The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Kevin J. Frankowski - One of the best experts on this subject based on the ideXlab platform.
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Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase
2015Co-Authors: Noreena L Sweeney, Kevin J. Frankowski, Jean Ndjomou, Frank J. Schoenen, Sourav Mukherjee, Alicia M. Hanson, Brian J. Geiss, Jordan J. Steel, David N FrickAbstract:The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye Primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%
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Primuline derivatives that mimic rna to stimulate hepatitis c virus ns3 helicase catalyzed atp hydrolysis
Journal of Biological Chemistry, 2013Co-Authors: Noreena L Sweeney, Kevin J. Frankowski, Frank J. Schoenen, Swagato Mukherjee, William R Shadrick, David N FrickAbstract:Abstract ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye Primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissocia-tion constants of about 2 μM. Molecular modeling and site-directed mutagenesis stud-ies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg393, Glu493, and Ser231.
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identification and analysis of hepatitis c virus ns3 helicase inhibitors using nucleic acid binding assays
Nucleic Acids Research, 2012Co-Authors: Sourav Mukherjee, Kevin J. Frankowski, Jean Ndjomou, Alicia M. Hanson, William R Shadrick, Noreena L Sweeney, Diana Bartczak, John J Hernandez, Kelin Li, Julie A HeckAbstract:Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF; Cisbio) and AlphaScreen (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4kM), suramin sodium salt (IC50 = 3.6kM), NF 023 hydrate (IC50 = 6.2kM) and tyrphostin AG 538 (IC50 = 3.6kM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye Primuline, it revealed that some Primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.
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fluorescent Primuline derivatives inhibit hepatitis c virus ns3 catalyzed rna unwinding peptide hydrolysis and viral replicase formation
Antiviral Research, 2012Co-Authors: Jean Ndjomou, Kevin J. Frankowski, Alicia M. Hanson, Frank J. Schoenen, Rajesh Kolli, Swagato Mukherjee, William R Shadrick, Noreena L Sweeney, Diana Bartczak, David N FrickAbstract:The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye Primuline. This study further characterizes a subset of these Primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the Primuline derivatives both in vitro and in cells are also described. The Primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective Primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
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Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and Primuline.
Journal of medicinal chemistry, 2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.
Jean Ndjomou - One of the best experts on this subject based on the ideXlab platform.
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Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase
2015Co-Authors: Noreena L Sweeney, Kevin J. Frankowski, Jean Ndjomou, Frank J. Schoenen, Sourav Mukherjee, Alicia M. Hanson, Brian J. Geiss, Jordan J. Steel, David N FrickAbstract:The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye Primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%
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identification and analysis of hepatitis c virus ns3 helicase inhibitors using nucleic acid binding assays
Nucleic Acids Research, 2012Co-Authors: Sourav Mukherjee, Kevin J. Frankowski, Jean Ndjomou, Alicia M. Hanson, William R Shadrick, Noreena L Sweeney, Diana Bartczak, John J Hernandez, Kelin Li, Julie A HeckAbstract:Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF; Cisbio) and AlphaScreen (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4kM), suramin sodium salt (IC50 = 3.6kM), NF 023 hydrate (IC50 = 6.2kM) and tyrphostin AG 538 (IC50 = 3.6kM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye Primuline, it revealed that some Primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.
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fluorescent Primuline derivatives inhibit hepatitis c virus ns3 catalyzed rna unwinding peptide hydrolysis and viral replicase formation
Antiviral Research, 2012Co-Authors: Jean Ndjomou, Kevin J. Frankowski, Alicia M. Hanson, Frank J. Schoenen, Rajesh Kolli, Swagato Mukherjee, William R Shadrick, Noreena L Sweeney, Diana Bartczak, David N FrickAbstract:The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye Primuline. This study further characterizes a subset of these Primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the Primuline derivatives both in vitro and in cells are also described. The Primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective Primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
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Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and Primuline.
Journal of medicinal chemistry, 2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.
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Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the Yellow Dyes Thioflavine S and Primuline
2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM
Hongwen Huang - One of the best experts on this subject based on the ideXlab platform.
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geographical pattern of isolation and diversification in karst habitat islands a case study in the primulina eburnea complex
Journal of Biogeography, 2015Co-Authors: Yong Gao, Hanghui Kong, Ming Kang, Hongwen HuangAbstract:Aim This study aimed to clarify species boundaries as well as phylogenetic relationships among species of the Primulina eburnea complex, a rapidly diversifying group of edaphic specialists confined to karst habitat islands in southern China. We investigated patterns of genetic isolation and diversification in this complex. Location Limestone karsts in southern China. Methods We analysed data from three chloroplast DNA (cpDNA) regions and eight nuclear microsatellite loci, representing five closely related species in the P. eburnea complex sampled from 62 populations across its entire distributional range. We conducted a Bayesian phylogenetic analysis, constructed maximum parsimony haplotype networks and assessed population genetic diversity, population differentiation and geographical structure within and between species. Results Of the 60 identified haplotypes, eight were shared between populations and only one was shared between species. Both cpDNA and the nuclear markers revealed a high level of population genetic differentiation and a strong phylogeographical structure. Four main genetic clusters were identified according to their geographical proximity, although geographical structuring differed slightly between the cpDNA and the nuclear loci. The pattern of genetic structure of both the cpDNA and microsatellite genomes can be explained by patterns of isolation by distance (IBD), across both the distribution of the complex and the populations of individual species. Main conclusions This is the first study to investigate geographical isolation and speciation in a clade associated with karst habitat islands in southern China. Geographically structured population differentiation of the P. eburnea complex suggests that divergence is mainly driven by genetic drift, with little evidence of gene flow. Allopatric speciation is the main mode of diversification in the group. These results highlight the importance of geographical isolation in promoting population differentiation in karst habitat islands.
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nitrogen limitation as a driver of genome size evolution in a group of karst plants
Scientific Reports, 2015Co-Authors: Ming Kang, Jing Wang, Hongwen HuangAbstract:Genome size is of fundamental biological importance with significance in predicting structural and functional attributes of organisms. Although abundant evidence has shown that the genome size can be largely explained by differential proliferation and removal of non-coding DNA of the genome, the evolutionary and ecological basis of genome size variation remains poorly understood. Nitrogen (N) and phosphorus (P) are essential elements of DNA and protein building blocks, yet often subject to environmental limitation in natural ecosystems. Using phylogenetic comparative methods, we test this hypothesis by determining whether leaf N and P availability affects genome sizes in 99 species of Primulina (Gesneriaceae), a group of soil specialists adapted to limestone karst environment in south China. We find that genome sizes in Primulina are strongly positively correlated with plant N content, but the correlation with plant P content is not significant when phylogeny history was taken into account. This study shows for the first time that N limitation might have been a plausible driver of genome size variation in a group of plants. We propose that competition for nitrogen nutrient between DNA synthesis and cellular functions is a possible mechanism for genome size evolution in Primulina under N-limitation.
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adaptive and nonadaptive genome size evolution in karst endemic flora of china
New Phytologist, 2014Co-Authors: Ming Kang, Junjie Tao, Jing Wang, Chen Ren, Qiuyun Xiang, Hongwen HuangAbstract:Summary Genome size variation is of fundamental biological importance and has been a longstanding puzzle in evolutionary biology. Several hypotheses for genome size evolution including neutral, maladaptive, and adaptive models have been proposed, but the relative importance of these models remains controversial. Primulina is a genus that is highly diversified in the Karst region of southern China, where genome size variation and the underlying evolutionary mechanisms are poorly understood. We reconstructed the phylogeny of Primulina using DNA sequences for 104 species and determined the genome sizes of 101 species. We examined the phylogenetic signal in genome size variation, and tested the fit to different evolutionary models and for correlations with variation in latitude and specific leaf area (SLA). The results showed that genome size, SLA and latitudinal variation all displayed strong phylogenetic signals, but were best explained by different evolutionary models. Furthermore, significant positive relationships were detected between genome size and SLA and between genome size and latitude. Our study is the first to investigate genome size evolution on such a comprehensive scale and in the Karst region flora. We conclude that genome size in Primulina is phylogenetically conserved but its variation among species is a combined outcome of both neutral and adaptive evolution.
Frank J. Schoenen - One of the best experts on this subject based on the ideXlab platform.
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Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase
2015Co-Authors: Noreena L Sweeney, Kevin J. Frankowski, Jean Ndjomou, Frank J. Schoenen, Sourav Mukherjee, Alicia M. Hanson, Brian J. Geiss, Jordan J. Steel, David N FrickAbstract:The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye Primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%
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Primuline derivatives that mimic rna to stimulate hepatitis c virus ns3 helicase catalyzed atp hydrolysis
Journal of Biological Chemistry, 2013Co-Authors: Noreena L Sweeney, Kevin J. Frankowski, Frank J. Schoenen, Swagato Mukherjee, William R Shadrick, David N FrickAbstract:Abstract ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye Primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissocia-tion constants of about 2 μM. Molecular modeling and site-directed mutagenesis stud-ies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg393, Glu493, and Ser231.
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fluorescent Primuline derivatives inhibit hepatitis c virus ns3 catalyzed rna unwinding peptide hydrolysis and viral replicase formation
Antiviral Research, 2012Co-Authors: Jean Ndjomou, Kevin J. Frankowski, Alicia M. Hanson, Frank J. Schoenen, Rajesh Kolli, Swagato Mukherjee, William R Shadrick, Noreena L Sweeney, Diana Bartczak, David N FrickAbstract:The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye Primuline. This study further characterizes a subset of these Primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the Primuline derivatives both in vitro and in cells are also described. The Primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective Primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
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Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and Primuline.
Journal of medicinal chemistry, 2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.
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Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the Yellow Dyes Thioflavine S and Primuline
2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM
Alicia M. Hanson - One of the best experts on this subject based on the ideXlab platform.
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identification and analysis of hepatitis c virus ns3 helicase inhibitors using nucleic acid binding assays
Nucleic Acids Research, 2012Co-Authors: Sourav Mukherjee, Kevin J. Frankowski, Jean Ndjomou, Alicia M. Hanson, William R Shadrick, Noreena L Sweeney, Diana Bartczak, John J Hernandez, Kelin Li, Julie A HeckAbstract:Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF; Cisbio) and AlphaScreen (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4kM), suramin sodium salt (IC50 = 3.6kM), NF 023 hydrate (IC50 = 6.2kM) and tyrphostin AG 538 (IC50 = 3.6kM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye Primuline, it revealed that some Primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.
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fluorescent Primuline derivatives inhibit hepatitis c virus ns3 catalyzed rna unwinding peptide hydrolysis and viral replicase formation
Antiviral Research, 2012Co-Authors: Jean Ndjomou, Kevin J. Frankowski, Alicia M. Hanson, Frank J. Schoenen, Rajesh Kolli, Swagato Mukherjee, William R Shadrick, Noreena L Sweeney, Diana Bartczak, David N FrickAbstract:The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye Primuline. This study further characterizes a subset of these Primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the Primuline derivatives both in vitro and in cells are also described. The Primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective Primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
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Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and Primuline.
Journal of medicinal chemistry, 2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.
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Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the Yellow Dyes Thioflavine S and Primuline
2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye Primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of Primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM