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1.
SLAS Discov ; : 2472555218796410, 2018 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-30142014

RESUMO

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at risk of contracting malaria and an estimated 450,000 deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector. This stage is a bottleneck for reproduction of the parasite, making it a target for small-molecule drug discovery. Targeting this stage, we used whole Plasmodium falciparum gametocytes from in vitro culture and implemented them into 1536-well plates to create a live/dead phenotypic antigametocyte assay. Using specialized equipment and upon further validation, we screened ~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified 100 primary screening hits that were tested for concentration response. Additional follow-up studies to determine specificity, potency, and increased efficacy of the antigametocyte candidate compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13 chemical analogs were subsequently tested as de novo powders, which confirmed original activity from the initial analysis and now provide a point of future engagement.

2.
Assay Drug Dev Technol ; 16(5): 278-288, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30019946

RESUMO

GPR119 drug discovery efforts in the pharmaceutical industry for the treatment of type 2 diabetes mellitus (T2DM) and obesity, were initiated based on its restricted distribution in pancreas and GI tract, and its possible role in glucose homeostasis. While a number of lead series have emerged, the pharmacological endpoints they provide have not been clear. In particular, many lead series have demonstrated loss of efficacy and significant toxic side effects. Thus, we sought to identify novel, potent, positive modulators of GPR119. In this study, we have successfully developed and optimized a high-throughput screening strategy to identify GPR119 modulators using a live cell assay format that utilizes a cyclic nucleotide-gated channel as a biosensor for cAMP production. Our high-throughput screening (HTS) approach is unique to that of previous HTS approaches targeting this receptor, as changes in cAMP were measured both in the presence and absence of an EC10 of the endogenous ligand, oleoylethanolamide, enabling detection of both agonists and potential allosteric modulators in a single assay. From these efforts, we have identified positive modulators of GPR119 with similar as well as unique scaffolds compared to existing compounds and similar as well as unique signaling properties. Our compounds will not only serve as novel molecular probes to better understand GPR119 pleiotropic signaling and the underlying physiological consequences of receptor activation, but are also well-suited for translation as potential therapeutic agents.

3.
SLAS Discov ; 23(2): 122-131, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28957636

RESUMO

Chemotaxis is the directional movement of cells in response to a chemical stimulus and is vital for many physiological processes, including immune responses, tumor metastasis, wound healing, and blood vessel formation. Therefore, modulation of chemotaxis is likely to be of therapeutic benefit. Hence, a high-throughput means to conduct chemotaxis assays is advantageous for lead evaluation and optimization in drug discovery. In this study, we have validated a novel approach for a higher-throughput, label-free, image-based IncuCyte chemotaxis assay encompassing various cell types, including T cells, B cells, mouse Th17, immature and mature dendritic cells, monocyte THP-1, CCRF-CEM, monocytes, neutrophils, macrophages, and MDA-MB-231. These assays enable us to visualize chemotactic cell migration in real time and perform kinetic cell motility studies on an automated platform, thereby allowing us to incorporate the quantitative studies of cell migration behavior into a routine drug discovery screening cascade.

4.
SLAS Discov ; 22(1): 21-31, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27628691

RESUMO

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

5.
SLAS Discov ; 22(1): 58-66, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27827304

RESUMO

Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z' factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

6.
Bioorg Med Chem Lett ; 26(17): 4282-6, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27476142

RESUMO

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.


Assuntos
Descoberta de Drogas , Cetonas/farmacocinética , Receptor Muscarínico M1/agonistas , Regulação Alostérica , Animais , Sistema Nervoso Central/metabolismo , Humanos , Cetonas/síntese química , Cetonas/química , Estrutura Molecular , Relação Estrutura-Atividade
8.
Bioorg Med Chem Lett ; 26(13): 3029-3033, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27185330

RESUMO

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.


Assuntos
Piperidinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Receptor Muscarínico M4/metabolismo , Tiofenos/farmacologia , Regulação Alostérica , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Piperidinas/síntese química , Piperidinas/metabolismo , Pirimidinas/síntese química , Pirimidinas/metabolismo , Quinazolinas/síntese química , Quinazolinas/metabolismo , Ratos , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inibidores , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismo
9.
J Biomol Screen ; 21(7): 713-21, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27146384

RESUMO

There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.


