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PubMed2002

Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV of Enterococcus faecalis.

We have cloned the DNA gyrase and topoisomerase IV genes of Enterococcus faecalis to examine the actions of quinolones against E. faecalis genetically and enzymatically. We first generated levofloxacin-resistant mutants of E. faecalis by stepwise selection with increasing drug concentrations and analyzed the quinolone resistance-determining regions of gyrA and parC from the resistant mutants. Isogenic mutants with low-level resistance contained a mutation in gyrA, whereas those with higher levels of resistance had mutations in both gyrA and parC. These results suggested that gyrA is the primary target for levofloxacin in E. faecalis. We then purified the recombinant DNA gyrase and topoisomerase IV enzymes of E. faecalis and measured the in vitro inhibitory activities of quinolones against these enzymes. The 50% inhibitory concentrations (IC(50)s) of levofloxacin, ciprofloxacin, sparfloxacin, tosufloxacin, and gatifloxacin for DNA gyrase were found to be higher than those for topoisomerase IV. In conflict with the genetic data, these results indicated that topoisomerase IV would be the primary target for quinolones in E. faecalis. Among the quinolones tested, the IC(50) of sitafloxacin (DU-6859a), which shows the greatest potency against enterococci, for DNA gyrase was almost equal to that for topoisomerase IV; its IC(50)s were the lowest among those of all the quinolones tested. These results indicated that other factors can modulate the effect of target affinity to determine the bacterial killing pathway, but the highest inhibitory actions against both enzymes correlated with good antienterococcal activities.

Anti-Infective Agents↗

PubMed2001

Flow cytometric evaluation of antiviral agents against human herpesvirus 6.

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.

Acyclovir↗

PubMed2004

Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon- alpha.

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Amino Acid Sequence↗

PubMed2003

Contribution of the 8-methoxy group to the activity of gatifloxacin against type II topoisomerases of Streptococcus pneumoniae.

The inhibitory activities (50% inhibitory concentrations [IC(50)s]) of gatifloxacin and other quinolones against both DNA gyrase and topoisomerase IV of the wild-type Streptococcus pneumoniae IID553 were determined. The IC(50)s of 10 compounds ranged from 4.28 to 582 microg/ml against DNA gyrase and from 1.90 to 35.2 microg/ml against topoisomerase IV. The inhibitory activity against DNA gyrase was more varied than that against topoisomerase IV among fluoroquinolones. The IC(50)s for DNA gyrase of the 8-methoxy quinolones gatifloxacin and AM-1147 were approximately seven times lower than those of their 8-H counterparts AM-1121 and ciprofloxacin, whereas the IC(50)s for topoisomerase IV were 1.5 times lower. Moreover, the IC(50) ratios (IC(50) for DNA gyrase/IC(50) for topoisomerase IV) of gatifloxacin, AM-1147, and moxifloxacin, which possess 8-methoxy groups, were almost the same. The 8-methoxy quinolones showed higher antibacterial activity and less mutant selectivity against IID553 than their 8-H counterparts. These results suggest that the 8-methoxy group enhances both target inhibition, especially for DNA gyrase, leading to potent antipneumococcal activity and dual inhibition against both DNA gyrase and topoisomerase IV in the bacterial cell.

Anti-Infective Agents↗

PubMed2000

Inhibitory effects of the quinolone antibiotics trovafloxacin, ciprofloxacin, and levofloxacin on osteoblastic cells in vitro.

We studied the inhibitory effects of the fluoroquinolones levofloxacin, ciprofloxacin, and trovafloxacin on growth and extracellular matrix mineralization in MC3T3-E1 osteoblast-like cell cultures. Levofloxacin had the least inhibitory effect on cell growth, with a 50% inhibitory concentration of approximately 80 microg/ml at 48 and 72 hours. Ciprofloxacin had an intermediate degree of inhibition, with a 50% inhibitory concentration of 40 microg/ml at 48 and 72 hours. Trovafloxacin exerted a profound inhibitory effect on cell growth, with a 50% inhibitory concentration of 0.5 microg/ml, lower than clinically achievable serum levels. The decreased cell counts with up to 2.5 microg/ml of trovafloxacin and with up to 40 microg/ml of ciprofloxacin were not associated with decreased rates of 5-bromo-2'-deoxyuridine incorporation per cell. Alatrovafloxacin, the L-alanyl-l-alanine prodrug of trovafloxacin, exerted effects on proliferation and 5-bromo-2'-deoxyuridine incorporation similar to those of the parent compound. The quinolones evaluated also inhibited extracellular matrix mineralization by MC3T3-E1 cells. Treatment of confluent cultures with trovafloxacin, ciprofloxacin, or levofloxacin resulted in strong inhibition of calcium deposition, as determined on day 14 by alizarin red staining and biochemical analysis. The effect was apparent with 2.5-5 microg/ml of each of the three antibiotics tested and progressively increased to more than a 90% decline in the calcium/protein ratio with 20-40 microg/ml antibiotic concentration. Further in vivo studies are advocated to evaluate the relevance of the in vitro cytotoxicity reported here to bone healing in orthopaedic patients.

Animals↗

PubMedDate not supplied

Ferulic acid dimer inhibits lipopolysaccharide-stimulated cyclooxygenase-2 expression in macrophages.

Phenylpropanoids may act as nonsteroidal anti-inflammatory drug (NSAID)-like compounds. 4-cis, 8-cis-Bis (4-hydroxy-3-methoxyphenyl)-3, 7-dioxabicyclo-[3.3.0]octane-2,6-dione (bis-FA, compound 2), a dimer of ferulic acid, was synthesized from ferulic acid (1), and its effect on lipopolysaccharide (LPS)-stimulated cyclooxygenase-2 (COX-2) expression in RAW 264.7 cells was compared with those of the parent ferulic acid (1) and of iso-ferulic acid (3-hydroxy-4-methoxycinnamic acid) (3). LPS-induced gene expression of COX-2 was markedly inhibited by compound 2 at a concentration of 10 microM and by compound 3 at 100 microM, but was not inhibited by compound 1 at 100 microM. This observation suggests that compound 2 may possess potent anti-inflammatory activity. These ferulic acid-related compounds were able to scavenge the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The 50% inhibitory concentration for DPPH radicals declined in the order 3 (40.20 mM) > 2 (3.16 mM) > 1 (0.145 mM). Compound 1 possessed potent anti-radical activity, but no COX-2 inhibitory activity, which may be a result of enhancement of its conjugate properties by abstraction of an H atom from the phenolic OH group, causing loss of phenolic function. In contrast, inhibition of COX-2 expression by compounds 2 and 3 could be caused by their increased phenolic function, which is associated with decreased anti-radical activity. Compounds 2 and 3, particularly 2, may have potential as NSAID-like compounds.

Actins↗

PubMed2006

Potent inhibitory effects of black tea theaflavins on off-odor formation from citral.

The present study was conducted to investigate the ability of black tea theaflavins to inhibit the off-odor formation from citral under acidic aqueous conditions. Acidic buffer solutions (pH 3.0) containing citral (10 mg/L) and an inhibitor (0-5 mg/L) were stored at 40 degrees C for 2 weeks. The formation of possible off-odorants p-cresol and p-methylacetophenone in the citral solutions was monitored by high-performance liquid chromatography. A mixture of the theaflavins showed inhibitory effects on the formation of both p-cresol and p-methylacetophenone with 50% inhibitory concentrations (IC(50)) of 0.18 and 0.10 mg/L, respectively. Individual theaflavins and a structurally related compound, purpurogallin, also inhibited the formation of both off-odorants, with the lowest IC(50) values for theaflavin 3,3'-digallate (0.17 and 0.06 mg/L for p-cresol and p-methylacetophenone, respectively). On the other hand, a mixture of green tea catechins and its major constituent, (-)-epigallocatechin gallate, showed relatively high IC(50) values for the formation of p-methylacetophenone (1.29 and 1.28 mg/L, respectively) and showed no inhibitory effect on the formation of p-cresol. The results of the sensory evaluation showed that the off-odor intensity of the stored citral solution was significantly decreased by the addition of the theaflavin mixture at concentrations of 0.5 mg/L and above. In addition, the calculation of the odor activity values (OAVs) for the volatile compounds detected by a gas chromatographic analysis indicated that the total OAVs of the major volatile compounds in the citral solution were significantly decreased by the addition of the theaflavins.

Acetophenones↗

PubMed1999

Synthesis and anti-influenza virus activity of novel pyrimidine derivatives.

