RESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough investigation for application in the treatment of multiple cancer types, including breast cancer. These studies have raised interest in combining these inhibitors, including CDK12/13 inhibitor THZ531, with a variety of other anti-cancer agents. However, the full scope of these potential synergistic interactions of transcriptional CDK inhibitors with kinase inhibitors has not been systematically investigated. Moreover, the mechanisms behind these previously described synergistic interactions remain largely elusive. METHODS: Kinase inhibitor combination screenings were performed to identify kinase inhibitors that synergize with CDK7 inhibitor THZ1 and CDK12/13 inhibitor THZ531 in TNBC cell lines. CRISPR-Cas9 knockout screening and transcriptomic evaluation of resistant versus sensitive cell lines were performed to identify genes critical for THZ531 resistance. RNA sequencing analysis after treatment with individual and combined synergistic treatments was performed to gain further insights into the mechanism of this synergy. Kinase inhibitor screening in combination with visualization of ABCG2-substrate pheophorbide A was used to identify kinase inhibitors that inhibit ABCG2. Multiple transcriptional CDK inhibitors were evaluated to extend the significance of the found mechanism to other transcriptional CDK inhibitors. RESULTS: We show that a very high number of tyrosine kinase inhibitors synergize with the CDK12/13 inhibitor THZ531. Yet, we identified the multidrug transporter ABCG2 as key determinant of THZ531 resistance in TNBC cells. Mechanistically, we demonstrate that most synergistic kinase inhibitors block ABCG2 function, thereby sensitizing cells to transcriptional CDK inhibitors, including THZ531. Accordingly, these kinase inhibitors potentiate the effects of THZ531, disrupting gene expression and increasing intronic polyadenylation. CONCLUSION: Overall, this study demonstrates the critical role of ABCG2 in limiting the efficacy of transcriptional CDK inhibitors and identifies multiple kinase inhibitors that disrupt ABCG2 transporter function and thereby synergize with these CDK inhibitors. These findings therefore further facilitate the development of new (combination) therapies targeting transcriptional CDKs and highlight the importance of evaluating the role of ABC transporters in synergistic drug-drug interactions in general.
Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Ciclina-Dependentes/genética , Pirimidinas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de NeoplasiasRESUMO
Each year, publicly available databases are updated with new compounds from different research institutions. Positive experimental outcomes are more likely to be reported; therefore, they account for a considerable fraction of these entries. Established publicly available databases such as ChEMBL allow researchers to use information without constrictions and create predictive tools for a broad spectrum of applications in the field of toxicology. Therefore, we investigated the distribution of positive and nonpositive entries within ChEMBL for a set of off-targets and its impact on the performance of classification models when applied to pharmaceutical industry data sets. Results indicate that models trained on publicly available data tend to overpredict positives, and models based on industry data sets predict negatives more often than those built using publicly available data sets. This is strengthened even further by the visualization of the prediction space for a set of 10,000 compounds, which makes it possible to identify regions in the chemical space where predictions converge. Finally, we highlight the utilization of these models for consensus modeling for potential adverse events prediction.
Assuntos
Aprendizado de Máquina , Bases de Dados FactuaisRESUMO
Neonicotinoid pesticides were initially designed in order to achieve species selectivity on insect nicotinic acetylcholine receptors (nAChRs). However, concerns arose when agonistic effects were also detected in human cells expressing nAChRs. In the context of next-generation risk assessments (NGRAs), new approach methods (NAMs) should replace animal testing where appropriate. Herein, we present a combination of in silico and in vitro methodologies that are used to investigate the potentially toxic effects of neonicotinoids and nicotinoid metabolites on human neurons. First, an ensemble docking study was conducted on the nAChR isoforms α7 and α3ß4 to assess potential crucial molecular initiating event (MIE) interactions. Representative docking poses were further refined using molecular dynamics (MD) simulations and binding energy calculations using implicit solvent models. Finally, calcium imaging on LUHMES neurons confirmed a key event (KE) downstream of the MIE. This method was also used to confirm the predicted agonistic effect of the metabolite descyano-thiacloprid (DCNT).
Assuntos
Cálcio , Receptores Nicotínicos , Animais , Humanos , Simulação de Acoplamento Molecular , Cálcio da Dieta , Neonicotinoides/farmacologiaRESUMO
The discovery of the first ATP-binding cassette (ABC) transporter, whose overexpression in cancer cells is responsible for exporting anticancer drugs out of tumor cells, initiated enormous efforts to overcome tumor cell multidrug resistance (MDR) by inhibition of ABC-transporter. Because of its many physiological functions, diverse studies have been conducted on the mechanism, function and regulation of this important group of transmembrane transport proteins. In this review, we will focus on the structural aspects of this transporter superfamily. Since the resolution revolution of electron microscope, experimentally solved structures increased rapidly. A summary of the structures available and an overview of recent structure-based studies are provided. More specifically, the artificial intelligence (AI)-based predictions from AlphaFold-2 will be discussed.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Inteligência Artificial , Resistencia a Medicamentos Antineoplásicos , Resistência a Múltiplos Medicamentos , Antineoplásicos/química , Neoplasias/tratamento farmacológicoRESUMO
Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.
Assuntos
Resistência a Medicamentos/genética , Proteínas Carreadoras de Solutos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antineoplásicos , Fenômenos Bioquímicos , Transporte Biológico/genética , Transporte Biológico/fisiologia , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Resistência a Medicamentos/fisiologia , Testes Genéticos , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Transporte Proteico/fisiologia , Proteínas Carreadoras de Solutos/fisiologia , Simportadores/genética , Simportadores/metabolismoRESUMO
Minimizing in vitro and in vivo testing in early drug discovery with the use of physiologically based pharmacokinetic (PBPK) modeling and machine learning (ML) approaches has the potential to reduce discovery cycle times and animal experimentation. However, the prediction success of such an approach has not been shown for a larger and diverse set of compounds representative of a lead optimization pipeline. In this study, the prediction success of the oral (PO) and intravenous (IV) pharmacokinetics (PK) parameters in rats was assessed using a "bottom-up" approach, combining in vitro and ML inputs with a PBPK model. More than 240 compounds for which all of the necessary inputs and PK data were available were used for this assessment. Different clearance scaling approaches were assessed, using hepatocyte intrinsic clearance and protein binding as inputs. In addition, a novel high-throughput PBPK (HT-PBPK) approach was evaluated to assess the scalability of PBPK predictions for a larger number of compounds in drug discovery. The results showed that bottom-up PBPK modeling was able to predict the rat IV and PO PK parameters for the majority of compounds within a 2- to 3-fold error range, using both direct scaling and dilution methods for clearance predictions. The use of only ML-predicted inputs from the structure did not perform well when using in vitro inputs, likely due to clearance miss predictions. The HT-PBPK approach produced comparable results to the full PBPK modeling approach but reduced the simulation time from hours to seconds. In conclusion, a bottom-up PBPK and HT-PBPK approach can successfully predict the PK parameters and guide early discovery by informing compound prioritization, provided that good in vitro assays are in place for key parameters such as clearance.
Assuntos
Descoberta de Drogas , Modelos Biológicos , Animais , Simulação por Computador , Descoberta de Drogas/métodos , Hepatócitos , Taxa de Depuração Metabólica/fisiologia , Farmacocinética , RatosRESUMO
Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.
Assuntos
Imidazolinas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Nitrocompostos/farmacologia , Piridinas/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Alcenos/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Neonicotinoides/metabolismo , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitrocompostos/metabolismo , Oócitos , Praguicidas/metabolismo , Praguicidas/farmacologia , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10-100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 µM) blunted the signaling response of nicotine. The pesticides (at 3-30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Neonicotinoides/toxicidade , Praguicidas/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Humanos , Neonicotinoides/administração & dosagem , Neuroblastoma/metabolismo , Técnicas de Patch-Clamp , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 µM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.
Assuntos
Anticonvulsivantes/uso terapêutico , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Estimulação Acústica/efeitos adversos , Animais , Anticonvulsivantes/farmacologia , Células CHO , Cricetulus , Epilepsia Reflexa/tratamento farmacológico , Epilepsia Reflexa/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/química , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Convulsões/etiologia , Resultado do TratamentoRESUMO
The drugs we use to cure our diseases can cause damage to the liver as it is the primary organ responsible for metabolism of environmental chemicals and drugs. To identify and eliminate potentially problematic drug candidates in the early stages of drug discovery, in silico techniques provide quick and practical solutions for toxicity determination. Deep learning has emerged as one of the solutions in recent years in the field of pharmaceutical chemistry. Generally, in the case of small data sets as used in toxicology, these data-hungry algorithms are prone to overfitting. We approach the problem from two sides. First, we use images of the three-dimensional conformations and benefit from convolutional neural networks which have fewer parameters than the standard deep neural networks with similar depth. Using images allows connecting various chemical features to the geometry of the compounds. Second, we employ the method COVER to up-sample the data set. It is used not only for increasing the size of the data set, but also for balancing the two classes, i.e., toxic and not toxic. The proof of concept is performed on the p53 end point from the Tox21 data set. The results, which are compatible with the winners of the data challenge, encouraged us to use our methods to predict liver toxicity. We use the most extensive publicly available liver toxicity data set by Mulliner et al. and obtain a sensitivity of 0.79 and a specificity of 0.52. These results demonstrate the applicability of image based toxicity prediction using deep neural networks.
Assuntos
Algoritmos , Redes Neurais de Computação , Descoberta de Drogas , FígadoRESUMO
P-glycoprotein (P-gp) is an ATP-dependent efflux pump that has a marked impact on the absorption, distribution, and excretion of therapeutic drugs. As P-gp inhibition can result in drug-drug interactions and altered drug bioavailability, identifying molecular properties that are linked to inhibition is of great interest in drug development. In this study, we combined chemical synthesis, in vitro testing, quantitative structure-activity relationship analysis, and docking studies to investigate the role of hydrogen bond (H-bond) donor/acceptor properties in transporter-ligand interaction. In a previous work, it has been shown that propafenone analogs with a 4-hydroxy-4-piperidine moiety exhibit a generally 10-fold higher P-gp inhibitory activity than expected based on their lipophilicity. Here, we specifically expanded the data set by introducing substituents at position 4 of the 4-phenylpiperidine moiety to assess the importance of H-bond donor/acceptor features in this region. The results suggest that indeed an H-bond acceptor, such as hydroxy and methoxy, increases the affinity by forming a H-bond with Tyr310.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Propafenona/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Células Cultivadas , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Estrutura Molecular , Propafenona/síntese química , Propafenona/química , Relação Quantitativa Estrutura-Atividade , Relação Estrutura-AtividadeRESUMO
Computational approaches currently assist medicinal chemistry through the entire drug discovery pipeline. However, while several computational tools and strategies are available to predict binding affinity, predicting the drug-target binding kinetics is still a matter of ongoing research. Here, we challenge scaled molecular dynamics simulations to assess the off-rates for a series of structurally diverse inhibitors of the heat shock protein 90 (Hsp90) covering 3 orders of magnitude in their experimental residence times. The derived computational predictions are in overall good agreement with experimental data. Aside from the estimation of exit times, unbinding pathways were assessed through dimensionality reduction techniques. The data analysis framework proposed in this work could lead to better understanding of the mechanistic aspects related to the observed kinetic behavior.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Simulação de Dinâmica Molecular , Preparações Farmacêuticas/metabolismo , Proteínas de Choque Térmico HSP90/química , Humanos , Cinética , Ligantes , Ligação Proteica , Conformação ProteicaRESUMO
The structural resolution of a bound ligand-receptor complex is a key asset to efficiently drive lead optimization in drug design. However, structural resolution of many drug targets still remains a challenging endeavor. In the absence of structural knowledge, scientists resort to structure-activity relationships (SARs) to promote compound development. In this study, we incorporated ligand-based knowledge to formulate a docking scoring function that evaluates binding poses for their agreement with a known SAR. We showcased this protocol by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216 at the benzodiazepine binding site of the GABAA receptor. Further evaluation of the final pose by molecular dynamics and free energy simulations revealed a close proximity between the pendent phenyl ring of the PQ and γ2D56, congruent with the low potency of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A mutation and in fact observed a 10-fold potency increase in the carboxyphenyl analogue, providing experimental evidence in favor of our binding hypothesis.
Assuntos
Pirazóis/farmacologia , Receptores de GABA-A/metabolismo , Benzodiazepinas/metabolismo , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pirazóis/química , Receptores de GABA-A/química , Software , Relação Estrutura-AtividadeRESUMO
Cheminformatics datasets used in classification problems, especially those related to biological or physicochemical properties, are often imbalanced. This presents a major challenge in development of in silico prediction models, as the traditional machine learning algorithms are known to work best on balanced datasets. The class imbalance introduces a bias in the performance of these algorithms due to their preference towards the majority class. Here, we present a comparison of the performance of seven different meta-classifiers for their ability to handle imbalanced datasets, whereby Random Forest is used as base-classifier. Four different datasets that are directly (cholestasis) or indirectly (via inhibition of organic anion transporting polypeptide 1B1 and 1B3) related to liver toxicity were chosen for this purpose. The imbalance ratio in these datasets ranges between 4:1 and 20:1 for negative and positive classes, respectively. Three different sets of molecular descriptors for model development were used, and their performance was assessed in 10-fold cross-validation and on an independent validation set. Stratified bagging, MetaCost and CostSensitiveClassifier were found to be the best performing among all the methods. While MetaCost and CostSensitiveClassifier provided better sensitivity values, Stratified Bagging resulted in high balanced accuracies.
Assuntos
Simulação por Computador , Conjuntos de Dados como Assunto , Fígado/efeitos dos fármacos , Algoritmos , Animais , Colestase/induzido quimicamente , Humanos , Fígado/metabolismo , Aprendizado de Máquina , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidoresRESUMO
The large neutral amino acid transporter 1 (LAT1, or SLC7A5) is a sodium- and pH-independent transporter, which supplies essential amino acids (e.g., leucine, phenylalanine) to cells. It plays an important role at the Bloodâ»Brain Barrier (BBB) where it facilitates the transport of thyroid hormones, pharmaceuticals (e.g., l-DOPA, gabapentin), and metabolites into the brain. Moreover, its expression is highly upregulated in various types of human cancer that are characterized by an intense demand for amino acids for growth and proliferation. Therefore, LAT1 is believed to be an important drug target for cancer treatment. With the crystallization of the arginine/agmatine antiporter (AdiC) from Escherichia Coli, numerous homology models of LAT1 have been built to elucidate the substrate binding site, ligandâ»transporter interaction, and structureâ»function relationship. The use of these models in combination with molecular docking and experimental testing has identified novel chemotypes of ligands of LAT1. Here, we highlight the structure, function, transport mechanism, and homology modeling of LAT1. Additionally, results from structureâ»function studies performed on LAT1 are addressed, which have enhanced our knowledge of the mechanism of substrate binding and translocation. This is followed by a discussion on ligand- and structure-based approaches, with an emphasis on elucidating the molecular basis of LAT1 inhibition. Finally, we provide an exhaustive summary of different LAT1 inhibitors that have been identified so far, including the recently discovered irreversible covalent inhibitors.
Assuntos
Descoberta de Drogas , Transportador 1 de Aminoácidos Neutros Grandes/química , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Sítios de Ligação , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Pró-Fármacos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure-activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC50 value in the sub-µM range.
Assuntos
Descoberta de Drogas , Transportador 1 de Aminoácidos Neutros Grandes/química , Sítios de Ligação , Simulação por Computador , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Fluxo de TrabalhoRESUMO
Cholestasis represents one out of three types of drug induced liver injury (DILI), which comprises a major challenge in drug development. In this study we applied a two-class classification scheme based on k-nearest neighbors in order to predict cholestasis, using a set of 93 two-dimensional (2D) physicochemical descriptors and predictions of selected hepatic transporters' inhibition (BSEP, BCRP, P-gp, OATP1B1, and OATP1B3). In order to assess the potential contribution of transporter inhibition, we compared whether the inclusion of the transporters' inhibition predictions contributes to a significant increase in model performance in comparison to the plain use of the 93 2D physicochemical descriptors. Our findings were in agreement with literature findings, indicating a contribution not only from BSEP inhibition but a rather synergistic effect deriving from the whole set of transporters. The final optimal model was validated via both 10-fold cross validation and external validation. It performs quite satisfactorily resulting in 0.686 ± 0.013 for accuracy and 0.722 ± 0.014 for area under the receiver operating characteristic curve (AUC) for 10-fold cross-validation (mean ± standard deviation from 50 iterations).
Assuntos
Colestase/induzido quimicamente , Colestase/metabolismo , Simulação por Computador , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Células HEK293 , HumanosRESUMO
With the public availability of large data sources such as ChEMBLdb and the Open PHACTS Discovery Platform, retrieval of data sets for certain protein targets of interest with consistent assay conditions is no longer a time consuming process. Especially the use of workflow engines such as KNIME or Pipeline Pilot allows complex queries and enables to simultaneously search for several targets. Data can then directly be used as input to various ligand- and structure-based studies. In this contribution, using in-house projects on P-gp inhibition, transporter selectivity, and TRPV1 modulation we outline how the incorporation of linked life science data in the daily execution of projects allowed to expand our approaches from conventional Hansch analysis to complex, integrated multilayer models.
Assuntos
Disciplinas das Ciências Biológicas , Biologia Computacional/métodos , Desenho de Fármacos , Indústria Farmacêutica , Software , Estrutura Molecular , Proteínas/química , Relação Estrutura-Atividade , Fluxo de TrabalhoRESUMO
The bile salt export pump (BSEP) actively transports conjugated monovalent bile acids from the hepatocytes into the bile. This facilitates the formation of micelles and promotes digestion and absorption of dietary fat. Inhibition of BSEP leads to decreased bile flow and accumulation of cytotoxic bile salts in the liver. A number of compounds have been identified to interact with BSEP, which results in drug-induced cholestasis or liver injury. Therefore, in silico approaches for flagging compounds as potential BSEP inhibitors would be of high value in the early stage of the drug discovery pipeline. Up to now, due to the lack of a high-resolution X-ray structure of BSEP, in silico based identification of BSEP inhibitors focused on ligand-based approaches. In this study, we provide a homology model for BSEP, developed using the corrected mouse P-glycoprotein structure (PDB ID: 4M1M). Subsequently, the model was used for docking-based classification of a set of 1212 compounds (405 BSEP inhibitors, 807 non-inhibitors). Using the scoring function ChemScore, a prediction accuracy of 81% on the training set and 73% on two external test sets could be obtained. In addition, the applicability domain of the models was assessed based on Euclidean distance. Further, analysis of the protein-ligand interaction fingerprints revealed certain functional group-amino acid residue interactions that could play a key role for ligand binding. Though ligand-based models, due to their high speed and accuracy, remain the method of choice for classification of BSEP inhibitors, structure-assisted docking models demonstrate reasonably good prediction accuracies while additionally providing information about putative protein-ligand interactions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Animais , Sítios de Ligação , Transporte Biológico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Simulação por Computador , Bases de Dados de Compostos Químicos , Humanos , Ligantes , Aprendizado de Máquina , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Bibliotecas de Moléculas Pequenas/classificaçãoRESUMO
Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.