RESUMO
OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.
Assuntos
Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Transporte Biológico , HumanosRESUMO
OATP2B1 is an intestinal and hepatic drug uptake transporter. Intestinal OATP2B1 has been elucidated as the mechanism of unexpected clinical drug-drug interactions (DDIs), where drug exposure was unexpectedly decreased with unchanged half-life. Hepatic OATP2B1 may be an understudied clinical DDI mechanism. The aim of the present work was to understand the prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors in marketed drug space. HEK293 cells stably overexpressing human OATP2B1 or vector control were generated and cultured for 72 h in a 96-well format. OATP2B1-mediated uptake of dibromofluorescein (DBF) was found to be optimal at 10 µM concentration and 30 min incubation time. A total of 294 drugs (top 300 marketed drugs, excluding biologics and restricted drugs, supplemented with â¼100 small-molecule drugs) were screened for OATP2B1 inhibition at 10 µM. Drugs demonstrating ≥50% inhibition in this screen were advanced for IC50 determination, which was extrapolated to clinical intestinal and hepatic OATP2B1 inhibition as per 2017 FDA DDI guidance. Of the 294 drugs screened, 67 elicited ≥50% inhibition of OATP2B1-mediated DBF uptake at 10 µM screening concentration. For the 67 drugs flagged in the single-concentration inhibition screen, upon evaluation of a full concentration range, IC50 values could be determined for 58 drugs. OATP2B1 IC50 values established for these 58 drugs were extrapolated as potentially clinically relevant at the intestinal level for 38 orally administered drugs (Igut/IC50 ≥ 10), and 17 were flagged as potential clinical inhibitors of hepatic OATP2B1 uptake (1 + Iin,max,u/IC50 ≥ 1.1). This analysis of 294 drugs demonstrated prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors to be 13 and 6%, respectively. As OATP2B1-inhibitor drugs are not exceedingly rare, these results suggest that clinical OATP2B1 DDIs have been rarely observed because OATP2B1 is uncommonly the predominant determinant of drug disposition.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Cloridrato de Erlotinib/farmacologia , Fluoresceínas/metabolismo , Células HEK293 , Meia-Vida , Humanos , Concentração Inibidora 50 , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , TransfecçãoRESUMO
We extended thermal proteome profiling to detect transmembrane protein-small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell-receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1.
Assuntos
Proteínas de Membrana/metabolismo , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Temperatura Alta , Humanos , Células Jurkat , Células K562 , Ligantes , Ligação Proteica , Estabilidade Proteica , Proteoma/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Vanadatos/farmacologiaRESUMO
The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.
Assuntos
Aldeído Oxidase/metabolismo , Inibidores de Janus Quinases/farmacocinética , Pulmão/metabolismo , Administração Intranasal , Administração Intravenosa , Animais , Sítios de Ligação , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Inibidores de Janus Quinases/administração & dosagem , Inibidores de Janus Quinases/síntese química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Simulação de Acoplamento Molecular , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound 19. Selectivity profiling of compound 19 showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound 19 was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound 41, which showed improved toxicological outcomes at similar exposure levels to compound 19.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacocinética , Animais , Ligação Competitiva , Disponibilidade Biológica , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Isoenzimas , Modelos Moleculares , Simulação de Acoplamento Molecular , Inibidores de Fosfoinositídeo-3 Quinase/toxicidade , Ratos , Relação Estrutura-AtividadeRESUMO
Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.
Assuntos
Dasatinibe/farmacologia , Janus Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Proteômica/métodos , Animais , Cromatografia de Afinidade/métodos , Cães , Células HEK293 , Humanos , Janus Quinases/metabolismo , Células K562 , Camundongos , Microesferas , Monócitos/metabolismo , Ligação Proteica , Proteoma/química , Sefarose/análogos & derivados , Especificidade por SubstratoRESUMO
The direct detection of drug-protein interactions in living cells is a major challenge in drug discovery research. Recently, we introduced an approach termed thermal proteome profiling (TPP), which enables the monitoring of changes in protein thermal stability across the proteome using quantitative mass spectrometry. We determined the intracellular thermal profiles for up to 7,000 proteins, and by comparing profiles derived from cultured mammalian cells in the presence or absence of a drug we showed that it was possible to identify direct and indirect targets of drugs in living cells in an unbiased manner. Here we demonstrate the complete workflow using the histone deacetylase inhibitor panobinostat. The key to this approach is the use of isobaric tandem mass tag 10-plex (TMT10) reagents to label digested protein samples corresponding to each temperature point in the melting curve so that the samples can be analyzed by multiplexed quantitative mass spectrometry. Important steps in the bioinformatic analysis include data normalization, melting curve fitting and statistical significance determination of compound concentration-dependent changes in protein stability. All analysis tools are made freely available as R and Python packages. The workflow can be completed in 2 weeks.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Espectrometria de Massas , Proteoma/genética , Humanos , Células K562 , Análise Serial de Proteínas , Estabilidade Proteica , TemperaturaRESUMO
The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity.
Assuntos
Antineoplásicos/farmacologia , Proteoma/efeitos dos fármacos , Proteômica/métodos , Adenosina Trifosfatases/metabolismo , Temperatura Alta , Humanos , Células K562 , Ligantes , Ligação Proteica , Desnaturação Proteica , Estabilidade ProteicaRESUMO
The handling of organic compounds in the laboratory requires the use of organic (co-) solvents to mediate solubility. Advantages and disadvantages of the widely used solvent dimethylsulfoxide (DMSO) are discussed, and guidelines for dissolution and storage of compounds are given. Finally, nephelometry is introduced as a fast method to determine the kinetic solubility of a compound.
Assuntos
Bioquímica/métodos , Bibliotecas de Moléculas Pequenas/química , Soluções/síntese química , Cinética , Luz , Análise de Regressão , Espalhamento de Radiação , SolubilidadeRESUMO
Proteolytic processing of the transmembrane domain of the amyloid precursor protein (APP) is a key component of Alzheimer's disease pathogenesis. Using C-terminally tagged APP derivatives, we have identified by amino-terminal sequencing a novel cleavage site of APP, at Leu-49, distal to the gamma-secretase site. This was termed -cleavage. Brefeldin A treatment and pulse-chase experiments indicate that this cleavage occurs late in the secretory pathway. The level of -cleavage is decreased by expression of presenilin-1 mutants known to impair Abeta formation, and it is sensitive to the gamma-secretase inhibitors MDL28170 and L-685,458. Remarkably, it shares similarities with site 3 cleavage of Notch-1: membrane topology, cleavage before a valine, dependence on presenilins, and inhibition profile.