RESUMEN

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

Asunto(s)

Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Quimioinformática/métodos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad

RESUMEN

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Asunto(s)

Técnicas de Cultivo de Célula , Ensayos Analíticos de Alto Rendimiento , Humanos , Línea Celular , Bibliotecas de Moléculas Pequeñas/farmacología

RESUMEN

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

RESUMEN

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)-including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)-activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow-derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

Asunto(s)

Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Ratones , Animales , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Mesilato de Imatinib , Leucocitos Mononucleares/metabolismo , Muerte Celular , Células Mieloides/metabolismo , Interleucina-1beta/metabolismo

RESUMEN

Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.

Asunto(s)

Descubrimiento de Drogas , Transcriptoma , Descubrimiento de Drogas/métodos , Humanos , ARN , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos

RESUMEN

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Asunto(s)

Proteínas de Ciclo Celular/genética , Epidermis/efectos de los fármacos , Repitelización/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Heridas no Penetrantes/tratamiento farmacológico , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Repitelización/genética , Úlcera Cutánea/genética , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Heridas no Penetrantes/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patología

RESUMEN

Multiplexed gene-signature-based phenotypic assays are increasingly used for the identification and profiling of small molecule-tool compounds and drugs. Here we introduce a method (provided as R-package) for the quantification of the dose-response potency of a gene-signature as EC50 and IC50 values. Two signaling pathways were used as models to validate our methods: beta-adrenergic agonistic activity on cAMP generation (dedicated dataset generated for this study) and EGFR inhibitory effect on cancer cell viability. In both cases, potencies derived from multi-gene expression data were highly correlated with orthogonal potencies derived from cAMP and cell growth readouts, and superior to potencies derived from single individual genes. Based on our results we propose gene-signature potencies as a novel valid alternative for the quantitative prioritization, optimization and development of novel drugs.

Asunto(s)

Agonistas Adrenérgicos beta/farmacología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/genética , Agonistas Adrenérgicos beta/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Humanos , Concentración 50 Inhibidora , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fenotipo , Transducción de Señal/efectos de los fármacos , Células THP-1

RESUMEN

Here we report Digital RNA with pertUrbation of Genes (DRUG-seq), a high-throughput platform for drug discovery. Pharmaceutical discovery relies on high-throughput screening, yet current platforms have limited readouts. RNA-seq is a powerful tool to investigate drug effects using transcriptome changes as a proxy, yet standard library construction is costly. DRUG-seq captures transcriptional changes detected in standard RNA-seq at 1/100th the cost. In proof-of-concept experiments profiling 433 compounds across 8 doses, transcription profiles generated from DRUG-seq successfully grouped compounds into functional clusters by mechanism of actions (MoAs) based on their intended targets. Perturbation differences reflected in transcriptome changes were detected for compounds engaging the same target, demonstrating the value of using DRUG-seq for understanding on and off-target activities. We demonstrate DRUG-seq captures common mechanisms, as well as differences between compound treatment and CRISPR on the same target. DRUG-seq provides a powerful tool for comprehensive transcriptome readout in a high-throughput screening environment.

Asunto(s)

Descubrimiento de Drogas/métodos , Perfilación de la Expresión Génica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos

RESUMEN

Using computational bioactivity prediction models we identified phosphodiesterase 3B (PDE3B) and cathepsin L as macromolecular targets of de novo designed compounds. By disclosing the most potent cathepsin L activator known to date, small molecule repurposing by target panel prediction represents a feasible route towards innovative leads for chemical biology and molecular medicine.

Asunto(s)

Catepsina L/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Inhibidores de Fosfodiesterasa 3/química , Inhibidores de Proteasas/química , Catepsina L/antagonistas & inhibidores , Simulación por Computador , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/química , Inhibidores de Fosfodiesterasa 3/metabolismo , Inhibidores de Proteasas/metabolismo , Unión Proteica , Relación Estructura-Actividad

RESUMEN

Fragment-based ligand and drug discovery predominantly employs sp(2)-rich compounds covering well-explored regions of chemical space. Despite the ease with which such fragments can be coupled, this focus on flat compounds is widely cited as contributing to the attrition rate of the drug discovery process. In contrast, biologically validated natural products are rich in stereogenic centres and populate areas of chemical space not occupied by average synthetic molecules. Here, we have analysed more than 180,000 natural product structures to arrive at 2,000 clusters of natural-product-derived fragments with high structural diversity, which resemble natural scaffolds and are rich in sp(3)-configured centres. The structures of the cluster centres differ from previously explored fragment libraries, but for nearly half of the clusters representative members are commercially available. We validate their usefulness for the discovery of novel ligand and inhibitor types by means of protein X-ray crystallography and the identification of novel stabilizers of inactive conformations of p38α MAP kinase and of inhibitors of several phosphatases.

