RESUMEN
We have previously characterised the histone lysine methyltransferase properties of PRDM9, a member of the PRDM family of putative transcriptional regulators. PRDM9 displays broad substrate recognition and methylates a range of histone substrates, including octamers, core histone proteins, and peptides. In the present study, we show that PRDM9 performs intramolecular automethylation on multiple lysine residues localised to a lysine-rich region on the post-SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain. PRDM9 automethylation is abolished by a single active-site mutation, C321P, also known to disrupt interactions with S-adenosylmethionine. We have taken an initial step towards tool compound generation through rational design of a substrate-mimic, peptidic inhibitor of PRDM9 automethylation. The discovery of automethylation in PRDM9 adds a new dimension to our understanding of PRDM9 enzymology.
Asunto(s)
Cisteína/química , N-Metiltransferasa de Histona-Lisina/química , Prolina/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Dominio Catalítico , Clonación Molecular , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Cinética , Ligandos , Metilación , Ratones , Modelos Moleculares , Mutación , Prolina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The mosquito-borne West Nile virus (WNV) causes a wide range of symptoms ranging from fever to the often fatal viral encephalitis. To date, no vaccine or drug therapy is available. The trypsin-like WNV NS2B-NS3 protease is deemed a plausible drug target and was shown to be inhibited by bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein isolated from bovine lung. Herein, we report a protein truncation study that resulted in a novel 14-residue cyclic peptide with equipotent inhibitory activity to native BPTI. We believe our truncation strategy can be further applied in the development of peptide-based inhibitors targeting trypsin-like proteases.
Asunto(s)
Inhibidores de Proteasas/farmacología , Inhibidores de Tripsina/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virus del Nilo Occidental/enzimología , Animales , Bovinos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Tripsina/metabolismo , Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/química , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/efectos de los fármacosRESUMEN
PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Cisteína/química , Cisteína/genética , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Histonas/genética , Humanos , Cinética , Mediciones Luminiscentes , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Prolina/química , Prolina/genética , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Xenopus laevisRESUMEN
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
Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Humanos , Modelos Moleculares , Fosforilación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-ActividadRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.
Asunto(s)
Crisis Blástica/tratamiento farmacológico , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Crisis Blástica/patología , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones SCID , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Técnicas de Química Sintética , Relación Dosis-Respuesta a Droga , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones SCID , Terapia Molecular Dirigida/métodos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The dengue virus is a mosquito-borne pathogen responsible for an estimated 50-100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor's ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 µM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 µM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay.
Asunto(s)
Antraquinonas/farmacología , Antivirales/farmacología , Virus del Dengue/enzimología , Inhibidores de Proteasas/farmacología , Carga Viral , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antraquinonas/toxicidad , Antivirales/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Pruebas de Sensibilidad Microbiana , Inhibidores de Proteasas/toxicidad , ARN Helicasas/antagonistas & inhibidores , Serina EndopeptidasasRESUMEN
Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/enzimología , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
The Murray Valley encephalitis virus (MVEV) and the West Nile virus (WNV) are mosquito-borne single-stranded RNA Flaviviruses responsible for many cases of viral encephalitis and deaths worldwide. The former is endemic in north Australia and Papua New Guinea while the latter has spread to different parts of the world and was responsible for a recent North American outbreak in 2012, resulting in 243 fatalities. There is currently no approved vaccines or drugs against MVEV and WNV viral infections. A plausible drug target is the viral non-structural NS2B/NS3 protease due to its role in viral replication. This trypsin-like serine protease recognizes and cleaves viral polyproteins at the C-terminal end of an arginine residue, opening an avenue for the development of peptide-based antivirals. This communication compares the P2 and P3 residue preferences of the MVEV and WNV NS2B/NS3 proteases using a series of C-terminal agmatine dipeptides. Our results revealed that both viral enzymes were highly specific toward lysines at the P2 and P3 positions, suggesting that a peptidomimetic viral protease inhibitor developed against one virus should also be active against the other.