Efficient synthetic routes of 2-amino-4-(omega-hydroxyalkylamino)pyrimidine derivatives were investigated in relation to the anti-influenza virus activity of these compounds. The derivatives in which cyclobutyl and cyclopentyl groups were introduced to the beta-position of the aminoalkyl group (especially the cyclobutyl group substituted by a phenylalkyl group at the 3'-position) resulted in improved antiviral potency: i.e. an average 50% effective concentration for inhibition of plaque formation (EC50, microM) of 0.1-0.01 microM for both types A and B influenza virus. The antiviral efficacies were in the order of amino group > hydroxyiminomethyl group > halogen substitution at the 5-position, and chlorine or methoxy group > hydrogen at the 6-position of the pyrimidine ring. The antiviral indices of these compounds were 2-6 with respect to the 50% inhibitory concentration for cell proliferation (IC50, microM) for growing cells, but > 500 to > 10(4) with respect to the IC50 for stationary cells, indicating that these compounds may be efficacious for the topical treatment of influenza virus infection.

Animals↗

PubMed2000

Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates.

Removal of 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC(50)] of approximately 0.5 microM), whereas removal of AZTMP is much less sensitive to this inhibition (IC(50) of >100 microM). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.

Adenosine Triphosphate↗

PubMed2001

Effects of ketamine on voltage-dependent calcium currents and membrane potentials in single bullfrog atrial cells.

PURPOSE: This study was undertaken to assess the effect of ketamine on L-type calcium channel current (I(Ca)) and membrane action potential in the bullfrog single atrial myocyte. METHODS: Bullfrog single atrial myocytes were prepared by enzymatic dispersion. Whole-cell voltage-clamp technique and current clamp technique were used to monitor I(Ca), membrane resting potential, and action potential. RESULTS: Ketamine (10(-5)-10(-3) M) showed dose-dependent inhibition of I(Ca) in a reversible manner. The 50% inhibitory concentration (IC(50)) of ketamine on I(Ca) was estimated to be 0.92 x 10(-5) M. Use-dependent block of I(Ca) was not observed. The resting membrane potential was depolarized at a high concentration (10(-4) M) of ketamine. Reduction of the plateau phase and prolonged duration of the action potential were observed in the presence of a high concentration of ketamine (10(-4) M). CONCLUSION: Ketamine has an inhibitory effect on I(Ca) in the bullfrog single atrial myocyte, and a high dose (10(-4) M) of ketamine prolonges the duration of the action potential. The mechanism of inhibition of I(Ca) seems to be a direct effect on the L-type calcium channel, not like an open channel blocker.

Journal Article↗

PubMed2004

Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity.

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC(50)s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC(50)s of these three quinazoline compounds (2.4 +/- 0.4, 3.4 +/- 0.6, and 3.9 +/- 1.1 microM, respectively) were in the range of the IC(50) of ganciclovir (1.2 +/- 0.2 microM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Antiviral Agents↗

PubMed1992

Ultrastructural changes associated with reduced mitochondrial DNA and impaired mitochondrial function in the presence of 2'3'-dideoxycytidine.

Incubation of Molt-4 cells in 4 microM 2'3'-dideoxycytidine did not produce a significant change in the mitochondrial ultrastructure after 4 days; however, by 12 days, the mitochondrial ultrastructure was distorted, with condensed cristae or vacuolization, or both. Concentration-dependent decreases in both cell growth (mean 50% inhibitory concentration, 4.70 +/- 0.5 microM) and mitochondrial DNA content (mean 50% inhibitory concentration, 0.46 +/- 0.06 microM) occurred after incubation with 2'3'-dideoxycytidine for 4 days.

Cells, Cultured↗

PubMed2004

Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx.

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2000

In vitro evaluation of the effect of temporary removal of HIV drug pressure.

We tried to establish whether MT-4 cells that were infected with HIV-1(HTLV-III(B)) at a high multiplicity of infection (m.o.i.=1), and subsequently treated with high concentrations of anti-HIV drugs for several days, would be able to resume virus production after the antivirals are washed away. The HIV inhibitors studied were the nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine and lamivudine, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, delavirdine and loviride, the acyclic nucleoside phosphonate RT inhibitor (R)-9-(2-phosphonylmethoxypropyl)adenine (tenofovir) and the protease inhibitors (PIs) saquinavir, indinavir and ritonavir. The compounds, at 50 and 500 times their 50% inhibitory concentration (IC(50), determined at a m.o.i.=0.01), were added immediately after virus adsorption and removed after an incubation period of 0 (wash control), 24, 48 or 72 h. Virus breakthrough was monitored by microscopical examination of cytopathicity, viral infectivity (yield) and p24 levels in the supernatant. The presence of HIV-1(HTLV-III(B)) proviral DNA was determined after a 72-h incubation period. None of the antiviral drugs studied was able to prevent resumption of viral growth after removal of the compound. Tenofovir, lamivudine and the NNRTIs nevirapine, delavirdine and loviride, at 500 times their respective IC(50), were able to delay viral breakthrough for approximately 2-3 days. The NRTI zidovudine and the PIs saquinavir, indinavir and ritonavir, under the same conditions, were not able to delay viral breakthrough at all. Virus recovered upon treatment proved as sensitive to the anti-HIV drugs as wild-type virus. Our results suggest that viral replication at the cellular level was not completely inhibited by drug monotherapy. Consequently, virus rebounded when drug therapy stopped. In conclusion, our findings suggest that drug holidays would result in viral breakthrough, even after virus replication has been previously suppressed by adequate drug levels.

Anti-HIV Agents↗

PubMed1989

Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W. N. Konings, J. Bacteriol. 170:5647-5653, 1988). The dependence of solute transport in R. sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.

Alanine↗

PubMed1991

Comparative activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine against rat cytomegalovirus infection in vitro and in vivo.

Two antiviral compounds, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [HPMPC] and 9-(1,3-dihydroxy-2-propoxymethyl)guanine [DHPG], were evaluated for their inhibitory effects on human cytomegalovirus (HCMV) replication in human embryonal fibroblasts and on rat cytomegalovirus (RCMV) replication in rat embryonal fibroblasts. The concentrations of HPMPC or DHPG required to inhibit HCMV plaque formation by 50% were 0.1 and 0.6 micrograms/ml, respectively. For RCMV, these values were 1.1 and 25 micrograms/ml, respectively. For HCMV, the selectivity indices of HPMPC and DHPG, as determined by the ratio of the 50% inhibitory concentration for cell growth to the 50% inhibitory concentration for virus plaque formation, were 1,250 and 140, respectively, and for RCMV, they were 500 and 76, respectively. HPMPC was far more active than DHPG against RCMV infection in vivo as measured by mortality, histopathological changes, and virus titers in organs of immunocompromised RCMV-infected rats. The minimal effective dosage required to prevent mortality from RCMV infection was a single dose of HPMPC at 2 mg/kg of body weight compared with DHPG therapy twice daily at 20 mg/kg/day for 5 days. Furthermore, HPMPC was more effective than DHPG in reducing virus titers in internal organs (P less than 0.01) and in RCMV-induced histopathologic lesions. In contrast to DHPG, which did not show activity when administered 1 day before infection, HPMPC was effective even when administered 7 days before RCMV infection.

Animals↗

PubMed2002

Identification of small-molecule inhibitors of nucleoside triphosphate hydrolase in Toxoplasma gondii.

Approximately 150,000 small-molecule compounds were tested by a robotic screening assay for their ability to inhibit nucleoside triphosphate hydrolase (NTPase), a novel enzyme of the tachyzoite form of Toxoplasma gondii. Five unrelated species of compounds were found to inhibit the activities of both NTPase isoforms (NTPase isoform I [NTPase-I] and NTPase-II). The 50% inhibitory concentrations (IC(50)s) ranged from 0.1 to 20 microM, and in general, the IC(50)s were similar for both NTPase isoforms. However, the activity of NTPase-I was 20 times more sensitive than the activity of NTPase-II to one of the inhibitors: 9-hydroxy-10-(pentachlorophenoxy)stearic acid. The five compounds identified also prevented tachyzoite replication in vitro, with IC(50)s ranging from approximately 7 to > or =50 microM. The most effective of these initial compounds, 2-phenylthio-indole, was used to identify six additional, structurally related compounds, which were tested for their inhibitory effects on enzyme activities and tachyzoite replication. Surprisingly, these compounds were competitive inhibitors of NTPase-I but noncompetitive inhibitors of NTPase-II. Modifications to the indole and phenol rings resulted in alterations of activity, thus providing insight into the structural features that are important for inhibition of T. gondii NTPases.

Acid Anhydride Hydrolases↗

PubMed2006

Rapid propagation of low-fitness drug-resistant mutants of human immunodeficiency virus type 1 by a streptococcal metabolite sparsomycin.

Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.

Cells, Cultured↗