2-chloro-3-substituted-1,4-naphthoquinone inactivators of human cytomegalovirus protease.

A random screening approach has identified 2-chloro-3-substituted-1,4-naphthoquinones as potent inactivators of HCMV protease. Enzyme inactivation is due to modification of Cys202. Two of the most potent compounds maintain activity against HCMV in a plaque reduction assay.     

Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay.     

Synergistic Effect of 5-Nitro-2′-deoxyuridine with Ganciclovir Against Human Cytomegalovirus In Vitro

Abstract

In this paper we describe a practical synthesis of 5-nitro-2′-deoxyuridine (4) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-nitrouracil (11). These compounds were then evaluated for their ability to inhibit the growth of human cytomegalovirus (HCMV, strain AD169) in MRC-5 cells using a plaque reduction assay. Compound 11 was unable to inhibit the growth of HCMV at the highest concentration tested (100 μg/mL). However, compound 4 (5-NO₂-dU) exhibited marginal activity against HCMV in vitro in a dose-dependent manner with a 50% inhibitory concentrations (IC₅₀) of 1 to 5 μg/mL. Combinations of 5-NO₂-dU with ganciclovir synergistically inhibited HCMV induced cell killing in culture.     

Salicylamide inhibitors of influenza virus fusion

Structural variation of the quinolizidine heterocycle of the influenza fusion inhibitor BMY-27709 was examined by several topological dissections in order to illuminate the critical features of the ring system. This exercise resulted in the identification of a series of synthetically more accessible decahydroquinolines that retained the structural elements of BMY-27709 important for antiviral activity. The 2-methyl-cis-decahydroquinoline 6f was the most potent influenza inhibitor identified that demonstrated an EC50 of 90 ng/mL in a plaque reduction assay.     

Antiviral activity of berberine and related compounds against human cytomegalovirus

Berberine chloride (1) and the structurally related compounds were assessed for the anti-human cytomegalovirus (HCMV) activity using the plaque assay. The anti-HCMV activity (IC(50) 0.68 microM) of 1 was equivalent to that (IC(50) 0.91 microM) of ganciclovir (GCV). The mechanism of action by which 1 inhibits the replication of HCMV is presumed to be different from that of GCV; 1 would interfere with intracellular events after virus penetration into the host cells and before viral DNA synthesis.     

Synthesis of 4-substituted imidazo[4,5-d][1,2,3]triazine (2-azapurine)nucleosides

Several methods for functionalization of the 4-position of imidazo[4,5-d][1,2,3]triazin-4-one were investigated. These investigations were successful and led to the preparation of 4-amino, 4-triazol-1-yl, 4-methoxy, 4-methylthio, 4-methylamino, 4-thio, 4-nitrobenzyl, and 4-unsubstituted 9-(beta-D-ribofuranosyl)-imidazo-[4,5-d][1,2,3]triazine (2-azapurine ribosides). The 4-unsubstituted compound (19) was slightly active against HCMV in plaque and yield reduction experiments and was not cytotoxic at 100 microM. The methylamino (15), hydrazino (16), and p-nitrobenzylthio (20) were inactive against HCMV but slightly cytotoxic. The thiomethyl-substituted analog (21) was the most active with activity comparable to ganciclovir but with greater cytotoxicity. We conclude that even though none of the tested compounds had antiviral activity superior to ganciclovir, the new synthetic methods will provide a route to more interesting compounds.     

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.     

Human cytomegalovirus: demonstration of permissive epithelial cells and nonpermissive fibroblastic cells in a survey of human cell lines.

To more clearly define the characteristics which render a cell permissive for human cytomegalovirus (HCMV), we screened a panel of human cell lines differing in morphology, ploidy, and extent of differentiation for the ability to sustain productive HCMV replication. Cells were exposed to HCMV at 5 to 20 PFU per cell and examined at 4 to 14 days postinfection to detect the production of infectious virus by a plaque assay and the assembly of progeny virions by electron microscopy. By these criteria, high-titer HCMV replication (10(6) to 10(7) PFU/ml) occurred in a well-differentiated, diploid, epithelial cell line, HCMC, which had been derived from normal human colonic mucosa. In contrast, all aneuploid human cell types proved to be nonpermissive, including a fibroblastic cell line designated HT-144. These results indicate that HCMV replication in cultures is not strictly limited to fibroblasts and conversely that not all human fibroblastic cells are permissive for HCMV. Nonpermissive cell types were further investigated by attempts to chemically induce HCMV replication. Treatment of nonpermissive cell types with 25 to 500 micrograms of 5-iodo-2'-deoxyuridine per ml prior to infection did not convert them to the permissive state. The implications of these findings for the possible mechanisms maintaining the nonpermissive state are discussed.     

Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.     

A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction

Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan. It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P. aeruginosa infections. The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S. aureus cell suspensions. This assay, however, does not permit kinetic studies and its reproducibility is poor. Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase. Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader. Activity of nanogram amounts of LasA protease could be determined within a few minutes. Furthermore, this assay permitted the determination of Km and kcat values for LasA protease, which were 0.46 mM and 11.8s(-1), respectively. Pseudomonas elastase was also active in the assay. However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon. The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.     

大蒜中抗人巨细胞病毒成份的筛选

临床试用大蒜制剂预防和治疗人巨细胞病毒感染取得了明显效果。在此基础上我们应用细胞病变(CPE)抑制试验和空斑抑制试验,对大蒜中的抗人巨细胞病毒成份进行了初步筛选和比较,证实新鲜大蒜中存在多种抗该病毒的有效成份,其中至少包括大蒜新素(二丙烯三硫)和Ajoene类似物。     

Engineered external guide sequences effectively block viral gene expression and replication in cultured cells

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. We have previously used an in vitro selection procedure to generate EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the mRNA encoding the protease of human cytomegalovirus (HCMV), which is essential for viral capsid formation and replication. The EGS variant was about 35-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 95% in the expression of the protease and a reduction of 4,000-fold in viral growth were observed in HCMV-infected cells that expressed the EGS variant, whereas a reduction of 80% in the protease expression and an inhibition of 150-fold in viral growth were detected in cells that expressed the EGS derived from a natural tRNA sequence. No significant reduction in viral protease expression or viral growth was observed in cells that either did not express an EGS or produced a "disabled" EGS, which carried nucleotide mutations that precluded RNase P recognition. Our results provide direct evidence that engineered EGS variant is highly effective in blocking HCMV expression and growth by targeting the viral protease. Furthermore, these results demonstrate the utility of engineered EGS RNAs in gene targeting applications, including the inhibition of HCMV infection by blocking the expression of virus-encoded essential proteins.     

Synthesis of 4-Substituted Imidazo[4,5-d][1,2,3]triazine (2-Azapurine)nucleosides

Abstract

Several methods for functionalization of the 4-position of imidazo[4,5-d][1,2,3]triazin-4-one were investigated. These investigations were successful and led to the preparation of 4-amino, 4-triazol-1-yl, 4-methoxy, 4-methylthio, 4-methylamino, 4-thio, 4-nitrobenzyl, and 4-unsubstituted 9-(β-D-ribofuranosyl)-imidazo-[4,5-d][1,2,3]triazine (2-azapurine ribosides). The 4-unsubstituted compound (19) was slightly active against HCMV in plaque and yield reduction experiments and was not cytotoxic at 100 μM. The methylamino (15), hydrazino (16), and p-nitrobenzylthio (20) were inactive against HCMV but slightly cytotoxic. The thiomethyl-substituted analog (21) was the most active with activity comparable to ganciclovir but with greater cytotoxicity. We conclude that even though none of the tested compounds had antiviral activity superior to ganciclovir, the new synthetic methods will provide a route to more interesting compounds.     

Study on synthesis,characterization and biological activity of some new nitrogen heterocycle porphyrins

