistema Inmunológico/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Linfocitos T/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Proteínas Portadoras/inmunología , Dominio Catalítico/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Desarrollo Embrionario/genética , Técnicas de Sustitución del Gen , Heterocigoto , Sistema Inmunológico/embriología , Sistema Inmunológico/crecimiento & desarrollo , Sistema Inmunológico/patología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Mutación , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Sirolimus/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Serina-Treonina Quinasas TOR , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores

RESUMEN

Peptidyl-prolyl cis-trans isomerases are a group of cytosolic enzymes initially characterized by their ability to catalyze the cis-trans isomerization of peptidyl-prolyl bonds. This represents a significant event for protein folding because cis-proline introduces critical bends within the protein conformation. FK506-binding proteins (FKBPs) represent one of the three families of enzymes sharing peptidyl-prolyl cis-trans isomerase activity. Inhibitors of FKBP12, in particular, have potent neurotrophic properties both in vivo and in vitro. Here, we describe a fragment-based unbiased nuclear magnetic resonance drug discovery approach for the identification of novel classes of chemical inhibitors against FKBP12. Compared to FK506, the fragment-based FKBP12 inhibitors developed herein possess significant advantages as drug candidates.

Asunto(s)

Morfolinas/síntesis química , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Animales , Línea Celular , Diseño de Fármacos , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Morfolinas/química , Morfolinas/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Ratas , Ratas Long-Evans , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Relación Estructura-Actividad , Tacrolimus/química , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/química

RESUMEN

Methotrexate (MTX), an inhibitor of dihydrofolate reductase, was tethered to an FKBP12 ligand (SLF), and the resulting bifunctional molecule (MTXSLF) potently inhibits either enzyme but not both simultaneously. MTXSLF is cytotoxic to fibroblasts derived from FKBP12-null mice but is detoxified 40-fold by FKBP12 in wild-type fibroblasts. These studies demonstrate that non-target proteins in an otherwise identical genetic background can be used to predictably regulate the biological activity of synthetic molecules.

Asunto(s)

Antagonistas del Ácido Fólico/farmacología , Metotrexato/química , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Tetrahidrofolato Deshidrogenasa/metabolismo , Ingeniería , Ambiente , Antagonistas del Ácido Fólico/química , Humanos , Metotrexato/farmacología , Proteína 1A de Unión a Tacrolimus/metabolismo