RESUMEN
The post-translational modification (PTM) ADP-ribosylation plays an important role in cell signalling and regulating protein function and has been implicated in the development of multiple diseases, including breast and ovarian cancers. Studying the underlying mechanisms through which this PTM contributes towards disease development, however, has been hampered by the lack of appropriate tools for reliable identification of physiologically relevant ADP-ribosylated proteins in a live-cell environment. Herein, we explore the application of an alkyne-tagged proprobe, 6Yn-ProTide-Ad (6Yn-Pro) as a chemical tool for the identification of intracellular ADP-ribosylated proteins through metabolic labelling. We applied targeted metabolomics and chemical proteomics in HEK293T cells treated with 6Yn-Pro to demonstrate intracellular metabolic conversion of the probe into ADP-ribosylation cofactor 6Yn-NAD+, and subsequent labelling and enrichment of PARP1 and multiple known ADP-ribosylated proteins in cells under hydrogen peroxide-induced stress. We anticipate that the approach and methodology described here will be useful for future identification of novel intracellular ADP-ribosylated proteins.
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Ubiquitin-specific protease 30 (USP30) is a deubiquitinating enzyme (DUB) localized at the mitochondrial outer membrane and involved in PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKß-USP30-ACLY-regulated lipogenesis/tumorigenesis. A USP30 inhibitor, MTX652, has recently entered clinical trials as a potential treatment for mitochondrial dysfunction. Small molecule activity-based probes (ABPs) for DUBs have recently emerged as powerful tools for in-cell inhibitor screening and DUB activity analysis, and here, we report the first small molecule ABPs (IMP-2587 and IMP-2586) which can profile USP30 activity in cells. Target engagement studies demonstrate that IMP-2587 and IMP-2586 engage active USP30 at nanomolar concentration after only 10 min incubation time in intact cells, dependent on the presence of the USP30 catalytic cysteine. Interestingly, proteomics analyses revealed that DESI1 and DESI2, small ubiquitin-related modifier (SUMO) proteases, can also be engaged by these probes, further suggesting a novel approach to develop DESI ABPs.
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Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest; however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report the structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1α binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or the androgen receptor without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design, expanding the substrate scope of covalent E3 ligase PROTACs.
Asunto(s)
Proteínas Nucleares , Ácidos Sulfínicos , Factores de Transcripción , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteolisis , LigandosRESUMEN
Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35â DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.
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Bioensayo , Complejo de la Endopetidasa Proteasomal , Citoplasma , Ubiquitina , Enzimas DesubicuitinizantesRESUMEN
Light-activable spatiotemporal control of PROTAC-induced protein degradation was achieved with novel arylazopyrazole photoswitchable PROTACs (AP-PROTACs). The use of a promiscuous kinase inhibitor in the design enables this unique photoswitchable PROTAC to selectively degrade four protein kinases together with on/off optical control using different wavelengths of light.
Asunto(s)
Luz , Ubiquitina-Proteína Ligasas , Proteínas Quinasas/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Pirazoles/química , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Despite much progress in developing better drugs, many patients with acute myeloid leukemia (AML) still die within a year of diagnosis. This is partly because it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Overcoming this block should allow for the identification of therapies that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify compounds that stimulate differentiation in genetically diverse AML cell lines. Lead compounds were shown to decrease tumor burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a function for tubulin disruptors in causing differentiation of AML cells.
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A therapeutic approach that holds the potential to treat all Duchenne muscular dystrophy (DMD) patient populations is utrophin modulation. Ezutromid, a first generation utrophin modulator which was later found to act via antagonism of the arylhydrocarbon receptor, progressed to Phase 2 clinical trials. Although interim data showed target engagement and functional improvements, ezutromid ultimately failed to meet its clinical endpoints. We recently described the identification of a new class of hydrazide utrophin modulators which has a different mechanism of action to ezutromid. In this study we report our early optimisation studies on this hydrazide series. The new analogues had significantly improved potency in cell-based assays, increased sp3 character and reduced lipophilicity, which also improved their physicochemical properties. A representative new analogue combining these attributes increased utrophin protein in dystrophic mouse cells showing it can be used as a chemical tool to reveal new insights regarding utrophin upregulation as a strategy for DMD therapeutic intervention.
