RESUMEN
KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Piperazinas/farmacología , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Inmunoterapia , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Fosforilación/efectos de los fármacos , Piperazinas/administración & dosificación , Piperazinas/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas/administración & dosificación , Piridinas/química , Pirimidinas/administración & dosificación , Pirimidinas/química , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Animales , Reactores Biológicos , Células CHO , Cricetinae , Cricetulus , Inhibidores Enzimáticos/farmacología , Proteínas Recombinantes/análisisRESUMEN
2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/química , Quinolinas/farmacología , Animales , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Humanos , Masculino , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Quinolinas/síntesis química , Quinolinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Based on lead compound 1, which was discovered from a high-throughput screen, a series of PI3Kα/mTOR inhibitors were evaluated that contained an imidazo[1,2-a]pyridine as a core replacement for the benzimidazole contained in 1. By exploring various ring systems that occupy the affinity pocket, two fragments containing a methoxypyridine were identified that gave <100 nM potency toward PI3Kα in enzyme and cellular assays with moderate stability in rat and human liver microsomes. With the two methoxypyridine groups selected to occupy the affinity pocket, analogs were prepared with various fragments intended to occupy the ribose pocket of PI3Kα and mTOR. From these analogs, tertiary alcohol 18 was chosen for in vivo pharmacodynamic evaluation based on its potency in the PI3Kα cellular assay, microsomal stability, and in vivo pharmacokinetic properties. In a mouse liver pharmacodynamic assay, compound 18 showed 56% inhibition of HFG-induced AKT (Ser473) phosphorylation at a 30 mg/kg dose.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Ratas , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.
Asunto(s)
Inmunoglobulina G/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Cadenas lambda de Inmunoglobulina/metabolismo , Sulfuros/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Inmunoglobulina G/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Replacement of the piperazine sulfonamide portion of the PI3Kα inhibitor AMG 511 (1) with a range of aliphatic alcohols led to the identification of a truncated gem-dimethylbenzylic alcohol analog, 2-(5-(4-amino-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)propan-2-ol (7). This compound possessed good in vitro efficacy and pharmacokinetic parameters and demonstrated an EC50 of 239 ng/mL in a mouse liver pharmacodynamic model measuring the inhibition of hepatocyte growth factor (HGF)-induced Akt Ser473 phosphorylation in CD1 nude mice 6 h post-oral dosing.
Asunto(s)
Alcoholes/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Sulfonamidas/química , Triazinas/síntesis química , Animales , Femenino , Semivida , Hígado/metabolismo , Masculino , Ratones , Ratones Desnudos , Conformación Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Piperazina , Piperazinas/metabolismo , Piperazinas/farmacocinética , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/metabolismo , Piridinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Triazinas/metabolismo , Triazinas/farmacocinéticaRESUMEN
Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.
Asunto(s)
Cinesinas , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Cinesinas/genética , Cinesinas/metabolismo , Mitosis/genética , Línea Celular , Puntos de Control de la Fase M del Ciclo CelularRESUMEN
γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aß42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aß42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Picolinas/farmacología , Enfermedad de Alzheimer/enzimología , Amidas/química , Animales , Células HEK293 , Humanos , Picolinas/química , Ratas , Ratas Sprague-DawleyRESUMEN
Indoleamine 2,3-dioxygenase (IDO1) catalyzes the first step in tryptophan breakdown along the kynurenine pathway. Therapeutic inhibition of IDO1 is receiving much attention due to its proposed role in the pathogenesis of several diseases including cancer, hypotension and neurodegenerative disorders. A related enzyme, IDO2 has recently been described. We report the first purification and kinetic characterization of human IDO2 using a facile l-tryptophan consumption assay amenable to high throughput screening. We found that the K(m) of human IDO2 for l-tryptophan is much higher than that of IDO1. We also describe the identification and characterization of a new IDO1 inhibitor compound, Amg-1, by high throughput screening, and compare the inhibition profiles of IDO1 and IDO2 with Amg-1 and previously described compounds. Our data indicate that human IDO1 and IDO2 have different kinetic parameters and different inhibition profiles. Docking of Amg-1 and related analogs to the known structure of IDO1 and to homology-modeled IDO2 suggests possible rationales for the different inhibition profiles of IDO1 and IDO2.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/aislamiento & purificación , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indolamina-Pirrol 2,3,-Dioxigenasa/aislamiento & purificación , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Phosphoinositide 3-kinase (PI3K) is an important target in oncology due to the deregulation of the PI3K/Akt signaling pathway in a wide variety of tumors. A series of 4-amino-6-methyl-1,3,5-triazine sulfonamides were synthesized and evaluated as inhibitors of PI3K. The synthesis, in vitro biological activities, pharmacokinetic and in vivo pharmacodynamic profiling of these compounds are described. The most promising compound from this investigation (compound 3j) was found to be a pan class I PI3K inhibitor with a moderate (>10-fold) selectivity over the mammalian target of rapamycin (mTOR) in the enzyme assay. In a U87 MG cellular assay measuring phosphorylation of Akt, compound 3j displayed low double digit nanomolar IC(50) and exhibited good oral bioavailability in rats (F(oral)=63%). Compound 3j also showed a dose dependent reduction in the phosphorylation of Akt in a U87 tumor pharmacodynamic model with a plasma EC(50)=193 nM (91 ng/mL).
Asunto(s)
Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Femenino , Humanos , Ratones , Simulación del Acoplamiento Molecular , Neoplasias/enzimología , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Triazinas/química , Triazinas/farmacocinética , Triazinas/farmacología , Triazinas/uso terapéuticoRESUMEN
Chromosomal instability (CIN) is a hallmark of cancer that results from errors in chromosome segregation during mitosis. Targeting of CIN-associated vulnerabilities is an emerging therapeutic strategy in drug development. KIF18A, a mitotic kinesin, has been shown to play a role in maintaining bipolar spindle integrity and promotes viability of CIN cancer cells. To explore the potential of KIF18A, a series of inhibitors was identified. Optimization of an initial hit led to the discovery of analogues that could be used as chemical probes to interrogate the role of KIF18A inhibition. Compounds 23 and 24 caused significant mitotic arrest in vivo, which was sustained for 24 h. This would be followed by cell death either in mitosis or in the subsequent interphase. Furthermore, photoaffinity labeling experiments reveal that this series of inhibitors binds at the interface of KIF18A and tubulin. This study represents the first disclosure of KIF18A inhibitors with in vivo activity.
Asunto(s)
Cinesinas , Neoplasias , Muerte Celular , Humanos , Mitosis , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The effects of fluorine substitution at the C-5 center of pyranosyl fluorides on the reactivity at the C-1 anomeric center was probed by studying a series of 5-fluoroxylosyl fluoride derivatives. X-ray structures of their per-O-acetates detailed the effects on the ground-state structures. First-order rate constants for spontaneous hydrolysis, in conjunction with computational studies, revealed that changes in the stereochemistry of the 5-fluorine had minimal effects on the solvolysis rate constants and that the observed rate reductions were broadly similar to those caused by additional fluorine substitution at C-1 but significantly less than those due to substitution at C-2. Differences in the trapping behavior of 5- versus 2-fluoro-substituted glycosyl fluorides with α- and ß-glycosidases arise more from differences in steric effects and hydrogen-bonding interactions than from intrinsic reactivity differences.
Asunto(s)
Fluoruros/química , Flúor/química , Xilosa/química , Cristalografía por Rayos X , Isomerismo , Modelos MolecularesRESUMEN
Affinity selection mass spectrometry (MS) or, simply, affinity mass spectrometry (AMS) is a label-free technology that has been used to identify high-affinity ligands of target proteins of interest by screening against small-molecule compound libraries and identifying molecules that are enriched in the presence of the target protein. We have previously applied Agilent Technology's (Santa Clara, CA) RapidFire solid-phase extraction (SPE)-based high-throughput MS technology to screen small-molecule libraries using AMS. However, SPE-based technologies rely on fluidics for desalting and separation prior to mass analysis with attendant high solvent consumption, relatively high sample volume requirements, risk of sample carryover, and frequent maintenance. To address these challenges, we have established an AMS platform using a laser diode thermal desorption-atmospheric pressure chemical ionization (LDTD-APCI) ionization source (Phytronix, Quebec, Canada) coupled with a SCIEX 5600+ TripleTOF MS (Framingham, MA). We also validated a data-independent acquisition (DIA) Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) method for the robust detection and analysis of small-molecule affinity hits. An informatics platform developed in-house has resulted in a streamlined data analysis workflow for high-throughput AMS screening campaigns and reduced data processing time without compromising data quality. Finally, 68,000 compounds were screened in a single plate and affinity selected hits were confirmed in an orthogonal enzyme activity assay.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Bibliotecas de Moléculas PequeñasRESUMEN
A comprehensive understanding of structure-reactivity relationships is critical to the design and optimization of cysteine-targeted covalent inhibitors. Herein, we report glutathione (GSH) reaction rates for N-phenyl acrylamides with varied substitutions at the α- and ß-positions of the acrylamide moiety. We find that the GSH reaction rates can generally be understood in terms of the electron donating or withdrawing ability of the substituent. When installed at the ß-position, aminomethyl substituents with amine pKa's > 7 accelerate, while those with pKa's < 7 slow the rate of GSH addition at pH 7.4, relative to a hydrogen substituent. Although a computational model was able to only approximately capture experimental reactivity trends, our calculations do not support a frequently invoked mechanism of concerted amine/thiol proton transfer and C-S bond formation and instead suggest that protonated aminomethyl functions as an electron-withdrawing group to reduce the barrier for thiolate addition to the acrylamide.
