RESUMEN
Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins, we developed and validated a structural model of human PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis, as well as the inhibition of its enzymatic activity, and can facilitate the development of improved inhibitors and the understanding of disease-relevant PORCN mutants. This article has an associated First Person interview with the joint first authors of the paper.
Asunto(s)
Hipoplasia Dérmica Focal , Preparaciones Farmacéuticas , Aciltransferasas/genética , Animales , Dominio Catalítico , Humanos , Proteínas de la Membrana/genética , Modelos EstructuralesRESUMEN
AXL is a member of the TAM (TYRO3, AXL, MER) subfamily of receptor tyrosine kinases. It is upregulated in a variety of cancers and its overexpression is associated with poor disease prognosis and acquired drug resistance. Utilizing a fragment-based lead discovery approach, a new indazole-based AXL inhibitor was obtained. The indazole fragment hit 11, identified through a high concentration biochemical screen, was expeditiously improved to fragment 24 by screening our in-house expanded library of fragments (ELF) collection. Subsequent fragment optimization guided by docking studies provided potent inhibitor 54 with moderate exposure levels in mice. X-ray crystal structure of analog 50 complexed with the I650M mutated kinase domain of Mer revealed the key binding interactions for the scaffold. The good potency coupled with reasonable kinase selectivity, moderate in vivo exposure levels, and availability of structural information for the series makes it a suitable starting point for further optimization efforts.
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Descubrimiento de Drogas , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/síntesis química , Indazoles/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Tirosina Quinasa del Receptor AxlRESUMEN
Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells.
Asunto(s)
Diseño de Fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Lisina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Lisina/síntesis química , Lisina/química , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Traditionally small molecules have mainly been used to inhibit biochemical activities of proteins, however such compounds can also be used to change the conformational energy landscape of proteins. Tool compounds that modulate protein conformations often reveal unexpected biological mechanisms, which have therapeutic potential. We discuss two examples where screening hits were found to bind to unexpected binding pockets on well known proteins, establishing new routes for the inhibition of proteins that were thought to be undruggable.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Conformación Proteica , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Proteínas/antagonistas & inhibidores , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/químicaRESUMEN
The oncoprotein YAP (Yes-associated protein) requires the TEAD family of transcription factors for the up-regulation of genes important for cell proliferation. Disrupting YAP-TEAD interaction is an attractive strategy for cancer therapy. Targeting TEADs using small molecules that either bind to the YAP-binding pocket or the palmitate-binding pocket is proposed to disrupt the YAP-TEAD interaction. There is a need for methodologies to facilitate robust and reliable identification of compounds that occupy either YAP-binding pocket or palmitate-binding pocket. Here, using NMR spectroscopy, we validated compounds that bind to these pockets and also identify the residues in mouse TEAD4 (mTEAD4) that interact with these compounds. Flufenamic acid (FA) was used as a positive control for validation of palmitate-binding pocket-occupying compounds by NMR. Furthermore, we identify a hit from a fragment screen and show that it occupies a site close to YAP-binding pocket on the TEAD surface. Our results also indicate that purified mTEAD4 can catalyze autopalmitoylation. NMR studies on mTEAD4 revealed that exchanges exist in TEAD as NMR signal broadening was observed for residues close to the palmitoylation site. Mutating the palmitoylated cysteine (C360S mutant) abolished palmitoylation, while no significant changes in the NMR spectrum were observed for the mutant which still binds to YAP. We also show that FA inhibits TEAD autopalmitoylation. Our studies highlight the utility of NMR spectroscopy in identifying small molecules that bind to TEAD pockets and reinforce the notion that both palmitate-binding pocket and YAP-binding pocket are targetable.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Unión al ADN/química , Proteínas Musculares/química , Fosfoproteínas/química , Factores de Transcripción/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ácido Flufenámico/química , Lipoilación , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of ß1-α2 and residues in the loop of ß3-ß4 might improve the inhibitor potency.
Asunto(s)
Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Inhibidores de Topoisomerasa/química , Humanos , Indoles/química , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 µM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.
Asunto(s)
Enterovirus Humano A/enzimología , Peptidomiméticos/farmacología , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Proteínas Virales/antagonistas & inhibidores , Proteasas Virales 3C , Antivirales/química , Antivirales/farmacología , Técnicas de Química Sintética , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Concentración 50 Inhibidora , Isoxazoles/química , Isoxazoles/farmacología , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Fenilalanina/análogos & derivados , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Pirrolidinonas/química , Pirrolidinonas/farmacología , Valina/análogos & derivados , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G(+) strain (Streptococcus pneumoniae (sParE)) and a G(-) strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between ß2 and α3, and the ß2 and ß6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.
Asunto(s)
Topoisomerasa de ADN IV/química , Secuencia de Aminoácidos , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Pseudomonas aeruginosa , Soluciones , Streptococcus pneumoniae , Resonancia por Plasmón de Superficie , TemperaturaRESUMEN
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.
Asunto(s)
Adenosina Trifosfato/metabolismo , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/farmacología , Escherichia coli/enzimología , Adenosina Trifosfato/antagonistas & inhibidores , Secuencia de Aminoácidos , Antibacterianos/farmacología , Unión Competitiva , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/química , Diseño de Fármacos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Over the past decade, academic groups and pharmaceutical companies have uncovered several components and targets for intervention in the Wnt pathway. One approach is to block Wnt signalling through the use of orally bioavailable small molecules that prevent Wnt ligand secretion. In recent years, the membrane bound O-acyl transferase (MBOAT) porcupine (PORCN) has emerged as a molecular target of interest in the search for clinical options to treat Wnt-driven cancers. This review shall provide an overview of the reported small molecule inhibitors for PORCN and discuss the progress made in identifying human disease models that are responsive to PORCN inhibitors.
Asunto(s)
Benzotiazoles/química , Proteínas de la Membrana/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Vía de Señalización Wnt , Aciltransferasas , Animales , Benzotiazoles/farmacología , Células HEK293 , Humanos , Estructura MolecularRESUMEN
Porcupine is a component of the Wnt pathway which regulates cell proliferation, migration, stem cell self-renewal, and differentiation. The Wnt pathway has been shown to be dysregulated in a variety of cancers. Porcupine is a membrane bound O-acyltransferase that palmitoylates Wnt. Inhibiting porcupine blocks the secretion of Wnt and effectively inhibits the Wnt pathway. Using high throughput screening, we have identified a number of novel porcupine inhibitors with diverse scaffolds. The pharmacophore requirements for our porcupine inhibitors were elucidated, and a pharmacophore model is proposed. Our compounds as well as all currently published porcupine inhibitors may be fitted to this model in low energy conformations with good superimposition of the pharmacophore elements. The model also explains the stereochemical requirements of our chiral porcupine inhibitors. The pharmacophore model was successfully used for designing 3 new series of porcupine inhibitors having a tricyclic xantine, a phtalimide, or a piperidine-maleimide scaffold.
Asunto(s)
Diseño de Fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Modelos Moleculares , Proteínas Wnt/antagonistas & inhibidores , Aciltransferasas , Células HEK293 , Humanos , Conformación Molecular , Estereoisomerismo , Especificidad por SustratoRESUMEN
The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.
Asunto(s)
Virus del Dengue/enzimología , Complejos Multienzimáticos/química , Proteínas no Estructurales Virales/química , Cristalografía por Rayos X , Virus del Dengue/genética , Humanos , Espectroscopía de Resonancia Magnética , Complejos Multienzimáticos/genética , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , ARN Helicasas/química , ARN Helicasas/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Wnts are lipid-modified glycoproteins that play key roles in both embryonic development and adult homeostasis. Wnt signaling is dysregulated in many cancers and preclinical data shows that targeting Wnt biosynthesis and secretion can be effective in Wnt-addicted cancers. An integral membrane protein known as Wntless (WLS/Evi) is essential for Wnt secretion. However, WLS remains undrugged thus far. The cryo-EM structure of WLS in complex with WNT8A shows that WLS has a druggable G-protein coupled receptor (GPCR) domain. Using Active Learning/Glide, we performed an ultra-large scale virtual screening from Enamine's REAL 350/3 Lead-Like library containing nearly 500 million compounds. 68 hits were examined after on-demand synthesis in cell-based Wnt reporter and other functional assays. ETC-451 emerged as a potential first-in-class WLS inhibitor. ETC-451 blocked WLS-WNT3A interaction and decreased Wnt-addicted pancreatic cancer cell line proliferation. The current hit provides a starting chemical scaffold for further structure or ligand-based drug discovery targeting WLS.
RESUMEN
The one-dimensional model of Hann et al. (JChem Inf Comput Sci 41(3):856864) has been extended to include reverse binding and wrap-around interaction modes between the protein and ligand to explore the complete combinatorial matrix of molecular recognition. The cumulative distribution function of the MaxwellBoltzmann distribution has been used to calculate the probability of measuring the sensitivity of the interactions as the asymptotic limits of the distribution better describe the behavior of the interactions under experimental conditions. Based on our model, we hypothesized that molecules of lower complexity are preferred for target based screening campaigns, while augmenting such a library with moieties of moderate complexities maybe better suited for phenotypic screens. The validity of the hypothesis has been assessed via the analysis of the hit rate profiles for four ChemBL datasets for enzymatic and phenotypic screens.
