RESUMEN
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
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
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.
Asunto(s)
Agmatina/química , Dipéptidos/química , Virus de la Encefalitis del Valle Murray/enzimología , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/química , Proteínas no Estructurales Virales , Virus del Nilo Occidental/enzimología , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/químicaRESUMEN
This communication describes the synthesis and inhibitory activities of thirty-seven novel C-terminal agmatine dipeptides used as screening compounds to study the structure-activity relationship between active-site peptidomimetics and the West Nile virus (WNV) NS2B/NS3 serine protease. Our efforts lead to the discovery of a novel agmatine dipeptide inhibitor (compound 33, IC50 2.6 ± 0.3 µM) with improved inhibitory activity in comparison to the most potent inhibitor described in our recent report [IC50 4.7 ± 1.2 µM; Lim et al., Eur. J. Med. Chem. 46 (2011) 3130-3134]. In addition, our study cleared the contention surrounding the previous X-ray co-crystallization study and an enzyme inhibition report on the binding conformation adopted by active-site peptide aldehydes. Our data should provide valuable insights into the design of future peptidomimetic antivirals against WNV infections.
Asunto(s)
Agmatina/farmacología , Dipéptidos/farmacología , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/efectos de los fármacos , Virus del Nilo Occidental/enzimología , Agmatina/síntesis química , Agmatina/química , Cristalografía por Rayos X , Dipéptidos/síntesis química , Dipéptidos/química , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , ARN Helicasas/efectos de los fármacos , Serina Endopeptidasas/efectos de los fármacos , Programas Informáticos , Relación Estructura-Actividad , Virus del Nilo Occidental/efectos de los fármacosRESUMEN
West Nile virus (WNV) NS2B-NS3 protease is an important drug target since it is an essential protein for the replication of the virus. In order to determine the minimum pharmacophore for protease inhibition, a series of dipeptide aldehydes were synthesized. The 50% inhibitory concentration (IC(50)) measurements revealed that a simple acetyl-KR-aldehyde was only threefold less active than 4-phenyl-phenylacetyl-KKR-aldehyde (1) (Stoermer et al., 2008) that was used as the reference compound. The ligand efficiency of 0.40 kcal/mol/HA (HA=heavy atom) for acetyl-KR-aldehyde is much improved compared to the reference compound 1 (0.23 kcal/mol/HA). The binding of the inhibitors was examined using (1)H-(15)N-HSQC experiments and differential chemical shifts were used to map the ligand binding sites. The biophysical studies show that the conformational mobility of WNV protease has a major impact on the design of novel inhibitors, since the protein conformation changes profoundly depending on the structure of the bound ligand.
Asunto(s)
Inhibidores de Proteasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Inhibidores de Proteasas/química , Unión Proteica , ARN Helicasas/química , ARN Helicasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/químicaRESUMEN
This communication reports the synthesis and inhibitory activities of novel non-covalent peptidomimetic inhibitors of the West Nile virus NS2B/NS3 protease containing a decarboxylated P1 arginine (agmatine; 4-aminobutylguanidine) and related analogues. One agmatine peptidomimetic (4-phenyl-phenacetyl-Lys-Lys-agmatine; compound 2) was shown to be a competitive inhibitor with a binding affinity of K(i) 2.05 ± 0.13 µM and was inactive against thrombin (IC(50) > 100 µM). Our results suggest that peptidomimetics with agmatine at the P1 position could potentially be employed as starting tools in the design of non-covalent competitive protease inhibitors due to their relative stability and ease of chemical synthesis compared to inhibitors containing reactive electrophilic warheads.