Assuntos
Inibidores Enzimáticos/isolamento & purificação , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Fosfolipases A2 Secretórias/antagonistas & inibidores , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Humanos , Cinética , Mastócitos/química , Mastócitos/metabolismo , Fosfolipases A2 Secretórias/química , Fosfolipídeos/química , Bibliotecas de Moléculas Pequenas/análise
10.
J Diabetes Sci Technol ; 10(6): 1216-1221, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27207890

RESUMO

BACKGROUND: We developed a system to suspend insulin pump delivery overnight when the glucose trend predicts hypoglycemia. This predictive low-glucose suspend (PLGS) system substantially reduces nocturnal hypoglycemia without an increase in morning ketosis. Evaluation of hypoglycemia risk factors that could potentially influence the efficacy of the system remains critical for understanding possible problems with the system and identifying patients that may have the greatest benefit when using the system. METHODS: The at-home randomized trial consisted of 127 study participants with hemoglobin A1c (A1C) of ≤8.5% (mmol/mol) for patients aged 4-14 years and ≤8.0% for patient aged 15-45 years. Factors assessed included age, gender, A1C, diabetes duration, daily percentage basal insulin, total daily dose of insulin (units/kg-day), bedtime BG, bedtime snack, insulin on board, continuous glucose monitor (CGM) rate of change (ROC), day of the week, time system activated, daytime exercise intensity, and daytime CGM-measured hypoglycemia. RESULTS: The PLGS system was effective in preventing hypoglycemia for each factor subgroup. There was no evidence that the PLGS system was more or less effective in preventing hypoglycemia in any one subgroup compared with the other subgroups based on that factor. In addition, the effect of the system on overnight hyperglycemia did not differ in subgroups. CONCLUSIONS: The PLGS system tested in this study effectively reduced hypoglycemia without a meaningful increase in hyperglycemia across a variety of factors.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemia/prevenção & controle , Hipoglicemiantes/administração & dosagem , Sistemas de Infusão de Insulina , Insulina/administração & dosagem , Adolescente , Adulto , Área Sob a Curva , Glicemia/análise , Automonitorização da Glicemia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Adulto Jovem
11.
J Biomol Screen ; 21(5): 468-79, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26838761

RESUMO

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores de N-Metil-D-Aspartato/metabolismo , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Regulação Alostérica/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Cálcio/química , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Plasticidade Neuronal/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
12.
ACS Chem Biol ; 11(1): 172-84, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26524379

RESUMO

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38 350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional ΔAPSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.


Assuntos
Antituberculosos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Enxofre/metabolismo , Animais , Antituberculosos/síntese química , Antituberculosos/química , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Reprodutibilidade dos Testes , Enxofre/química , Compostos de Enxofre/metabolismo , Tuberculose/tratamento farmacológico
13.
Assay Drug Dev Technol ; 14(1): 58-66, 2016 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26669516

RESUMO

In the light of emerging antibiotic resistance mechanisms found in bacteria throughout the world, discovery of drugs that potentiate the effect of currently available antibiotics remains an important aspect of pharmaceutical research in the 21st century. Well-established clinical tests exist to determine synergy in vitro, but these are only optimal for low-throughput experimentation while leaving analysis of results and interpretation of high-throughput microscale assays poorly standardized. Here, we describe a miniaturized broth microdilution checkerboard assay and data analysis method in 384-well plate format that conforms to the Clinical Laboratory and Standards Institute (CLSI) methods. This method has been automated and developed to rapidly determine the synergism of current antibiotics with various beta-lactamase inhibitors emerging from our antimicrobial research efforts. This technique increases test throughput and integrity of results, and saves test compound and labor. We facilitated the interpretation of results with an automated analysis tool allowing us to rapidly qualify inter- and intraplate robustness, determine efficacy of multiple antibiotics at the same time, and standardize the results of synergy interpretation. This procedure should enhance high-throughput antimicrobial drug discovery and supersedes former techniques.


