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1.
JACS Au ; 4(2): 760-770, 2024 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-38425914

RESUMEN

We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.

2.
bioRxiv ; 2024 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-38370689

RESUMEN

While efforts to identify microglial subtypes have recently accelerated, the relation of transcriptomically defined states to function has been largely limited to in silico annotations. Here, we characterize a set of pharmacological compounds that have been proposed to polarize human microglia towards two distinct states - one enriched for AD and MS genes and another characterized by increased expression of antigen presentation genes. Using different model systems including HMC3 cells, iPSC-derived microglia and cerebral organoids, we characterize the effect of these compounds in mimicking human microglial subtypes in vitro. We show that the Topoisomerase I inhibitor Camptothecin induces a CD74high/MHChigh microglial subtype which is specialized in amyloid beta phagocytosis. Camptothecin suppressed amyloid toxicity and restored microglia back to their homeostatic state in a zebrafish amyloid model. Our work provides avenues to recapitulate human microglial subtypes in vitro, enabling functional characterization and providing a foundation for modulating human microglia in vivo.

3.
Bioorg Med Chem Lett ; 92: 129409, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37453616

RESUMEN

Phosphodiesterase 5 (PDE5) is a cyclic guanosine monophosphate-degrading enzyme involved in numerous biological pathways. Inhibitors of PDE5 are important therapeutics for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD). We previously reported the first generation of quinoline-based PDE5 inhibitors for the treatment of AD. However, the short in vitro microsomal stability rendered them unsuitable drug candidates. Here we report a series of new quinoline-based PDE5 inhibitors. Among them, compound 4b, 8-cyclopropyl-3-(hydroxymethyl)-4-(((6-methoxypyridin-3-yl)methyl)amino)quinoline-6-carbonitrile, shows a PDE5 IC50 of 20 nM and improved in vitro microsomal stability (t1/2 = 44.6 min) as well as excellent efficacy in restoring long-term potentiation, a type of synaptic plasticity to underlie memory formation, in electrophysiology experiments with a mouse model of AD. These results provide an insight into the development of a new class of PDE5 inhibitors for the treatment of AD.


Asunto(s)
Enfermedad de Alzheimer , Quinolinas , Ratones , Animales , Inhibidores de Fosfodiesterasa 5/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Plasticidad Neuronal , Enfermedad de Alzheimer/tratamiento farmacológico , Quinolinas/farmacología , Quinolinas/uso terapéutico
4.
Science ; 380(6648): 942-948, 2023 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-37262137

RESUMEN

Aptameric receptors are important biosensor components, yet our ability to identify them depends on the target structures. We analyzed the contributions of individual functional groups on small molecules to binding within 27 target-aptamer pairs, identifying potential hindrances to receptor isolation-for example, negative cooperativity between sterically hindered functional groups. To increase the probability of aptamer isolation for important targets, such as leucine and voriconazole, for which multiple previous selection attempts failed, we designed tailored strategies focused on overcoming individual structural barriers to successful selections. This approach enables us to move beyond standardized protocols into functional group-guided searches, relying on sequences common to receptors for targets and their analogs to serve as anchors in regions of vast oligonucleotide spaces wherein useful reagents are likely to be found.


Asunto(s)
Antifúngicos , Aptámeros de Nucleótidos , Técnicas Biosensibles , Leucina , Técnica SELEX de Producción de Aptámeros , Voriconazol , Aptámeros de Nucleótidos/química , Técnica SELEX de Producción de Aptámeros/métodos , Leucina/sangre , Voriconazol/análisis , Antifúngicos/análisis
5.
Nat Med ; 27(4): 601-615, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33753937