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Distrofia Muscular de Duchenne , Animales , Hidrazinas/farmacología , Hidrazinas/uso terapéutico , Ratones , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Relación Estructura-Actividad , Regulación hacia Arriba , Utrofina/genética , Utrofina/metabolismo , Utrofina/uso terapéuticoRESUMEN
C-type natriuretic peptide (CNP) is involved in the regulation of vascular homeostasis, which is at least partly mediated through agonism of natriuretic peptide receptor C (NPR-C), and loss of this signaling has been associated with vascular dysfunction. As such, NPR-C is a novel therapeutic target to treat cardiovascular diseases. A series of novel small molecules have been designed and synthesized, and their structure-activity relationships were evaluated by a surface plasmon resonance binding assay. The biological activity of hit compounds was confirmed through organ bath assays measuring vascular relaxation and inhibition of cAMP production, which was shown to be linked to its NPR-C activity. Lead compound 1 was identified as a potent agonist (EC50 â¼ 1 µM) with promising in vivo pharmacokinetic properties.
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Péptido Natriurético Tipo-C , Transducción de Señal , Homeostasis , Péptido Natriurético Tipo-C/metabolismo , Péptido Natriurético Tipo-C/farmacologíaRESUMEN
Drug resistant tuberculsosis (TB) is global health crisis that demands novel treatment strategies. Bacterial ATP synthase inhibitors such as bedaquiline and next-generation analogues (such as TBAJ-876) have shown promising efficacy in patient populations and preclinical studies, respectively, suggesting that selective targeting of this enzyme presents a validated therapeutic strategy for the treatment of TB. In this work, we report tetrahydronaphthalene amides (THNAs) as a new class of ATP synthase inhibitors that are effective in preventing the growth of Mycobacterium tuberculosis (M.tb) in culture. Design, synthesis and comprehensive structure-activity relationship studies for approximately 80 THNA analogues are described, with a small selection of compounds exhibiting potent (in some cases MIC90 <1 µg/mL) in vitro M.tb growth inhibition taken forward to pharmacokinetic and off-target profiling studies. Ultimately, we show that some of these THNAs possess reduced lipophilic properties, decreased hERG liability, faster mouse/human liver microsomal clearance rates and shorter plasma half-lives compared with bedaquiline, potentially addressing of the main concerns of persistence and phospholipidosis associated with bedaquiline.
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Amidas/química , Antituberculosos/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Tetrahidronaftalenos/síntesis química , Animales , Antituberculosos/efectos adversos , Antituberculosos/farmacocinética , Diarilquinolinas/farmacología , Diarilquinolinas/normas , Descubrimiento de Drogas , Humanos , Hígado , Ratones , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Tetrahidronaftalenos/efectos adversos , Tetrahidronaftalenos/farmacocinéticaRESUMEN
Breast cancer has the highest incidence and death rate among cancers in women worldwide. In particular, metastatic estrogen receptor negative (ER-) breast cancer and triple-negative breast cancer (TNBC) subtypes have very limited treatment options, with low survival rates. Ubiquitin carboxyl terminal hydrolase L1 (UCHL1), a ubiquitin C-terminal hydrolase belonging to the deubiquitinase (DUB) family of enzymes, is highly expressed in these cancer types, and several key reports have revealed emerging and important roles for UCHL1 in breast cancer. However, selective and potent small-molecule UCHL1 inhibitors have been disclosed only very recently, alongside chemical biology approaches to detect regulated UHCL1 activity in cancer cells. These tools will enable novel insights into oncogenic mechanisms driven by UCHL1, and identification of substrate proteins deubiquitinated by UCHL1, with the ultimate goal of realising the potential of UCHL1 as a drug target in breast cancer.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Terapia Molecular Dirigida/métodos , Neoplasias de la Mama Triple Negativas/patología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Duchenne muscular dystrophy is a fatal disease with no cure, caused by lack of the cytoskeletal protein dystrophin. Upregulation of utrophin, a dystrophin paralogue, offers a potential therapy independent of mutation type. The failure of first-in-class utrophin modulator ezutromid/SMT C1100 in Phase II clinical trials necessitates development of compounds with better efficacy, physicochemical and ADME properties and/or complementary mechanisms. We have discovered and performed a preliminary optimisation of a novel class of utrophin modulators using an improved phenotypic screen, where reporter expression is derived from the full genomic context of the utrophin promoter. We further demonstrate through target deconvolution studies, including expression analysis and chemical proteomics, that this compound series operates via a novel mechanism of action, distinct from that of ezutromid.
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Descubrimiento de Drogas , Hidrazinas/farmacología , Distrofia Muscular de Duchenne/tratamiento farmacológico , Pirimidinas/farmacología , Utrofina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Hidrazinas/síntesis química , Hidrazinas/química , Estructura Molecular , Distrofia Muscular de Duchenne/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , ARN Mensajero/metabolismo , Relación Estructura-ActividadRESUMEN
Protein methylation is a key post-translational modification whose effects on gene expression have been intensively studied over the last two decades. Recently, renewed interest in non-histone protein methylation has gained momentum for its role in regulating important cellular processes and the activity of many proteins, including transcription factors, enzymes, and structural complexes. The extensive and dynamic role that protein methylation plays within the cell also highlights its potential for bioengineering applications. Indeed, while synthetic histone protein methylation has been extensively used to engineer gene expression, engineering of non-histone protein methylation has not been fully explored yet. Here, we report the latest findings, highlighting how non-histone protein methylation is fundamental for certain cellular functions and is implicated in disease, and review recent efforts in the engineering of protein methylation.
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Proteínas/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Bioingeniería , Ciclo Celular/fisiología , Humanos , Metilación , Mitocondrias/metabolismo , Neoplasias/fisiopatología , Procesamiento Proteico-PostraduccionalRESUMEN
DNA-encoded combinatorial libraries (DECLs) represent an exciting new technology for high-throughput screening, significantly increasing its capacity and cost-effectiveness. Historically, DECLs have been the domain of specialized academic groups and industry; however, there has recently been a shift toward more drug discovery academic centers and institutes adopting this technology. Key to this development has been the simplification, characterization and standardization of various DECL subprotocols, such as library design, affinity screening and data analysis of hits. This review examines the feasibility of implementing DECL screening technology as a first-time user, particularly in academia, exploring the some important considerations for this, and outlines some applications of the technology that academia could contribute to the field.
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ADN/química , Bibliotecas de Moléculas Pequeñas/química , Avidina/química , Biotina/química , Técnicas Químicas Combinatorias , Reactivos de Enlaces Cruzados/química , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Microfluídica , Fotólisis , Relación Estructura-ActividadRESUMEN
A series of 5,8-disubstituted tetrahydroisoquinolines were shown to be effective inhibitors of M. tb in culture and modest inhibitors of M. tb ATP synthase. There was a broad general trend of improved potency with higher lipophilicity. Large substituents (e.g., Bn) at the tetrahydroquinoline 5-position were well-tolerated, while N-methylpiperazine was the preferred 8-substituent. Structure-activity relationships for 7-linked side chains showed that the nature of the 7-linking group was important; -CO- and -COCH2- linkers were less effective than -CH2- or -CONH- ones. This suggests that the positioning of a terminal aromatic ring is important for target binding. Selected compounds showed much faster rates of microsomal clearance than did the clinical ATP synthase inhibitor bedaquiline, and modest inhibition of mycobacterial ATP synthase.
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Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tetrahidroisoquinolinas/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Tetrahidroisoquinolinas/síntesis química , Tetrahidroisoquinolinas/químicaRESUMEN
Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.
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Neoplasias de la Mama/terapia , Isotiocianatos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anticarcinógenos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/metabolismo , Transducción de Señal/genética , Sulfóxidos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Bedaquiline is a novel drug approved in 2012 by the FDA for treatment of drug-resistant tuberculosis (TB). Although it shows high efficacy towards drug-resistant forms of TB, its use has been limited by the potential for significant side effects. In particular, bedaquiline is a very lipophilic compound with an associated long terminal half-life and shows potent inhibition of the cardiac potassium hERG channel, resulting in QTc interval prolongation in humans that may result in cardiac arrhythmia. To address these issues, we carried out a drug discovery programme to develop an improved second generation analogue of bedaquiline. From this medicinal chemistry program, a candidate (TBAJ-876) has been selected to undergo further preclinical evaluation. During this evaluation, three major metabolites arising from TBAJ-876 were observed in several preclinical animal models. We report here our synthetic efforts to unequivocally structurally characterize these three metabolites through their independent directed synthesis.