Assuntos
Anti-Infecciosos/análise , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana/métodos , Miniaturização/métodos
14.
Clin Toxicol (Phila) ; 54(1): 14-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26653952

RESUMO

CONTEXT: Synthetic cannabinoid use has increased in many states, and medicinal and/or recreational marijuana use has been legalized in some states. These changes present challenges to law enforcement drug recognition experts (DREs) who determine whether drivers are impaired by synthetic cannabinoids or marijuana, as well as to clinical toxicologists who care for patients with complications from synthetic cannabinoids and marijuana. Our goal was to compare what effects synthetic cannabinoids and marijuana had on performance and behavior, including driving impairment, by reviewing records generated by law enforcement DREs who evaluated motorists arrested for impaired driving. METHODS: Data were from a retrospective, convenience sample of de-identified arrest reports from impaired drivers suspected of using synthetic cannabinoids (n = 100) or marijuana (n = 33). Inclusion criteria were arrested drivers who admitted to using either synthetic cannabinoids or marijuana, or who possessed either synthetic cannabinoids or marijuana; who also had a DRE evaluation at the scene; and whose blood screens were negative for alcohol and other drugs. Exclusion criteria were impaired drivers arrested with other intoxicants found in their drug or alcohol blood screens. Blood samples were analyzed for 20 popular synthetic cannabinoids by using liquid chromatography-tandem mass spectrometry. Delta-9-tetrahydrocannabinol (THC) and THC-COOH were quantified by gas chromatography-mass spectrometry. Statistical significance was determined by using Fisher's exact test or Student's t-test, where appropriate, to compare the frequency of characteristics of those in the synthetic cannabinoid group versus those in the marijuana group. RESULTS: 16 synthetic cannabinoid and 25 marijuana records met selection criteria; the drivers of these records were arrested for moving violations. Median age for the synthetic cannabinoid group (n = 16, 15 males) was 20 years (IQR 19-23 years). Median age for the marijuana group (n = 25, 21 males) was 20 years (IQR 19-24 years) (p = 0.46). In the synthetic cannabinoid group, 94% (15/16) admitted to using synthetic cannabinoids. In the marijuana group, 96% (24/25) admitted to using marijuana. Blood was available for testing in 96% (24/25) of the marijuana group; 21 of these 24 had quantitative levels of THC (mean + SD = 10.7 + 5 ng/mL) and THC-COOH (mean + SD = 57.8 + 3 ng/mL). Blood was available for testing in 63% (10/16) of the synthetic cannabinoid group, with 80% (8/10) of these positive for synthetic cannabinoids. Those in the synthetic cannabinoid group were more frequently confused (7/16 [44%] vs. 0/25 [0%], p ≤ 0.003) and disoriented (5/16 [31%] vs. 0/25 [0%], p ≤ 0.003), and more frequently had incoherent, slurred speech (10/16 [63%] vs. 3/25 [12%], p = 0.0014) and horizontal gaze nystagmus (8/16 [50%] vs. 3/25 [12%], p = 0.01) than those in the marijuana group. CONCLUSION: Drivers under the influence of synthetic cannabinoids were more frequently impaired with confusion, disorientation, and incoherent, slurred speech than drivers under the influence of marijuana in this population evaluated by DREs.


Assuntos
Condução de Veículo , Canabinoides/farmacologia , Cannabis , Crime , Abuso de Maconha/psicologia , Fumar Maconha/psicologia , Extratos Vegetais/farmacologia , Psicotrópicos/farmacologia , Detecção do Abuso de Substâncias/métodos , Canabinoides/sangue , Canabinoides/síntese química , Canabinoides/isolamento & purificação , Cromatografia Líquida , Confusão/induzido quimicamente , Confusão/psicologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Abuso de Maconha/sangue , Abuso de Maconha/complicações , Abuso de Maconha/diagnóstico , Fumar Maconha/efeitos adversos , Fumar Maconha/sangue , Nistagmo Patológico/induzido quimicamente , Extratos Vegetais/sangue , Extratos Vegetais/isolamento & purificação , Valor Preditivo dos Testes , Psicotrópicos/sangue , Psicotrópicos/síntese química , Psicotrópicos/isolamento & purificação , Estudos Retrospectivos , Percepção Espacial/efeitos dos fármacos , Inteligibilidade da Fala/efeitos dos fármacos , Espectrometria de Massas em Tandem , Adulto Jovem
15.
Cell Metab ; 22(5): 851-60, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26411340

RESUMO

Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-ß hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas de Transporte/metabolismo , Lipólise/genética , Fosfoproteínas/metabolismo , Proteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Células 3T3-L1 , Acil Coenzima A/metabolismo , Adipócitos/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Ligantes , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Perilipina-1 , Perilipina-5 , Fosfoproteínas/genética , Proteínas/genética
16.
J Biomol Screen ; 20(7): 858-68, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25877150

RESUMO

Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer's disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M(4) (M(4)), we developed a homogenous, multiparametric, 1536-well assay to measure M(4) receptor agonism, positive allosteric modulation (PAM), and antagonism in a single well. This assay yielded a Z' of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M(1) and M(5) subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M(4) selective primary agonists, 1450 PAMs, and 2389 antagonist hits. Concentration-response analysis identified 25 selective agonists, 4 PAMs, and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.


Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M4/metabolismo , Regulação Alostérica , Animais , Linhagem Celular , Descoberta de Drogas/métodos , Expressão Gênica , Humanos , Agonistas Muscarínicos/química , Antagonistas Muscarínicos/química , Receptor Muscarínico M4/genética , Bibliotecas de Moléculas Pequenas
17.
PLoS One ; 10(3): e0121833, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25811598

RESUMO

Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 µM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Biflavonoides/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fusão bcr-abl/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Células K562 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , Fatores de Tempo
18.
Clin Toxicol (Phila) ; 53(1): 60-70, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25511795

RESUMO

BACKGROUND: The Gila monster (Heloderma suspectum) is a venomous lizard native to the deserts of southwestern United States (US) and northern Mexico. The purpose of this study was to describe human exposures to Gila monsters reported to US poison control centers (PCCs) with a focus on Arizona cases. METHODS: The American Association of Poison Control Centers' National Poison Data System (NPDS) was used to access and retrospectively review all calls to US PCCs, concerning Gila monsters between January 1, 2000 and October 31, 2011. In addition, detailed records from the two Arizona PCCs were reviewed for the same time period. RESULTS: A total of 319 calls regarding Gila monsters were identified in the NPDS. Of these, 105 (33%) were human exposures; most (79%) occurred in males. A total of 71 (68%) of these 105 cases were referred to a health care facility (HCF); 30 (29%) were managed on-site. Of the 71 HCF referrals, 36 (51%) were discharged home and 17 (24%) were admitted. Most (65%) admissions were to an intensive care unit (ICU). Arizona's PCCs received 70 unique reports of Gila monster bite. Most (77%) of the bites in Arizona involved an upper extremity. Eight (11%) involved patients under the age of 18 years. Eleven (16%) Arizona cases were work-related. Twenty-eight (40%) of the 70 bites in Arizona were evaluated in a HCF, but not admitted. Eleven (16%) were admitted, of which five were to an ICU. Six patients had edema of airway structures; three required emergent airway management, one by cricothyrotomy. There were no deaths. CONCLUSION: Gila monster bites are uncommon. Many cases did not require hospitalization. Edema of airway structures is an infrequent, but life-threatening complication.


Assuntos
Mordeduras e Picadas/diagnóstico , Mordeduras e Picadas/terapia , Lagartos , Peçonhas/toxicidade , Animais , Arizona , Bases de Dados Factuais , Feminino , Humanos , Masculino , Centros de Controle de Intoxicações , Estados Unidos
19.
Bioorg Med Chem Lett ; 25(2): 384-8, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25435150

RESUMO

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.


Assuntos
Descoberta de Drogas , Pirazóis/farmacologia , Quinolinas/farmacologia , Quinolonas/química , Receptor Muscarínico M1/química , Receptor Muscarínico M1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica , Sítio Alostérico , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/química , Quinolinas/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
20.
Assay Drug Dev Technol ; 12(8): 470-80, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25383721

RESUMO

The kinesin superfamily of motor proteins are involved in the active transport of a large number of cargos such as organelles, proteins, and RNAs from the neuronal cell body to distal neuronal processes. Previously, we have shown that kinesin-mediated axonal transport of proteins and RNAs are important for long-term memory storage. Identification of small molecules that can activate or inhibit kinesins is of specific interest due to the significance of kinesin-mediated functions in neuronal health and plasticity. Here, we describe a high-throughput screening assay designed to specifically identify compounds that inhibit or activate adenosine triphosphatase activity of the kinesin 5B of humans. The luminescence-based assay that we developed is highly reproducible and robust. Using this approach, we screened a pharmacologically characterized compound collection and have identified small molecules with either activator or inhibitor-like activity. To further characterize screening hits, we also developed an orthogonal assay based on absorbance and a counter screen assay based on luminescence. Development of such assays is important to help identify small molecules that can be used in potential drug development efforts targeted at modulating the function of kinesin.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Cinesina/efeitos dos fármacos , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Indicadores e Reagentes , Luminescência , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas
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