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the coronavirus disease 2019 (COVID-19) pandemic, which has resulted in global healthcare crises and strained health resources. As the population of patients recovering from COVID-19 grows, it is paramount to establish an understanding of the healthcare issues surrounding them. COVID-19 is now recognized as a multi-organ disease with a broad spectrum of manifestations. Similarly to post-acute viral syndromes described in survivors of other virulent coronavirus epidemics, there are increasing reports of persistent and prolonged effects after acute COVID-19. Patient advocacy groups, many members of which identify themselves as long haulers, have helped contribute to the recognition of post-acute COVID-19, a syndrome characterized by persistent symptoms and/or delayed or long-term complications beyond 4 weeks from the onset of symptoms. Here, we provide a comprehensive review of the current literature on post-acute COVID-19, its pathophysiology and its organ-specific sequelae. Finally, we discuss relevant considerations for the multidisciplinary care of COVID-19 survivors and propose a framework for the identification of those at high risk for post-acute COVID-19 and their coordinated management through dedicated COVID-19 clinics.


Asunto(s)
COVID-19/complicaciones , SARS-CoV-2 , Enfermedad Aguda , COVID-19/epidemiología , COVID-19/etnología , COVID-19/terapia , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/terapia , Humanos , Defensa del Paciente , Síndrome , Síndrome de Respuesta Inflamatoria Sistémica/epidemiología , Síndrome de Respuesta Inflamatoria Sistémica/terapia , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/prevención & control
6.
iScience ; 23(12): 101884, 2020 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-33354662

RESUMEN

We previously identified the N-quinoline-benzenesulfonamide (NQBS) scaffold as a potent inhibitor of nuclear factor-κB (NF-κB) translocation. Now, we report the structure-activity relationship of compounds with the NQBS scaffold in models of diffuse large B-cell lymphoma (DLBCL). We identified CU-O42, CU-O47, and CU-O75 as NQBS analogs with the most potent cytotoxic activity in DLBCL lines. Their anti-lymphoma effect was mediated by NF-κB sequestration to the cytoplasm of DLBCL cells. Internal Coordinates Mechanics analysis suggested direct binding between CU-O75 and IκBα/p50/p65 which leads to the stabilization of the NF-κB trimer. A whole cellular thermal shift assay confirmed direct binding of the NQBS to IκBα, an inhibitory component of the IκBα/p50/p65 trimer. Lymphoma cell line sequencing revealed CU-O75 induced downregulation of NF-κB-dependent genes and DeMAND analysis identified IκBα as one of the top protein targets for CU-O75. CU-O42 was potent in inhibiting tumor growth in two mouse models of aggressive lymphomas.

7.
Front Physiol ; 11: 573492, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33013487

RESUMEN

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

8.
Eur J Endocrinol ; 183(5): R133-R147, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32755992

RESUMEN

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has generated an explosion of interest both in the mechanisms of infection leading to dissemination and expression of this disease, and in potential risk factors that may have a mechanistic basis for disease propagation or control. Vitamin D has emerged as a factor that may be involved in these two areas. The focus of this article is to apply our current understanding of vitamin D as a facilitator of immunocompetence both with regard to innate and adaptive immunity and to consider how this may relate to COVID-19 disease. There are also intriguing potential links to vitamin D as a factor in the cytokine storm that portends some of the most serious consequences of SARS-CoV-2 infection, such as the acute respiratory distress syndrome. Moreover, cardiac and coagulopathic features of COVID-19 disease deserve attention as they may also be related to vitamin D. Finally, we review the current clinical data associating vitamin D with SARS-CoV-2 infection, a putative clinical link that at this time must still be considered hypothetical.


Asunto(s)
Inmunidad Adaptativa/inmunología , Infecciones por Coronavirus/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Inmunidad Innata/inmunología , Inmunocompetencia/inmunología , Pulmón/inmunología , Neumonía Viral/inmunología , Linfocitos T/inmunología , Vitamina D/inmunología , Péptidos Catiónicos Antimicrobianos/inmunología , Autofagia/inmunología , Betacoronavirus , COVID-19 , Defensinas/inmunología , Humanos , Pandemias , SARS-CoV-2 , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Vitamina D/análogos & derivados , Catelicidinas
10.
Nat Med ; 26(7): 1017-1032, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32651579

RESUMEN

Although COVID-19 is most well known for causing substantial respiratory pathology, it can also result in several extrapulmonary manifestations. These conditions include thrombotic complications, myocardial dysfunction and arrhythmia, acute coronary syndromes, acute kidney injury, gastrointestinal symptoms, hepatocellular injury, hyperglycemia and ketosis, neurologic illnesses, ocular symptoms, and dermatologic complications. Given that ACE2, the entry receptor for the causative coronavirus SARS-CoV-2, is expressed in multiple extrapulmonary tissues, direct viral tissue damage is a plausible mechanism of injury. In addition, endothelial damage and thromboinflammation, dysregulation of immune responses, and maladaptation of ACE2-related pathways might all contribute to these extrapulmonary manifestations of COVID-19. Here we review the extrapulmonary organ-specific pathophysiology, presentations and management considerations for patients with COVID-19 to aid clinicians and scientists in recognizing and monitoring the spectrum of manifestations, and in developing research priorities and therapeutic strategies for all organ systems involved.


Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/patología , Especificidad de Órganos , Neumonía Viral/patología , Inmunidad Adaptativa/fisiología , Betacoronavirus/fisiología , COVID-19 , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/terapia , Progresión de la Enfermedad , Endotelio Vascular/patología , Endotelio Vascular/virología , Humanos , Inflamación/etiología , Inflamación/patología , Inflamación/virología , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/inmunología , Neumonía Viral/terapia , Sistema Renina-Angiotensina/fisiología , SARS-CoV-2 , Trombosis/etiología , Trombosis/patología , Trombosis/virología , Internalización del Virus
11.
Neuropsychopharmacology ; 45(9): 1545-1556, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32417852

RESUMEN

Enhancing stress resilience in at-risk populations could significantly reduce the incidence of stress-related psychiatric disorders. We have previously reported that the administration of (R,S)-ketamine prevents stress-induced depressive-like behavior in male mice, perhaps by altering α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated transmission in hippocampal CA3. However, it is still unknown whether metabolites of (R,S)-ketamine can be prophylactic in both sexes. We administered (R,S)-ketamine or its metabolites (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) and (2S,6S)-hydroxynorketamine ((2S,6S)-HNK) at various doses 1 week before one of a number of stressors in male and female 129S6/SvEv mice. Patch clamp electrophysiology was used to determine the effect of prophylactic drug administration on glutamatergic activity in CA3. To examine the interaction between ovarian hormones and stress resilience, female mice also underwent ovariectomy (OVX) surgery and a hormone replacement protocol prior to drug administration. (2S,6S)-HNK and (2R,6R)-HNK protected against distinct stress-induced behaviors in both sexes, with (2S,6S)-HNK attenuating learned fear in male mice, and (2R,6R)-HNK preventing stress-induced depressive-like behavior in both sexes. (R,S)-ketamine and (2R,6R)-HNK, but not (2S,6S)-HNK, attenuated large-amplitude AMPAR-mediated bursts in hippocampal CA3. All three compounds reduced N-methyl-D-aspartate receptor (NMDAR)-mediated currents 1 week after administration. Furthermore, ovarian-derived hormones were necessary for and sufficient to restore (R,S)-ketamine- and (2R,6R)-HNK-mediated prophylaxis in female mice. Our data provide further evidence that resilience-enhancing prophylactics may alter AMPAR-mediated glutamatergic transmission in CA3. Moreover, we show that prophylactics against stress-induced depressive-like behavior can be developed in a sex-specific manner and demonstrate that ovarian hormones are necessary for the prophylactic efficacy of (R,S)-ketamine and (2R,6R)-HNK in female mice.


Asunto(s)
Ketamina , Animales , Fenómenos Electrofisiológicos , Femenino , Hipocampo/metabolismo , Ketamina/análogos & derivados , Ketamina/farmacología , Masculino , Ratones , Receptores de N-Metil-D-Aspartato/metabolismo
12.
Biochem Pharmacol ; 176: 113818, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-31978378

RESUMEN

Nitric oxide (NO) is a gaseous molecule that plays a multifactorial role in several cellular processes. In the central nervous system, the NO dual nature in neuroprotection and neurotoxicity has been explored to unveil its involvement in Alzheimer's disease (AD). A growing body of research shows that the activation of the NO signaling pathway leading to the phosphorylation of the transcription factor cyclic adenine monophosphate responsive element binding protein (CREB) (so-called NO/cGMP/PKG/CREB signaling pathway) ameliorates altered neuroplasticity and memory deficits in AD animal models. In addition to NO donors, several other pharmacological agents, such as phosphodiesterase 5 (PDE5) inhibitors have been used to activate the pathway and rescue memory disorders. PDE5 inhibitors, including sildenafil, tadalafil and vardenafil, are marketed for the treatment of erectile dysfunction and arterial pulmonary hypertension due to their vasodilatory properties. The ability of PDE5 inhibitors to interfere with the NO/cGMP/PKG/CREB signaling pathway by increasing the levels of cGMP has prompted the hypothesis that PDE5 inhibition might be used as an effective therapeutic strategy for the treatment of AD. To this end, newly designed PDE5 inhibitors belonging to different chemical classes with improved pharmacologic profile (e.g. higher potency, improved selectivity, and blood-brain barrier penetration) have been synthesized and evaluated in several animal models of AD. In addition, recent medicinal chemistry effort has led to the development of agents concurrently acting on the PDE5 enzyme and a second target involved in AD. Both marketed and investigational PDE5 inhibitors have shown to reverse cognitive defects in young and aged wild type mice as well as transgenic mouse models of AD and tauopathy using a variety of behavioral tasks. These studies confirmed the therapeutic potential of PDE5 inhibitors as cognitive enhancers. However, clinical studies assessing cognitive functions using marketed PDE5 inhibitors have not been conclusive. Drug discovery efforts by our group and others are currently directed towards the development of novel PDE5 inhibitors tailored to AD with improved pharmacodynamic and pharmacokinetic properties. In summary, the present perspective reports an overview of the correlation between the NO signaling and AD, as well as an outline of the PDE5 inhibitors used as an alternative approach in altering the NO pathway leading to an improvement of learning and memory. The last two sections describe the preclinical and clinical evaluation of PDE5 inhibitors for the treatment of AD, providing a comprehensive analysis of the current status of the AD drug discovery efforts involving PDE5 as a new therapeutic target.


Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Modelos Animales de Enfermedad , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Transducción de Señal/efectos de los fármacos , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Humanos , Óxido Nítrico/metabolismo
13.
Toxicol Lett ; 291: 1-10, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29614332

RESUMEN

Organophosphate compounds (OPCs) are commonly used as pesticides and were developed as nerve agents for chemical warfare. Exposure to OPCs results in toxicity due to their covalent binding and inhibition of acetylcholinesterase (AChE). Treatment for toxicity due to OPC exposure has been largely focused on the reactivation of AChE by oxime-based compounds via direct nucleophilic attack on the phosphorous center. However, due to the disadvantages to existing oxime-based reactivators for treatment of OPC poisoning, we considered non-oxime mechanisms of reactivation. A high throughput screen of compound libraries was performed to discover previously unidentified reactivation compounds, followed by studies on their analogs. In the process, we discovered multiple non-oxime classes of compounds, the most robust of which we have already reported [1]. Herein, we report other classes of compounds we identified in our screen that are efficient at reactivation. During biochemical characterization, we also found some compounds with other activities that may inspire novel therapeutic approaches to OPC toxicity. Specifically, we found compounds that [1] increase the rate of substrate hydrolysis by AChE and, [2] protect the enzyme from inhibition by OPC. Further, we discovered that a subset of reactivator compounds recover activity from both AChE and the related enzyme butyrylcholinesterase (BuChE). We now report these compounds, their activities and discuss how each relates to therapeutic approaches that would provide alternatives to traditional oxime-based reactivation.


Asunto(s)
Reactivadores de la Colinesterasa/uso terapéutico , Intoxicación por Organofosfatos/tratamiento farmacológico , Acetilcolinesterasa/metabolismo , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/toxicidad , Donepezilo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrólisis , Imidazoles/farmacología , Indanos/química , Indanos/farmacología , Cinética , Oximas/uso terapéutico , Piperazinas/farmacología , Piperidinas/química , Piperidinas/farmacología , Piridinas/farmacología , Relación Estructura-Actividad
15.
J Med Chem ; 60(21): 8858-8875, 2017 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-28985058

RESUMEN

Phosphodiesterase 5 (PDE5) hydrolyzes cyclic guanosine monophosphate (cGMP) leading to increased levels of the cAMP response element binding protein (CREB), a transcriptional factor involved with learning and memory processes. We previously reported potent quinoline-based PDE5 inhibitors (PDE5Is) for the treatment of Alzheimer's disease (AD). However, the low aqueous solubility rendered them undesirable drug candidates. Here we report a series of novel PDE5Is with two new scaffolds, 1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine and 2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one. Among them, compound 6c, 2-acetyl-10-((3-chloro-4-methoxybenzyl)amino)-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile, the most potent compound, has an excellent in vitro IC50 (0.056 nM) and improved aqueous solubility as well as good efficacy in a mouse model of AD. Furthermore, we are proposing two plausible binding modes obtained through in silico docking, which provide insights into the structural basis of the activity of the two series of compounds reported herein.


Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Naftiridinas/síntesis química , Inhibidores de Fosfodiesterasa 5/síntesis química , Animales , Sitios de Unión , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Humanos , Ratones , Simulación del Acoplamiento Molecular , Naftiridinas/uso terapéutico , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Quinolinas , Solubilidad , Relación Estructura-Actividad
16.
Pain ; 158(5): 822-832, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28059868

RESUMEN

Activating PKG-1α induces a long-term hyperexcitability (LTH) in nociceptive neurons. Since the LTH correlates directly with chronic pain in many animal models, we tested the hypothesis that inhibiting PKG-1α would attenuate LTH-mediated pain. We first synthesized and characterized compound N46 (N-((3R,4R)-4-(4-(2-fluoro-3-methoxy-6-propoxybenzoyl)benzamido)pyrrolidin-3-yl)-1H-indazole-5-carboxamide). N46 inhibits PKG-1α with an IC50 of 7.5 nmol, was highly selective when tested against a panel of 274 kinases, and tissue distribution studies indicate that it does not enter the CNS. To evaluate its antinociceptive potential, we used 2 animal models in which the pain involves both activated PKG-1α and LTH. Injecting complete Freund's adjuvant (CFA) into the rat hind paw causes a thermal hyperalgesia that was significantly attenuated 24 hours after a single intravenous injection of N46. Next, we used a rat model of osteoarthritic knee joint pain and found that a single intra-articular injection of N46 alleviated the pain 14 days after the pain was established and the relief lasted for 7 days. Thermal hyperalgesia and osteoarthritic pain are also associated with the activation of the capsaicin-activated transient receptor protein vanilloid-1 (TRPV1) channel. We show that capsaicin activates PKG-1α in nerves and that a subcutaneous delivery of N46 attenuated the mechanical and thermal hypersensitivity elicited by exposure to capsaicin. Thus, PKG-1α appears to be downstream of the transient receptor protein vanilloid-1. Our studies provide proof of concept in animal models that a PKG-1α antagonist has a powerful antinociceptive effect on persistent, already existing inflammatory pain. They further suggest that N46 is a valid chemotype for the further development of such antagonists.


Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Inflamación/complicaciones , Osteoartritis/complicaciones , Osteoartritis/enzimología , Umbral del Dolor/fisiología , Dolor/enzimología , Dolor/etiología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacocinética , Animales , Compuestos de Bifenilo/uso terapéutico , Enfermedad Crónica , GMP Cíclico/análogos & derivados , GMP Cíclico/uso terapéutico , Modelos Animales de Enfermedad , Método Doble Ciego , Inhibidores Enzimáticos/uso terapéutico , Adyuvante de Freund/toxicidad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Masculino , Modelos Moleculares , Osteoartritis/tratamiento farmacológico , Dolor/tratamiento farmacológico , Umbral del Dolor/efectos de los fármacos , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Tionucleótidos/uso terapéutico , Factores de Tiempo
17.
Blood ; 129(1): 88-99, 2017 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-27784673

RESUMEN

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc-dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc.


Asunto(s)
Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Neoplasias Hematológicas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Ratones , Oligopéptidos/farmacología , Biosíntesis de Proteínas , Distribución Aleatoria , Ensayos Antitumor por Modelo de Xenoinjerto
18.
Chembiochem ; 16(15): 2205-2215, 2015 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-26350723

RESUMEN

Acetylcholinesterase (AChE) that has been covalently inhibited by organophosphate compounds (OPCs), such as nerve agents and pesticides, has traditionally been reactivated by using nucleophilic oximes. There is, however, a clearly recognized need for new classes of compounds with the ability to reactivate inhibited AChE with improved in vivo efficacy. Here we describe our discovery of new functional groups--Mannich phenols and general bases--that are capable of reactivating OPC--inhibited AChE more efficiently than standard oximes and we describe the cooperative mechanism by which these functionalities are delivered to the active site. These discoveries, supported by preliminary in vivo results and crystallographic data, significantly broaden the available approaches for reactivation of AChE.


Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Descubrimiento de Drogas , Organofosfatos/farmacología , Fenoles/química , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Estructura Molecular , Organofosfatos/síntesis química , Organofosfatos/química , Relación Estructura-Actividad
19.
Protein Eng Des Sel ; 28(11): 501-6, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26243887

RESUMEN

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.


Asunto(s)
Cisteína/química , Organofosfatos/metabolismo , Hidrolasas de Triéster Fosfórico/química , Hidrolasas de Triéster Fosfórico/metabolismo , Cisteína/genética , Cisteína/metabolismo , Fluoresceínas/química , Fluoresceínas/metabolismo , Cinética , Modelos Moleculares , Mutación , Organofosfatos/análisis , Paraoxon/análisis , Paraoxon/metabolismo , Hidrolasas de Triéster Fosfórico/genética , Ingeniería de Proteínas
20.
Diabetes ; 64(10): 3396-405, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26068544

RESUMEN

The prevalence of obesity-induced type 2 diabetes (T2D) is increasing worldwide, and new treatment strategies are needed. We recently discovered that obesity activates a previously unknown pathway that promotes both excessive hepatic glucose production (HGP) and defective insulin signaling in hepatocytes, leading to exacerbation of hyperglycemia and insulin resistance in obesity. At the hub of this new pathway is a kinase cascade involving calcium/calmodulin-dependent protein kinase II (CaMKII), p38α mitogen-activated protein kinase (MAPK), and MAPKAPK2/3 (MK2/3). Genetic-based inhibition of these kinases improves metabolism in obese mice. Here, we report that treatment of obese insulin-resistant mice with an allosteric MK2/3 inhibitor, compound (cmpd) 28, ameliorates glucose homeostasis by suppressing excessive HGP and enhancing insulin signaling. The metabolic improvement seen with cmpd 28 is additive with the leading T2D drug, metformin, but it is not additive with dominant-negative MK2, suggesting an on-target mechanism of action. Allosteric MK2/3 inhibitors represent a potentially new approach to T2D that is highly mechanism based, has links to human T2D, and is predicted to avoid certain adverse effects seen with current T2D drugs.


Asunto(s)
Glucemia/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Masculino , Ratones , Ratones Noqueados , Ratones Obesos
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