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Técnicas Químicas Combinatorias , Diseño de Fármacos , Descubrimiento de Drogas , Preparaciones Farmacéuticas/análisis , Bibliotecas de Moléculas Pequeñas/química , Evaluación Preclínica de Medicamentos , Humanos , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.
Asunto(s)
Antivirales/química , Virus del Dengue/enzimología , Inhibidores Enzimáticos/química , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/química , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/química , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Dengue/tratamiento farmacológico , Dengue/enzimología , Dengue/genética , Virus del Dengue/genética , Inhibidores Enzimáticos/farmacología , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , S-Adenosilmetionina/farmacología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/metabolismo , Dengue/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Viremia/tratamiento farmacológico , Adenosina/química , Animales , Antivirales/farmacocinética , Antivirales/uso terapéutico , Chlorocebus aethiops , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Masculino , Ratones , Estructura Molecular , Nivel sin Efectos Adversos Observados , Ratas , Células VeroRESUMEN
During the synthesis of the new antimalarial drug candidate NITD609, a high degree of diastereoselectivity was observed in the Pictet-Spengler reaction. By isolating both the 4E and 4Z imine intermediates, a systematic mechanistic study of the reaction under both kinetic and thermodynamic conditions was conducted. This study provides insight into the source of the diastereoselectivity for this important class of compounds.
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Antimaláricos/química , Indoles/química , Compuestos de Espiro/química , Antimaláricos/síntesis química , Descubrimiento de Drogas , Indoles/síntesis química , Cinética , Estructura Molecular , Compuestos de Espiro/síntesis química , EstereoisomerismoRESUMEN
Human eyes absent (EYA) proteins possess Tyr phosphatase activity, which is critical for numerous cancer and metastasis promoting activities, making it an attractive target for cancer therapy. In this work, we demonstrate that the inhibitor-bound form of EYA2 does not favour binding to Mg2+ , which is indispensable for the Tyr phosphatase activity. We further describe characterization and optimization of this class of allosteric inhibitors. A series of analogues were synthesized to improve potency of the inhibitors and to elucidate structure-activity relationships. Two co-crystal structures confirm the binding modes of this class of inhibitors. Our medicinal chemical, structural, biochemical, and biophysical studies provide insight into the molecular interactions of EYA2 with these allosteric inhibitors. The compounds derived from this study are useful for exploring the function of the Tyr phosphatase activity of EYA2 in normal and cancerous cells and serve as reference compounds for screening or developing allosteric phosphatase inhibitors. Finally, the co-crystal structures reported in this study will aid in structure-based drug discovery against EYA2.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares , Proteínas Tirosina Fosfatasas , Inhibidores Enzimáticos/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Relación Estructura-ActividadRESUMEN
We describe a novel translation inhibitor that has anti-dengue virus (DENV) activity in vitro and in vivo. The inhibitor was identified through a high-throughput screening using a DENV infection assay. The compound contains a benzomorphan core structure. Mode-of-action analysis indicated that the compound inhibits protein translation in a viral RNA sequence-independent manner. Analysis of the stereochemistry demonstrated that only one enantiomer of the racemic compound inhibits viral RNA translation. Medicinal chemistry was performed to eliminate a metabolically labile glucuronidation site of the compound to improve its in vivo stability. Pharmacokinetic analysis showed that upon a single subcutaneous dosing of 25 mg/kg of body weight in mice, plasma levels of the compound reached a C(max) (maximum plasma drug concentration) above the protein-binding-adjusted 90% effective concentration (EC(90)) value of 0.96 µM. In agreement with the in vivo pharmacokinetic results, treatment of DENV-infected mice with 25 mg/kg of compound once per day reduced peak viremia by about 40-fold. However, mice treated with 75 mg/kg of compound per day exhibited adverse effects. Collectively, our results demonstrate that the benzomorphan compounds inhibit DENV through suppression of RNA translation. The therapeutic window of the current compounds needs to be improved for further development.
Asunto(s)
Antivirales/farmacología , Antivirales/farmacocinética , Virus del Dengue/efectos de los fármacos , Animales , Antivirales/efectos adversos , Antivirales/química , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cromatografía Líquida de Alta Presión , Cricetinae , Virus del Dengue/genética , Femenino , Humanos , Ratones , Estructura Molecular , ARN Viral/genética , Ratas , Células VeroRESUMEN
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.