Asunto(s)
Acrilamidas/síntesis química , Glutatión/química , Acrilamidas/química , Aminas/química , Cisteína/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirimidinonas/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Ensayos Clínicos como Asunto , Perros , Descubrimiento de Drogas , Humanos , Isomerismo , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Piperazinas/química , Piperazinas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C, which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRASG12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRASG12C with submicromolar inhibition of downstream signaling in a KRASG12C-specific manner.
RESUMEN
Silent information regulator or sirtuin (SIRT) enzymes are beta-nicotinamide adenine dinucleotide (oxidized) (NAD(+))-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD(+). The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD(+) bioluminescence assay couples NAD(+) consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD(+) consumption and peptide deacetylation was shown to be 1:1 by the NAD(+) bioluminescence assay. Kinetic parameters of peptide and NAD(+) cosubstrates and IC(50) values of standard reference inhibitors determined in either assay were similar. With robust Z' values (0.7), both assays are amenable to high-throughput screening.
Asunto(s)
Bioensayo/métodos , Mediciones Luminiscentes/métodos , Microfluídica/métodos , Sirtuinas/análisis , Sirtuinas/metabolismo , Acetilación , Secuencia de Aminoácidos , Catálisis , Activación Enzimática , Inhibidores Enzimáticos/química , Humanos , Datos de Secuencia Molecular , NAD/análisis , NAD/metabolismo , Péptidos/química , Péptidos/metabolismo , Sirtuina 1 , Sirtuinas/antagonistas & inhibidoresRESUMEN
Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a "click chemistry" approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase-PROTAC-target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.
Asunto(s)
Química Clic , Evaluación Preclínica de Medicamentos , Proteínas Nucleares , Proteolisis , Factores de Transcripción , Humanos , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Química Clic/métodos , Evaluación Preclínica de Medicamentos/métodos , Ligandos , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Péptidos/farmacología , Proteolisis/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Acetyl-coenzyme A carboxylase (ACC) enzymes exist as two isoforms, ACC1 and ACC2, which play critical roles in fatty acid biosynthesis and oxidation. Though each isoform differs in tissue and subcellular localization, both catalyze the biotin- and ATP-dependent carboxylation of acetyl-coenzyme A to generate malonyl-coenzyme A, a key metabolite in the control of fatty acid synthesis and oxidation. The cytosolic ACC1 is expressed primarily in liver and adipose tissue, and uses malonyl-coenzyme A as a key building block in fatty acid biosynthesis. The mitochondrial ACC2 is primarily expressed in heart and skeletal muscle, where it is involved in the regulation of fatty acid oxidation. Inhibitors of ACC enzymes may therefore be useful therapeutics for diabetes, obesity, and metabolic syndrome. Two assay formats for these ATP-utilizing enzymes amenable to high-throughput screening are compared: a fluorescence intensity-based assay to detect inorganic phosphate and a fluorescence polarization-based assay to detect ADP. Acetyl-coenzyme A carboxylase inhibitors were identified by these high-throughput screening methods and were confirmed in a radiometric high performance liquid chromatography assay of malonyl-coenzyme A production.
Asunto(s)
Acetil-CoA Carboxilasa/antagonistas & inhibidores , Adenosina Difosfato/análisis , Inhibidores Enzimáticos/farmacología , Fosfatos/análisis , Cromatografía Líquida de Alta Presión , Dimetilsulfóxido/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Fluorescencia , Inmunoensayo de Polarización Fluorescente , Colorantes Fluorescentes , Isoenzimas/antagonistas & inhibidores , Cinética , Malonil Coenzima A/metabolismoRESUMEN
SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.