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1.
Cell ; 161(6): 1252-65, 2015 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-26046436

RESUMEN

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.


Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , National Institutes of Health (U.S.) , Estados Unidos
2.
Bioorg Med Chem Lett ; 26(17): 4282-6, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27476142

RESUMEN

This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.


Asunto(s)
Descubrimiento de Drogas , Cetonas/farmacocinética , Receptor Muscarínico M1/agonistas , Regulación Alostérica , Animales , Sistema Nervioso Central/metabolismo , Humanos , Cetonas/síntesis química , Cetonas/química , Estructura Molecular , Relación Estructura-Actividad
3.
Bioorg Med Chem Lett ; 26(13): 3029-3033, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27185330

RESUMEN

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.


Asunto(s)
Piperidinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Receptor Muscarínico M4/metabolismo , Tiofenos/farmacología , Regulación Alostérica , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Humanos , Microsomas Hepáticos/metabolismo , Piperidinas/síntesis química , Piperidinas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Quinazolinas/síntesis química , Quinazolinas/metabolismo , Ratas , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismo
4.
Proc Natl Acad Sci U S A ; 110(17): 7044-9, 2013 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-23576755

RESUMEN

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP-FRET-enabled high throughput assay (PrP-FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood-brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.


Asunto(s)
Astemizol/farmacología , Autofagia/efectos de los fármacos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Enfermedades por Prión/tratamiento farmacológico , Priones/metabolismo , Tacrolimus/farmacología , Animales , Astemizol/uso terapéutico , Western Blotting , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Proc Natl Acad Sci U S A ; 110(29): 12072-7, 2013 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-23818586

RESUMEN

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to µ-δ opioid receptor (µOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the µOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting µOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in µOR-δOR cells but not µOR or δOR cells alone. Among them, CYM51010 was found to be a µOR-δOR-biased ligand, because its activity is blocked by the µOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a µOR-δOR-biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.


Asunto(s)
Analgésicos/farmacología , Encéfalo/metabolismo , Piperidinas/farmacología , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Analgésicos/metabolismo , Análisis de Varianza , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular , Dimerización , Tolerancia a Medicamentos/fisiología , Ensayos Analíticos de Alto Rendimiento , Masculino , Ratones , Ratones Endogámicos C57BL , Piperidinas/metabolismo , Ensayo de Unión Radioligante , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Bibliotecas de Moléculas Pequeñas
6.
J Biol Chem ; 289(27): 18893-903, 2014 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-24817118

RESUMEN

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.


Asunto(s)
Quinasa Idelta de la Caseína/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/química , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica/efectos de los fármacos , Proteínas Tirosina Quinasas/química , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología
7.
Bioorg Med Chem Lett ; 25(2): 384-8, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25435150

RESUMEN

Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure.


Asunto(s)
Descubrimiento de Drogas , Pirazoles/farmacología , Quinolinas/farmacología , Quinolonas/química , Receptor Muscarínico M1/química , Receptor Muscarínico M1/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Sitio Alostérico , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirazoles/química , Quinolinas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad
8.
J Biol Chem ; 288(51): 36703-16, 2013 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-24187130

RESUMEN

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and ßarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through ßarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from ßarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit ßarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.


Asunto(s)
Receptores Opioides kappa/agonistas , Animales , Arrestinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Descubrimiento de Drogas , Proteínas de Unión al GTP/metabolismo , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Quinolonas/síntesis química , Quinolonas/farmacología , Receptores Opioides kappa/metabolismo , Transducción de Señal , Triazoles/síntesis química , Triazoles/farmacología , beta-Arrestinas
9.
Biopolymers ; 102(5): 396-406, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25048711

RESUMEN

Zinc metalloproteinases meprin α and meprin ß are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin α and meprin ß and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin ß. Kinetic studies revealed competitive (PPNDS) and mixed competitive/noncompetitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin ß active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin ß reported to the date. These results demonstrate the ability of meprin α and ß assays to identify selective compounds and discard artifacts of primary screening.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de la Metaloproteinasa de la Matriz/análisis , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Bioensayo , Bases de Datos de Compuestos Químicos , Humanos , Metaloendopeptidasas/química , Proyectos Piloto , Reproducibilidad de los Resultados , Especificidad por Sustrato/efectos de los fármacos , Factores de Tiempo
10.
Pharmacol Res ; 83: 38-51, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24373832

RESUMEN

A pharmacoperone (from "pharmacological chaperone") is a small molecule that enters cells and serves as molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell. Pharmacoperones have broad therapeutic applicability since a large number of diseases have their genesis in the misfolding of proteins and resultant misrouting within the cell. Misrouting may result in loss-of-function and, potentially, the accumulation of defective mutants in cellular compartments. Most known pharmacoperones were initially derived from receptor antagonist screens and, for this reason, present a complex pharmacology, although these are highly target specific. In this summary, we describe efforts to produce high throughput screens that identify these molecules from chemical libraries as well as a mouse model which provides proof-of-principle for in vivo protein rescue using existing pharmacoperones.


Asunto(s)
Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Transporte de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química
11.
Proc Natl Acad Sci U S A ; 108(12): 4834-9, 2011 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-21383145

RESUMEN

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.


Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Autofagia/efectos de los fármacos , Retículo Endoplásmico/enzimología , Inhibidores Enzimáticos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Quinazolinas/farmacología , Ubiquitina/metabolismo , Adenosina Trifosfatasas/genética , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Línea Celular , Retículo Endoplásmico/genética , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Quinazolinas/síntesis química , Quinazolinas/química , Ubiquitina/genética
12.
Proc Natl Acad Sci U S A ; 108(17): 6811-6, 2011 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-21398589

RESUMEN

National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-ß-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.


Asunto(s)
Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Inhibidores Enzimáticos , Animales , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Ratones , Ratones Noqueados , National Institutes of Health (U.S.) , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Estados Unidos
13.
SLAS Discov ; 29(4): 100159, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38723666

RESUMEN

To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.


Asunto(s)
Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Resonancia por Plasmón de Superficie , Flujo de Trabajo , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/química , Descubrimiento de Drogas/métodos , Humanos
14.
Nat Chem Biol ; 8(2): 185-96, 2011 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-22198733

RESUMEN

Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting this mechanism as a promising therapeutic approach. We describe the results of a screen comprised of ∼900,000 small molecules that identified new classes of small-molecule proteostasis regulators that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. These beneficial effects to proteome stability are mediated by HSF-1, FOXO, Nrf-2 and the chaperone machinery through mechanisms that are distinct from current known small-molecule activators of the heat shock response. We suggest that modulation of the proteostasis network by proteostasis regulators may be a promising therapeutic approach for the treatment of a variety of protein conformational diseases.


Asunto(s)
Evaluación Preclínica de Medicamentos , Chaperonas Moleculares/efectos de los fármacos , Proteínas/efectos de los fármacos , Deficiencias en la Proteostasis/tratamiento farmacológico , Factores de Transcripción/efectos de los fármacos , Animales , Caenorhabditis elegans , Línea Celular , Proteínas de Unión al ADN/efectos de los fármacos , Factores de Transcripción Forkhead/efectos de los fármacos , Factores de Transcripción del Choque Térmico , Homeostasis/efectos de los fármacos , Humanos , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Proteínas/química , Proteínas/fisiología , Ratas
15.
Bioorg Med Chem Lett ; 23(23): 6346-9, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24135724

RESUMEN

Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R-S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P3-R allosteric agonists.


Asunto(s)
Inmunosupresores/farmacología , Receptores de Lisoesfingolípidos/agonistas , Amidas/farmacología , Animales , Azoles/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Ratones , Modelos Moleculares , Unión Proteica , Receptores de Lisoesfingolípidos/biosíntesis , Relación Estructura-Actividad
16.
Bioorg Med Chem Lett ; 23(22): 6172-7, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-24080461

RESUMEN

Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.


Asunto(s)
Acetamidas/química , Acetamidas/farmacología , Antivirales/química , Antivirales/farmacología , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Acetamidas/síntesis química , Antivirales/síntesis química , Humanos , Modelos Moleculares , Síndrome Respiratorio Agudo Grave/virología , Relación Estructura-Actividad
17.
Bioorg Med Chem Lett ; 23(10): 2996-3000, 2013 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-23562060

RESUMEN

This Letter describes the further chemical optimization of the M5 PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M5 PAMs (M5 EC50 >10µM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M5 PAM, ML326 (VU0467903), (human and rat M5 EC50s of 409nM and 500nM, respectively) with excellent mAChR selectivity (M1-M4 EC50s >30µM) and a robust 20-fold leftward shift of the ACh CRC.


Asunto(s)
Descubrimiento de Drogas , Indoles/farmacología , Receptores Muscarínicos/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Estructura Molecular , Ratas , Relación Estructura-Actividad
18.
Bioorg Med Chem Lett ; 23(3): 839-43, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23260346

RESUMEN

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.


Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/antagonistas & inhibidores , Piperazinas/química , Quinolinas/química , Animales , Benzaldehídos/farmacología , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Estructura Molecular , Oximas/farmacología , Piperazinas/farmacología , Quinolinas/farmacología
19.
Bioorg Med Chem ; 21(11): 3138-46, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23591260

RESUMEN

Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z' factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z' factor of ≥0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC-MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.


Asunto(s)
Antibacterianos/química , Nitroprusiato/química , Zinc/química , Inhibidores de beta-Lactamasas , Ácido p-Cloromercuribenzoico/química , Dominio Catalítico , Cisteína/química , Cisteína/genética , Enterobacteriaceae/química , Enterobacteriaceae/enzimología , Enterobacteriaceae/genética , Ensayos Analíticos de Alto Rendimiento , Pruebas de Sensibilidad Microbiana , Mutación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masas en Tándem , Resistencia betalactámica , beta-Lactamasas/química , beta-Lactamasas/genética
20.
Bioorg Med Chem ; 21(21): 6642-9, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23993674

RESUMEN

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.


Asunto(s)
Hidroxiquinolinas/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Piperazinas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Sitios de Unión , Biomarcadores/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Células HL-60 , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidroxiquinolinas/síntesis química , Hidroxiquinolinas/toxicidad , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Piperazinas/síntesis química , Piperazinas/toxicidad , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismo
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