Asunto(s)

Productos Biológicos/química , Descubrimiento de Drogas , Ligandos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Algoritmos , Cristalografía por Rayos X , Concentración 50 Inhibidora , Estructura Molecular , Quinonas/química , Quinonas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/farmacología

RESUMEN

In the search for new bioactive compounds, there is a trend toward increasingly complex compound libraries aiming to target the demanding targets of the future. In contrast, medicinal chemistry and traditional library design rely mainly on a small set of highly established and robust reactions. Here, we probe a set of 58 such reactions for their ability to sample the chemical space of known bioactive molecules, and the potential to create new scaffolds. Combined with ~26,000 common available building blocks, the reactions retrieve around 9% of a scaffold-diverse set of compounds active on human target proteins covering all major pharmaceutical target classes. Almost 80% of generated scaffolds from virtual one-step synthesis products are not present in a large set of known bioactive molecules for human targets, indicating potential for new discoveries. The results suggest that established synthesis resources are well suited to cover the known bioactivity-relevant chemical space and that there are plenty of unexplored regions accessible by these reactions, possibly providing valuable "low-hanging fruit" for hit discovery.

Asunto(s)

Sistemas de Liberación de Medicamentos , Bibliotecas de Moléculas Pequeñas/síntesis química , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química

RESUMEN

A focused collection of organic synthesis reactions for computer-based molecule construction is presented. It is inspired by real-world chemistry and has been compiled in close collaboration with medicinal chemists to achieve high practical relevance. Virtual molecules assembled from existing starting material connected by these reactions are supposed to have an enhanced chance to be amenable to real chemical synthesis. About 50% of the reactions in the dataset are ring-forming reactions, which fosters the assembly of novel ring systems and innovative chemotypes. A comparison with a recent survey of the reactions used in early drug discovery revealed considerable overlaps with the collection presented here. The dataset is available encoded as computer-readable Reaction SMARTS expressions from the Supporting Information presented for this paper.

Asunto(s)

Técnicas de Química Sintética , Diseño de Fármacos , Técnicas de Química Sintética/métodos , Diseño Asistido por Computadora , Bases de Datos Factuales

RESUMEN

A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.

Asunto(s)

Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Lactonas/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Animales , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lactonas/síntesis química , Lactonas/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Especificidad por Sustrato , Tioléster Hidrolasas/metabolismo

Asunto(s)

Diseño Asistido por Computadora , Diseño de Fármacos , Química Farmacéutica/tendencias , Metodologías Computacionales

RESUMEN

Identification of meaningful chemical patterns in the increasing amounts of high-throughput-generated bioactivity data available today is an increasingly important challenge for successful drug discovery. Herein, we present the scaffold network as a novel approach for mapping and navigation of chemical and biological space. A scaffold network represents the chemical space of a library of molecules consisting of all molecular scaffolds and smaller "parent" scaffolds generated therefrom by the pruning of rings, effectively leading to a network of common scaffold substructure relationships. This algorithm provides an extension of the scaffold tree algorithm that, instead of a network, generates a tree relationship between a heuristically rule-based selected subset of parent scaffolds. The approach was evaluated for the identification of statistically significantly active scaffolds from primary screening data for which the scaffold tree approach has already been shown to be successful. Because of the exhaustive enumeration of smaller scaffolds and the full enumeration of relationships between them, about twice as many statistically significantly active scaffolds were identified compared to the scaffold-tree-based approach. We suggest visualizing scaffold networks as islands of active scaffolds.

Asunto(s)

Biología Computacional , Modelos Biológicos , Bibliotecas de Moléculas Pequeñas/química , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Modelos Moleculares , Estructura Molecular , Antagonistas del Receptor de Serotonina 5-HT3/química

RESUMEN

Structural simplification of an antimitotic natural product podophyllotoxin with mimetic heterocyclic scaffolds constructed using multicomponent reactions led to the identification of compounds exhibiting low nanomolar antiproliferative and apoptosis-inducing properties. The most potent compounds were found in the dihydropyridopyrazole, dihydropyridonaphthalene, dihydropyridoindole, and dihydropyridopyrimidine scaffold series. Biochemical mechanistic studies performed with dihydropyridopyrazole compounds showed that these heterocycles inhibit in vitro tubulin polymerization and disrupt the formation of mitotic spindles in dividing cells at low nanomolar concentrations, in a manner similar to podophyllotoxin itself. Separation of a racemic dihydropyridonaphthalene into individual enantiomers demonstrated that only the optical antipode matching the absolute configuration of podophyllotoxin possessed potent anticancer activity. Computer modeling, performed using the podophyllotoxin binding site on ß-tubulin, provided a theoretical understanding of these successful experimental findings.

Asunto(s)

Antineoplásicos/química , Apoptosis/efectos de los fármacos , Compuestos Heterocíclicos/química , Podofilotoxina/química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Sitios de Unión , Simulación por Computador , Células HeLa , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Humanos , Indoles , Modelos Moleculares , Imitación Molecular , Naftalenos , Pirazoles , Piridinas , Bibliotecas de Moléculas Pequeñas , Estereoisomerismo , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacología

RESUMEN

The design of a high-quality screening collection is of utmost importance for the early drug-discovery process and provides, in combination with high-quality assay systems, the foundation of future discoveries. Herein, we review recent trends and observations to successfully expand the access to bioactive chemical space, including the feedback from hit assessment interviews of high-throughput screening campaigns; recent successes with chemogenomics target family approaches, the identification of new relevant target/domain families, diversity-oriented synthesis and new emerging compound classes, and non-classical approaches, such as fragment-based screening and DNA-encoded chemical libraries. The role of in silico library design approaches are emphasized.

Asunto(s)

Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento/tendencias , Bibliotecas de Moléculas Pequeñas/química , Técnicas Químicas Combinatorias , Simulación por Computador , ADN/química , Descubrimiento de Drogas/tendencias , Proteínas/química

RESUMEN

The Scaffold Tree algorithm (J Chem Inf Model 47:47-58, 2007) allows to organize large molecular data sets by arranging sets of molecules into a unique tree hierarchy based on their scaffolds, with scaffolds forming leaf nodes of such tree. The hierarchy is created by iterative removal of rings from more complex scaffolds using chemically meaningful set of rules, until a single, root ring is obtained. The classification is deterministic, data set independent, and scales linearly with the number of compounds included in the data set. In this review we summarize the basic principles of the Scaffold Tree methodology and review its applications, which appeared in recent medicinal chemistry literature, including the use of Scaffold Trees for visualization of large chemical data sets, compound clustering, and the identification of novel bioactive molecules. References to several computer programs, including also free tools available on the Internet, allowing to perform classification and visualization of molecules based on their scaffolds are also provided.

Asunto(s)

Simulación por Computador , Bases de Datos Factuales , Algoritmos , Química Farmacéutica/métodos , Análisis por Conglomerados , Internet , Modelos Moleculares , Estructura Molecular , Preparaciones Farmacéuticas/química , Programas Informáticos

RESUMEN

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Propiolactona/análogos & derivados , Tioléster Hidrolasas/antagonistas & inhibidores , Tioléster Hidrolasas/química , Proteínas ras/fisiología , Animales , Línea Celular , Perros , Regulación hacia Abajo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Riñón/efectos de los fármacos , Riñón/fisiología , Ligandos , Lipasa/química , Lipasa/metabolismo , Lipoilación/efectos de los fármacos , Modelos Moleculares , Propiolactona/síntesis química , Propiolactona/química , Propiolactona/farmacología , Conformación Proteica , Proto-Oncogenes Mas , Transducción de Señal , Estómago/enzimología , Tioléster Hidrolasas/genética , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismo