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DNA-binding proteins are promising therapeutic targets but are notoriously difficult to drug. Here, we evaluate a chemoproteomic DNA interaction platform as a complementary strategy for parallelized compound profiling. To enable this approach, we determined the proteomic binding landscape of 92 immobilized DNA sequences. Perturbation-induced activity changes of captured transcription factors in disease-relevant settings demonstrated functional relevance of the enriched subproteome. Chemoproteomic profiling of >300 cysteine-directed compounds against a coverage optimized bead mixture, which specifically captures >150 DNA binders, revealed competition of several DNA-binding proteins, including the transcription factors ELF1 and ELF2. We also discovered the first compound that displaces the DNA-repair complex MSH2-MSH3 from DNA. Compound binding to cysteine 252 on MSH3 was confirmed using chemoproteomic reactive cysteine profiling. Overall, these results suggested that chemoproteomic DNA bead pull-downs enable the specific readout of transcription factor activity and can identify functional "hotspots" on DNA binders toward expanding the druggable proteome.
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Cisteína , Proteínas de Unión al ADN , Proteómica , Factores de Transcripción , ProteomaRESUMEN
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
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Infecciones por VIH , VIH-1 , Alquinos , Benzoxazinas , Proteínas Adaptadoras de Señalización CARD/metabolismo , Ciclopropanos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , Humanos , Inflamasomas/metabolismo , Leucocitos Mononucleares , Proteínas de Neoplasias/metabolismoRESUMEN
Determination of target engagement for candidate drug molecules in the native cellular environment is a significant challenge for drug discovery programs. The cellular thermal shift assay (CETSA) has emerged as a powerful tool for determining compound target engagement through measurement of changes to a protein's thermal stability upon ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA) that deploys a quantitative peptide tag for determination of compound target engagement in the native cellular environment using a high throughput, plate-based luminescence readout. We demonstrate that BiTSA can rapidly assess cellular target engagement of small molecule ligands against their cognate targets and highlight two applications of BiTSA for differentiating small molecules targeting mutant KRAS and TP53.
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By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Mass spectrometry-based discovery proteomics is an essential tool for the proximal readout of cellular drug action. Here, we apply a robust proteomic workflow to rapidly profile the proteomes of five lung cancer cell lines in response to more than 50 drugs. Integration of millions of quantitative protein-drug associations substantially improved the mechanism of action (MoA) deconvolution of single compounds. For example, MoA specificity increased after removal of proteins that frequently responded to drugs and the aggregation of proteome changes across cell lines resolved compound effects on proteostasis. We leveraged these findings to demonstrate efficient target identification of chemical protein degraders. Aggregating drug response across cell lines also revealed that one-quarter of compounds modulated the abundance of one of their known protein targets. Finally, the proteomic data led us to discover that inhibition of mitochondrial function is an off-target mechanism of the MAP2K1/2 inhibitor PD184352 and that the ALK inhibitor ceritinib modulates autophagy.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Proteómica/métodos , Antineoplásicos/farmacología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Espectrometría de Masas , ProteomaRESUMEN
The therapeutic potential of interfering with dysregulated proteins by inducing its selective degradation has been pursued using different mechanisms. In the present article, we review representative examples of monovalent protein-degraders that, contrary to the proteolysis targeting chimeras, achieve target degradation without displaying recognition motifs for the recruitment of E3 ubiquitin ligases. We also highlight new technologies and assays that may brought to bear on the discovery of common elements that could predict and enable the selective degradation of pathogenic targets by monovalent protein-degraders. The successful application of these methods would pave the way to the advancement of new drugs with unique efficacy and tolerability properties.
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Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Técnicas Biosensibles , Cinamatos/farmacología , Descubrimiento de Drogas , Fulvestrant/farmacología , Humanos , Indoles/farmacología , Ligandos , Unión Proteica , Receptores de Estrógenos/metabolismo , Estilbenos/farmacología , Tamoxifeno/farmacologíaRESUMEN
Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 familyâ 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.
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Cardiomiopatías/prevención & control , Familia 1 del Citocromo P450/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Cardiomiopatías/inducido químicamente , Cardiomiopatías/patología , Familia 1 del Citocromo P450/antagonistas & inhibidores , Familia 1 del Citocromo P450/genética , Modelos Animales de Enfermedad , Doxorrubicina/toxicidad , Insuficiencia Cardíaca , Mutagénesis , Fenotipo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Dorsal root ganglion (DRG) are a key tissue in the nervous system that have a role in neurological disease, particularly pain. Despite the importance of this tissue, the proteome of DRG is poorly understood, and it is unknown whether the proteome varies between organisms or different DRG along the spine. Therefore, we profiled the proteome of human and rat DRG. We identified 5,245 proteins in human DRG and 4959 proteins in rat DRG. Across species the proteome is largely conserved with some notable differences. While the most abundant proteins in both rat and human DRG played a role in extracellular functions and myelin sheeth, proteins detected only in humans mapped to roles in immune function whereas those detected only in rat mapped to roles in localization and transport. The DRG proteome between human T11 and L2 vertebrae was nearly identical indicating DRG from different vertebrae are representative of one another. Finally, we asked if this data could be used to enhance translatability by identifying mechanisms that modulate cellular phenotypes representative of pain in different species. Based on our data we tested and discovered that MAP4K4 inhibitor treatment increased neurite outgrowth in rat DRG as in human SH-SY5Y cells.
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Ganglios Espinales/citología , Neuronas/metabolismo , Proteoma/química , Animales , Línea Celular Tumoral , Células Cultivadas , Humanos , Proteoma/metabolismo , Ratas , Especificidad de la EspecieRESUMEN
Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.
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Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pirimidinas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Dihidroorotato Deshidrogenasa , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ratones , Ratones SCID , Mutación , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirimidinas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Células Tumorales CultivadasRESUMEN
Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.
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Cardiotoxicidad/prevención & control , Familia 1 del Citocromo P450/antagonistas & inhibidores , Doxorrubicina/antagonistas & inhibidores , Corazón/efectos de los fármacos , Khellin/farmacología , Animales , Apoptosis , Cardiotoxicidad/patología , Línea Celular , Doxorrubicina/toxicidad , Khellin/administración & dosificación , Khellin/química , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Miocitos Cardíacos/efectos de los fármacos , Relación Estructura-Actividad , Xenobióticos , Pez CebraRESUMEN
Compound potency is a key metric that is often used to drive medicinal chemistry programs. Compound potency is also taken into account when identifying the mechanism of action of compounds whose pharmacological target is unknown, particularly when these compounds are identified in phenotypic screens. Often compound potency is determined from assays using recombinantly generated, purified protein. It is well understood in the medicinal chemistry community that potency measured with recombinant enzyme and potency measured in cell may not entirely coincide. Decreases in cellular vs recombinant potency are often anticipated or explainable. What is less often realized is that compound potency can increase in a cellular environment due to several factors including cellular metabolism of compounds, protein-protein interactions, post-translational modifications, and asymmetric intracellular localization of compound. Here we discuss these factors and highlight examples where increases in cellular compound potency were critical to the development of probes or drugs.
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Química Farmacéutica/métodos , Composición de Medicamentos , Equivalencia Terapéutica , Animales , Humanos , Preparaciones FarmacéuticasRESUMEN
Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition.
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Fosforilación Oxidativa/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tiadiazoles/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/químicaRESUMEN
Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models.
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Loss-of-function studies are valuable for elucidating kinase function and the validation of new drug targets. While genetic techniques, such as RNAi and genetic knockouts, are highly specific and easy to implement, in many cases post-translational perturbation of kinase activity, specifically pharmacological inhibition, is preferable. However, due to the high degree of structural similarity between kinase active sites and the large size of the kinome, identification of pharmacological agents that are sufficiently selective to probe the function of a specific kinase of interest is challenging, and there is currently no systematic method for accomplishing this goal. Here, we present a modular chemical genetic strategy that uses antibody mimetics as highly selective targeting components of bivalent kinase inhibitors. We demonstrate that it is possible to confer high kinase selectivity to a promiscuous ATP-competitive inhibitor by tethering it to an antibody mimetic fused to the self-labeling protein SNAPtag. With this approach, a potent bivalent inhibitor of the tyrosine kinase Abl was generated. Profiling in complex cell lysates, with competition-based quantitative chemical proteomics, revealed that this bivalent inhibitor possesses greatly enhanced selectivity for its target, BCR-Abl, in K562 cells. Importantly, we show that both components of the bivalent inhibitor can be assembled in K562 cells to block the ability of BCR-Abl to phosphorylate a direct cellular substrate. Finally, we demonstrate the generality of using antibody mimetics as components of bivalent inhibitors by generating a reagent that is selective for the activated state of the serine/threonine kinase ERK2.
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Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Sitios de Unión , Activación Enzimática/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Concentración 50 Inhibidora , Células K562 , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Modelos Moleculares , Estructura Molecular , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , ProteómicaRESUMEN
Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.
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Autofagia/fisiología , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Ferritinas/metabolismo , Homeostasis/fisiología , Hierro/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Animales , Autofagia/efectos de los fármacos , Células Cultivadas , Humanos , Lisosomas/metabolismo , Ratones , Fagosomas/metabolismo , Unión ProteicaRESUMEN
Quantitative chemoproteomics has recently emerged as an experimental approach to determine protein interaction profiles of small molecules in a given cell line or tissue. In contrast to standard biochemical and biophysical kinase assays, application of this method to kinase inhibitors determines compound binding to endogenously expressed kinases under conditions approximating the physiological situation with regard to the molecular state of the kinase and presence of required cofactors and regulatory proteins. Using a dose-dependent, competition-based experimental design in combination with quantitative mass spectrometry approaches, such as the use of tandem mass tags (TMT) for isobaric labeling described here, allows to rank-order interactions of inhibitors to kinase by binding affinity.
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Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/química , Fosfotransferasas/metabolismo , Proteómica/métodos , Unión Competitiva , Línea Celular Tumoral , Cromatografía de Afinidad , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Células K562 , Espectrometría de Masas , Péptidos/metabolismo , Fosfotransferasas/antagonistas & inhibidores , Unión Proteica/efectos de los fármacos , Coloración y EtiquetadoRESUMEN
Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.
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Productos Biológicos/farmacología , Colestenonas/farmacología , Neoplasias/metabolismo , Fenazinas/farmacología , Receptores de Esteroides/metabolismo , Saponinas/farmacología , Compuestos de Espiro/farmacología , Esteroides/farmacología , Productos Biológicos/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colestenonas/antagonistas & inhibidores , Humanos , Hidroxicolesteroles/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Fenazinas/antagonistas & inhibidores , Receptores de Esteroides/genética , Saponinas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Esfingomielinas/biosíntesis , Compuestos de Espiro/antagonistas & inhibidores , Esteroides/antagonistas & inhibidores , Estilbenos/antagonistas & inhibidores , Estilbenos/farmacologíaRESUMEN
TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, with 2 different fluorophores [5(6) carboxytetramethylrhodamine and Alexa Fluor 488] and monitored their intracellular localization in human plasmacytoid dendritic cells (pDCs). All fluorescent compounds induced the production of IFN-α, TNF-α, and IL-6 and the up-regulation of CD80 and CD86 by pDCs showing they retained TLR7-stimulating activity. Confocal imaging of pDCs showed that, similar to CpG-B, all compounds concentrated in the MHC class II loading compartment (MIIC), identified as lysosome-associated membrane protein 1(+), CD63, and HLA-DR(+) endosomes. Treatment of pDCs with bafilomycin A, an antagonist of the vacuolar-type proton ATPase controlling endosomal acidification, prevented the accumulation of small molecule TLR7 agonists, but not of CpG-B, in the MIIC. These results indicate that a pH-driven concentration of small molecule TLR7 agonists in the MIIC is required for pDC activation.
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Adenina/análogos & derivados , Aminoquinolinas/farmacocinética , Células Dendríticas/metabolismo , Colorantes Fluorescentes , Genes MHC Clase II/fisiología , Imidazoles/farmacocinética , Receptor Toll-Like 7/agonistas , Adenina/farmacocinética , Antineoplásicos/farmacocinética , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Imiquimod , Macrólidos/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Quinolinas/química , Quinolinas/farmacocinética , Receptor Toll-Like 7/metabolismoRESUMEN
The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.