Seven new nitrogen heterocycle porphyrins, 5,10,15,20-tetra[4-(N-pyrrolidinyl)phenyl]porphine (TBPPH(2)), 5,10,15,20-tetra[4-(4'-ethylpiperazinyl)phenyl]porphine (TEPPH(2)), 5,10,15,20-tetra [4-(4'-butylpiperazinyl)phenyl]porphine (TUPPH(2)), 5,10,15,20-tetra[4-(4'-heptylpiperazinyl) phenyl]porphine (THPPH(2)), 5-[4-(4'-ethylpiperazinyl)phenyl]-10,15,20-triphenylporphine (MEPPH(2)), 5-[4-(4'-buthylpiperazinyl)phenyl]-10,15,20-triphenylporphine (MUPPH(2)) and piperazine bridge porphine dimer N,N'-di(5,10,15,20-tetraphenylporphinato)piperazine (DiPPH(2)) have been synthesized by the direct condensation of nitrogen heterocycle substituted benzaldehydes with pyrrole. Each porphine bears one or four substituted pyrrolidine or piperazine moieties that have been used as drugs. Their structures were characterized by elementary analysis, MS, 1H NMR, IR and UV-vis. These nitrogen heterocycle porphyrins aggregates in water and THF solution were studied by the spectrophotofluorimetry. The anticancer activity of these porphines for the liver cancer cells, the stomach tumor cells and the nasopharyngeal carcinoma cancer cells were tested by the MTT assay. Compared with cis-platinum (cis-Pt) and 5-Fluorouracil (5-Fu), the nitrogen heterocycle porphyrins have the better biological activity and might have potential application in medicine.     

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.     

Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease

Biochemical studies indicate that dimerization is required for the catalytic activity of herpesvirus proteases, whereas structural studies show a complete active site in each monomer, away from the dimer interface. Here we report kinetic, biophysical and crystallographic characterizations of structure-based mutants in the dimer interface of human cytomegalovirus (HCMV) protease. Such mutations can produce a 1,700-fold reduction in the kcat while having minimal effects on the K(m). Dimer stability is not affected by these mutations, suggesting that dimerization itself is insufficient for activity. There are large changes in monomer conformation and dimer organization of the apo S225Y mutant enzyme. However, binding of an activated peptidomimetic inhibitor induced a conformation remarkably similar to the wild type protease. Our studies suggest that appropriate dimer formation may be required to indirectly stabilize the protease oxyanion hole, revealing a novel mechanism for dimerization to regulate enzyme activity.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.     

Comparison of disc diffusion, Etest and broth microdilution for testing susceptibility of carbapenem-resistant P. aeruginosa to polymyxins

Background

New prophylactic and therapeutic tools are needed for the treatment of herpes simplex virus infections. Several essential oils have shown to possess antiviral activity in vitro against a wide spectrum of viruses.

Aim

The present study was assess to investigate the activities of the essential oil obtained from leaves of Artemisia arborescens against HSV-1 and HSV-2

Methods

The cytotoxicity in Vero cells was evaluated by the MTT reduction method. The IC₅₀ values were determined by plaque reduction assay. In order to characterize the mechanism of action, yield reduction assay, inhibition of plaque development assay, attachment assay, penetration assay and post-attachment virus neutralization assay were also performed.

Results

The IC₅₀ values, determined by plaque reduction assay, were 2.4 and 4.1 μg/ml for HSV-1 and HSV-2, respectively, while the cytotoxicity assay against Vero cells, as determined by the MTT reduction method, showed a CC₅₀ value of 132 μg/ml, indicating a CC₅₀/IC₅₀ ratio of 55 for HSV-1 and 32.2 for HSV-2. The antiviral activity of A. arborescens essential oil is principally due to direct virucidal effects. A poor activity determined by yield reduction assay was observed against HSV-1 at higher concentrations when added to cultures of infected cells. No inhibition was observed by attachment assay, penetration assay and post-attachment virus neutralization assay. Furthermore, inhibition of plaque development assay showed that A. arborescens essential oil inhibits the lateral diffusion of both HSV-1 and HSV-2.

Conclusion

This study demonstrates the antiviral activity of the essential oil in toto obtained from A. arborescens against HSV-1 and HSV-2. The mode of action of the essential oil as antiherpesvirus agent seems to be particularly interesting in consideration of its ability to inactivate the virus and to inhibit the cell-to-cell virus diffusion.     

Synthesis and anti-HCMV activity of 1-acyl-beta-lactams and 1-acylazetidines derived from phenylalanine

Different Phe-derived 1-acyl-beta-lactams, analogous to a series of 2-azetidinones acting as HCMV serine protease inhibitors, were synthesized. Some of these compounds were modest inhibitors of the HCMV replication. Interestingly, removal of the carbonyl group of the beta-lactam ring, most likely acting as the serine trap, resulted in an azetidine derivative with anti-HCMV activity comparable to that of the reference compound ganciclovir.