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Diarilquinolinas/química , Diarilquinolinas/farmacología , Animales , Antituberculosos/química , Antituberculosos/farmacología , Fenómenos Químicos , Técnicas de Química Sintética , Diarilquinolinas/síntesis química , Desarrollo de Medicamentos , Humanos , Estructura Molecular , Análisis EspectralRESUMEN
Neuropilin-1 (NRP1) is emerging as an important molecule in immune signaling where it has been shown to modulate the actions of TGF-ß1 in macrophages and regulatory T cells. The development of cost-effective and reliable assays for NRP1 binding is therefore important. We synthesized three new NRP1 small molecule fluorophores and examined their performance as fluorescent polarization probes. One molecule DS108 exhibited favorable binding and fluorescent characteristics and allowed us to establish a simple assay suitable for medium to high throughput screening of small molecules.
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Colorantes Fluorescentes/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Neuropilina-1/metabolismo , Colorantes Fluorescentes/síntesis química , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Bedaquiline is a new drug of the diarylquinoline class that has proven to be clinically effective against drug-resistant tuberculosis, but has a cardiac liability (prolongation of the QT interval) due to its potent inhibition of the cardiac potassium channel protein hERG. Bedaquiline is highly lipophilic and has an extremely long terminal half-life, so has the potential for more-than-desired accumulation in tissues during the relatively long treatment durations required to cure TB. The present work is part of a program that seeks to identify a diarylquinoline that is as potent as bedaquiline against Mycobacterium tuberculosis, with lower lipophilicity, higher clearance, and lower risk for QT prolongation. Previous work led to the identification of compounds with greatly-reduced lipophilicity compounds that retain good anti-tubercular activity in vitro and in mouse models of TB, but has not addressed the hERG blockade. We now present compounds where the C-unit naphthalene is replaced by a 3,5-dialkoxy-4-pyridyl, demonstrate more potent in vitro and in vivo anti-tubercular activity, with greatly attenuated hERG blockade. Two examples of this series are in preclinical development.
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Antituberculosos/farmacología , Diarilquinolinas/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Piridinas/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Diarilquinolinas/síntesis química , Diarilquinolinas/química , Relación Dosis-Respuesta a Droga , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
The ATP-synthase inhibitor bedaquiline is effective against drug-resistant tuberculosis but is extremely lipophilic (clogP 7.25) with a very long plasma half-life. Additionally, inhibition of potassium current through the cardiac hERG channel by bedaquiline, is associated with prolongation of the QT interval, necessitating cardiovascular monitoring. Analogues were prepared where the naphthalene C-unit was replaced with substituted pyridines to produce compounds with reduced lipophilicity, anticipating a reduction in half-life. While there was a direct correlation between in vitro inhibitory activity against M. tuberculosis (MIC90) and compound lipophilicity, potency only fell off sharply below a clogP of about 4.0, providing a useful lower bound for analogue design. The bulk of the compounds remained potent inhibitors of the hERG potassium channel, with notable exceptions where IC50 values were at least 5-fold higher than that of bedaquiline. Many of the compounds had desirably higher rates of clearance than bedaquiline, but this was associated with lower plasma exposures in mice, and similar or higher MICs resulted in lower AUC/MIC ratios than bedaquiline for most compounds. The two compounds with lower potency against hERG exhibited similar clearance to bedaquiline and excellent efficacy in vivo, suggesting further exploration of C-ring pyridyls is worthwhile.
Asunto(s)
Antituberculosos/farmacología , Diarilquinolinas/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Piridinas/farmacología , Animales , Antituberculosos/síntesis química , Antituberculosos/química , Diarilquinolinas/síntesis química , Diarilquinolinas/química , Relación Dosis-Respuesta a Droga , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
Endothelium-derived C-type natriuretic peptide possesses cytoprotective and anti-atherogenic functions that regulate vascular homeostasis. The vasoprotective effects of C-type natriuretic peptide are somewhat mediated by the natriuretic peptide receptor C, suggesting that this receptor represents a novel therapeutic target for the treatment of cardiovascular diseases. In order to facilitate our drug discovery efforts, we have optimized an array of biophysical methods including surface plasmon resonance, fluorescence polarization and thermal shift assays to aid in the design, assessment and characterization of small molecule agonist interactions with natriuretic peptide receptors. Assay conditions are investigated to explore the feasibility and dynamic range of each method, and peptide-based agonists and antagonists are used as controls to validate these conditions. Once established, each technique was compared and contrasted with respect to their drug discovery utility. We foresee that such techniques will facilitate the discovery and development of potential therapeutic agents for NPR-C and other large extracellular domain membrane receptors.