David Frick - Academia.edu (original) (raw)
Papers by David Frick
ABSTRACTThe virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous ... more ABSTRACTThe virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymers, helicase and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those seen in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses, and potential therapeutic target.
The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologo... more The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologous DNA strand exchange. Single-stranded DNA oligonucleotides complementary to either the short or long strand of a partially duplex DNA substrate affected the rate and extent of unwinding catalyzed by either the full-length HCV NS3 protein fused to a portion of HCV NS4A, or a truncated NS3 protein lacking the protease domain. The oligonucleotides did not, however, sequester HCV helicase and prevent it from separating the original duplex after a single binding cycle. Furthermore, when DNA oligonucleotides were pre-incubated with HCV helicase, they did not prevent subsequent duplex separation, indicating that the enzyme catalyzes strand exchange. The protease portion of NS3 was not needed for this strand exchange. Fluorescent DNA substrates were further used to directly monitor both this ssDNA assisted unwinding and homologous strand exchange, and the effect of a protein trap (poly(U) RNA) on HCV helicase-catalyzed strand exchange was examined. The results demonstrate that HCV helicase can simultaneously bind at least three DNA strands and imply that HCV NS3 helicase could play an important role not only in viral RNA unwinding, but also in the folding of the HCV genome.
Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C... more Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interactionwith NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111–22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously pro...
The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologo... more The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologous DNA strand exchange. Single-stranded DNA oligonucleotides complementary to either the short or long strand of a partially duplex DNA substrate affected the rate and extent of unwinding catalyzed by either the full-length HCV NS3 protein fused to a portion of HCV NS4A, or a truncated NS3 protein lacking the protease domain. The oligonucleotides did not, however, sequester HCV helicase and prevent it from separating the original duplex after a single binding cycle. Furthermore, when DNA oligonucleotides were pre-incubated with HCV helicase, they did not prevent subsequent duplex separation, indicating that the enzyme catalyzes strand exchange. The protease portion of NS3 was not needed for this strand exchange. Fluorescent DNA substrates were further used to directly monitor both this ssDNA assisted unwinding and homologous strand exchange, and the effect of a protein trap (poly(U) RNA)...
This study examines the role of the p12 subunit in the function of the human DNA polymerase δ (Po... more This study examines the role of the p12 subunit in the function of the human DNA polymerase δ (Pol δ) holoenzyme by comparing the kinetics of DNA synthesis and degradation catalyzed by the foursubunit complex, the three-subunit complex lacking p12, and site-directed mutants of each lacking proofreading exonuclease activity. Results show that p12modulates the rate and fidelity ofDNA synthesis by Pol δ. All four complexes synthesize DNA in a rapid burst phase and a slower, more linear phase. In the presence of p12, the burst rates of DNA synthesis are ∼5 times faster, while the affinity of the enzyme for its DNA and dNTP substrates appears unchanged. The p12 subunit alters Pol δ fidelity by modulating the proofreading 30 to 50 exonuclease activity. In the absence of p12, Pol δ is more likely to proofread DNA synthesis because it cleaves single-stranded DNA twice as fast and transfers mismatched DNA from the polymerase to the exonuclease sites 9 times faster. Pol δ also extends mismatc...
SLAS DISCOVERY: Advancing the Science of Drug Discovery
Small molecules that bind the SARS-CoV-2 nonstructural protein 3 Mac1 domain in place of ADP-ribo... more Small molecules that bind the SARS-CoV-2 nonstructural protein 3 Mac1 domain in place of ADP-ribose could be useful as molecular probes or scaffolds for COVID-19 antiviral drug discovery because Mac1 has been linked to the ability of coronaviruses to evade cellular detection. A high-throughput assay based on differential scanning fluorimetry (DSF) was therefore optimized and used to identify possible Mac1 ligands in small libraries of drugs and drug-like compounds. Numerous promising compounds included nucleotides, steroids, β-lactams, and benzimidazoles. The main drawback to this approach was that a high percentage of compounds in some libraries were found to influence the observed Mac1 melting temperature. To prioritize DSF screening hits, the shapes of the observed melting curves and initial assay fluorescence were examined, and the results were compared with virtual screens performed using AutoDock Vina. The molecular basis for alternate ligand binding was also examined by deter...
Biochemistry
The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins... more The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymerase, helicase, and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those observed in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses and a potential therapeutic target.
Advances in Experimental Medicine and Biology
This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and... more This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and its application to cellular receptors. The method is based on the kinetic theory of FRET, which can be used to predict FRET not only in dimers, but also higher order oligomers of donor and acceptor fluorophores. Models based on such FRET predictions can be fit to observed FRET efficiency histograms (also called FRET spectrograms) and used to estimate intracellular binding constants, free energy values, and stoichiometries. These "FRET spectrometry" methods have been used to analyze oligomers formed by various receptors in cell signaling pathways, but until recently such studies were limited to receptors residing on the cell surface. To study complexes residing inside the cell, a technique called Quantitative Micro-Spectroscopic Imaging (Q-MSI) was developed. Q-MSI combines determination of quaternary structure from pixel-level apparent FRET spectrograms with the determination of both donor and acceptor concentrations at the organelle level. This is done by resolving and analyzing the spectrum of a third fluorescent marker, which does not participate in FRET. Q-MSI was first used to study the interaction of a class of cytoplasmic receptors that bind viral RNA and signal an antiviral response via complexes formed mainly on mitochondrial membranes. Q-MSI revealed previously unknown RNA mitochondrial receptor orientations, and the interaction between the viral RNA receptor called LGP2 with the RNA helicase encoded by the hepatitis virus. The biological importance of these new observations is discussed.
European Journal of Medicinal Chemistry, 2017
A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helica... more A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesized, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly bound NS3 with a dissociation constant of 570 ± 270 nM. Highlights Virtual screening studies on the HCV NS3 helicase. Identification of a substituted piperazine as new anti-HCV scaffold. Synthesis of different series of novel piperazine-based structures. New inhibitors of the subgenomic HCV replicon 1b genotype identified.
Antiviral Chemistry and Chemotherapy, 2016
Background Despite the great progress made in the last 10 years, alternative strategies might hel... more Background Despite the great progress made in the last 10 years, alternative strategies might help improving definitive treatment options against hepatitis C virus infection. Methods With the aim of identifying novel inhibitors of the hepatitis C virus-1b replication targeting the viral NS3 helicase, the structures of previously reported symmetrical inhibitors of this enzyme were rationally modified, and according to docking-based studies, four novel scaffolds were selected for synthesis and evaluation in the hepatitis C virus-1b subgenomic replicon assay. Results Among the newly designed compounds, one new structural family was found to inhibit the hepatitis C virus-1b replication in the micromolar range. This scaffold was chosen for further exploration and different novel analogues were synthesised and evaluated. Conclusions Different new inhibitors of the hepatitis C virus genotype 1b replication were identified. Some of the new compounds show mild inhibition of the NS3 helicase ...
European journal of medicinal chemistry, Jan 10, 2016
A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, w... more A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with the aim to find novel inhibitors of the HCV replication. A library of ∼450000 commercially available compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV replicon assay. One hit characterized by a substituted thieno-pyrimidine scaffold was found to inhibit the viral replication with an EC50 value in the sub-micromolar range and a good selectivity index. Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new structures showed antiviral activity in the low or sub-micromolar range.
European journal of medicinal chemistry, Jan 23, 2016
We report the discovery of the bicyclic octahydrocyclohepta[b]pyrrol-4(1H)-one scaffold as a new ... more We report the discovery of the bicyclic octahydrocyclohepta[b]pyrrol-4(1H)-one scaffold as a new chemotype with anti-HCV activity on genotype 1b and 2a subgenomic replicons. The most potent compound 34 displayed EC50 values of 1.8 μM and 4.5 μM in genotype 1b and 2a, respectively, coupled with the absence of any antimetabolic effect (gt 1b SI = 112.4; gt 2a SI = 44.2) in a cell-based assay. Compound 34 did not target HCV NS5B, IRES, NS3 helicase, or selected host factors, and thus future work will involve the unique mechanism of action of these new antiviral compounds.
PLOS ONE, 2015
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people... more The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.
ACS National Meeting Book of Abstracts
ACS Chemical Biology, 2015
This study examines the specificity and mechanism of action of a recently reported hepatitis C vi... more This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) non-structural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevirresistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety of wildtype and mutant NS3 proteins suggested that HPI forms a bridge between the NS3 RNA-binding cleft and an allosteric site previously shown to bind other protease inhibitors. In most combinations, the antiviral effect of HPI was additive with telaprevir, boceprevir, minor synergy was observed with danoprevir and modest synergy was observed with grazoprevir.
European Journal of Medicinal Chemistry, 2015
Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is ... more Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is now a reality, today's HCV drugs are expensive, and more affordable drugs are still urgently needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected representative set of derivatives as well as their chemical synthesis are described herein. The most potent compound 39 displayed EC 50 values of 7.9 and 2.6 mM in genotype 1b and 2a, respectively. Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3 helicase, IRES mediated translation and selected host factors. Thus, future work will involve both the chemical optimization and target identification of 2-phenyl-4,5,6,7-Tetrahydro-1H-indoles as new anti-HCV agents.
The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologo... more The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologous DNA strand exchange. Single-stranded DNA oligonucleotides complementary to either the short or long strand of a partially duplex DNA substrate affected the rate and extent of unwinding catalyzed by either the full-length HCV NS3 protein fused to a portion of HCV NS4A, or a truncated NS3 protein lacking the protease domain. The oligonucleotides did not, however, sequester HCV helicase and prevent it from separating the original duplex after a single binding cycle. Furthermore, when DNA oligonucleotides were pre-incubated with HCV helicase, they did not prevent subsequent duplex separation, indicating that the enzyme catalyzes strand exchange. The protease portion of NS3 was not needed for this strand exchange. Fluorescent DNA substrates were further used to directly monitor both this ssDNA assisted unwinding and homologous strand exchange, and the effect of a protein trap (poly(U) RNA)...
Current issues in molecular biology, 2007
The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three dom... more The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.
ABSTRACTThe virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous ... more ABSTRACTThe virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymers, helicase and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those seen in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses, and potential therapeutic target.
The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologo... more The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologous DNA strand exchange. Single-stranded DNA oligonucleotides complementary to either the short or long strand of a partially duplex DNA substrate affected the rate and extent of unwinding catalyzed by either the full-length HCV NS3 protein fused to a portion of HCV NS4A, or a truncated NS3 protein lacking the protease domain. The oligonucleotides did not, however, sequester HCV helicase and prevent it from separating the original duplex after a single binding cycle. Furthermore, when DNA oligonucleotides were pre-incubated with HCV helicase, they did not prevent subsequent duplex separation, indicating that the enzyme catalyzes strand exchange. The protease portion of NS3 was not needed for this strand exchange. Fluorescent DNA substrates were further used to directly monitor both this ssDNA assisted unwinding and homologous strand exchange, and the effect of a protein trap (poly(U) RNA) on HCV helicase-catalyzed strand exchange was examined. The results demonstrate that HCV helicase can simultaneously bind at least three DNA strands and imply that HCV NS3 helicase could play an important role not only in viral RNA unwinding, but also in the folding of the HCV genome.
Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C... more Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interactionwith NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111–22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously pro...
The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologo... more The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologous DNA strand exchange. Single-stranded DNA oligonucleotides complementary to either the short or long strand of a partially duplex DNA substrate affected the rate and extent of unwinding catalyzed by either the full-length HCV NS3 protein fused to a portion of HCV NS4A, or a truncated NS3 protein lacking the protease domain. The oligonucleotides did not, however, sequester HCV helicase and prevent it from separating the original duplex after a single binding cycle. Furthermore, when DNA oligonucleotides were pre-incubated with HCV helicase, they did not prevent subsequent duplex separation, indicating that the enzyme catalyzes strand exchange. The protease portion of NS3 was not needed for this strand exchange. Fluorescent DNA substrates were further used to directly monitor both this ssDNA assisted unwinding and homologous strand exchange, and the effect of a protein trap (poly(U) RNA)...
This study examines the role of the p12 subunit in the function of the human DNA polymerase δ (Po... more This study examines the role of the p12 subunit in the function of the human DNA polymerase δ (Pol δ) holoenzyme by comparing the kinetics of DNA synthesis and degradation catalyzed by the foursubunit complex, the three-subunit complex lacking p12, and site-directed mutants of each lacking proofreading exonuclease activity. Results show that p12modulates the rate and fidelity ofDNA synthesis by Pol δ. All four complexes synthesize DNA in a rapid burst phase and a slower, more linear phase. In the presence of p12, the burst rates of DNA synthesis are ∼5 times faster, while the affinity of the enzyme for its DNA and dNTP substrates appears unchanged. The p12 subunit alters Pol δ fidelity by modulating the proofreading 30 to 50 exonuclease activity. In the absence of p12, Pol δ is more likely to proofread DNA synthesis because it cleaves single-stranded DNA twice as fast and transfers mismatched DNA from the polymerase to the exonuclease sites 9 times faster. Pol δ also extends mismatc...
SLAS DISCOVERY: Advancing the Science of Drug Discovery
Small molecules that bind the SARS-CoV-2 nonstructural protein 3 Mac1 domain in place of ADP-ribo... more Small molecules that bind the SARS-CoV-2 nonstructural protein 3 Mac1 domain in place of ADP-ribose could be useful as molecular probes or scaffolds for COVID-19 antiviral drug discovery because Mac1 has been linked to the ability of coronaviruses to evade cellular detection. A high-throughput assay based on differential scanning fluorimetry (DSF) was therefore optimized and used to identify possible Mac1 ligands in small libraries of drugs and drug-like compounds. Numerous promising compounds included nucleotides, steroids, β-lactams, and benzimidazoles. The main drawback to this approach was that a high percentage of compounds in some libraries were found to influence the observed Mac1 melting temperature. To prioritize DSF screening hits, the shapes of the observed melting curves and initial assay fluorescence were examined, and the results were compared with virtual screens performed using AutoDock Vina. The molecular basis for alternate ligand binding was also examined by deter...
Biochemistry
The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins... more The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymerase, helicase, and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those observed in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses and a potential therapeutic target.
Advances in Experimental Medicine and Biology
This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and... more This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and its application to cellular receptors. The method is based on the kinetic theory of FRET, which can be used to predict FRET not only in dimers, but also higher order oligomers of donor and acceptor fluorophores. Models based on such FRET predictions can be fit to observed FRET efficiency histograms (also called FRET spectrograms) and used to estimate intracellular binding constants, free energy values, and stoichiometries. These "FRET spectrometry" methods have been used to analyze oligomers formed by various receptors in cell signaling pathways, but until recently such studies were limited to receptors residing on the cell surface. To study complexes residing inside the cell, a technique called Quantitative Micro-Spectroscopic Imaging (Q-MSI) was developed. Q-MSI combines determination of quaternary structure from pixel-level apparent FRET spectrograms with the determination of both donor and acceptor concentrations at the organelle level. This is done by resolving and analyzing the spectrum of a third fluorescent marker, which does not participate in FRET. Q-MSI was first used to study the interaction of a class of cytoplasmic receptors that bind viral RNA and signal an antiviral response via complexes formed mainly on mitochondrial membranes. Q-MSI revealed previously unknown RNA mitochondrial receptor orientations, and the interaction between the viral RNA receptor called LGP2 with the RNA helicase encoded by the hepatitis virus. The biological importance of these new observations is discussed.
European Journal of Medicinal Chemistry, 2017
A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helica... more A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesized, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly bound NS3 with a dissociation constant of 570 ± 270 nM. Highlights Virtual screening studies on the HCV NS3 helicase. Identification of a substituted piperazine as new anti-HCV scaffold. Synthesis of different series of novel piperazine-based structures. New inhibitors of the subgenomic HCV replicon 1b genotype identified.
Antiviral Chemistry and Chemotherapy, 2016
Background Despite the great progress made in the last 10 years, alternative strategies might hel... more Background Despite the great progress made in the last 10 years, alternative strategies might help improving definitive treatment options against hepatitis C virus infection. Methods With the aim of identifying novel inhibitors of the hepatitis C virus-1b replication targeting the viral NS3 helicase, the structures of previously reported symmetrical inhibitors of this enzyme were rationally modified, and according to docking-based studies, four novel scaffolds were selected for synthesis and evaluation in the hepatitis C virus-1b subgenomic replicon assay. Results Among the newly designed compounds, one new structural family was found to inhibit the hepatitis C virus-1b replication in the micromolar range. This scaffold was chosen for further exploration and different novel analogues were synthesised and evaluated. Conclusions Different new inhibitors of the hepatitis C virus genotype 1b replication were identified. Some of the new compounds show mild inhibition of the NS3 helicase ...
European journal of medicinal chemistry, Jan 10, 2016
A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, w... more A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with the aim to find novel inhibitors of the HCV replication. A library of ∼450000 commercially available compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV replicon assay. One hit characterized by a substituted thieno-pyrimidine scaffold was found to inhibit the viral replication with an EC50 value in the sub-micromolar range and a good selectivity index. Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new structures showed antiviral activity in the low or sub-micromolar range.
European journal of medicinal chemistry, Jan 23, 2016
We report the discovery of the bicyclic octahydrocyclohepta[b]pyrrol-4(1H)-one scaffold as a new ... more We report the discovery of the bicyclic octahydrocyclohepta[b]pyrrol-4(1H)-one scaffold as a new chemotype with anti-HCV activity on genotype 1b and 2a subgenomic replicons. The most potent compound 34 displayed EC50 values of 1.8 μM and 4.5 μM in genotype 1b and 2a, respectively, coupled with the absence of any antimetabolic effect (gt 1b SI = 112.4; gt 2a SI = 44.2) in a cell-based assay. Compound 34 did not target HCV NS5B, IRES, NS3 helicase, or selected host factors, and thus future work will involve the unique mechanism of action of these new antiviral compounds.
PLOS ONE, 2015
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people... more The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.
ACS National Meeting Book of Abstracts
ACS Chemical Biology, 2015
This study examines the specificity and mechanism of action of a recently reported hepatitis C vi... more This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) non-structural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevirresistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety of wildtype and mutant NS3 proteins suggested that HPI forms a bridge between the NS3 RNA-binding cleft and an allosteric site previously shown to bind other protease inhibitors. In most combinations, the antiviral effect of HPI was additive with telaprevir, boceprevir, minor synergy was observed with danoprevir and modest synergy was observed with grazoprevir.
European Journal of Medicinal Chemistry, 2015
Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is ... more Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is now a reality, today's HCV drugs are expensive, and more affordable drugs are still urgently needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected representative set of derivatives as well as their chemical synthesis are described herein. The most potent compound 39 displayed EC 50 values of 7.9 and 2.6 mM in genotype 1b and 2a, respectively. Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3 helicase, IRES mediated translation and selected host factors. Thus, future work will involve both the chemical optimization and target identification of 2-phenyl-4,5,6,7-Tetrahydro-1H-indoles as new anti-HCV agents.
The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologo... more The helicase encoded by the hepatitis C virus (HCV) is shown in this chapter to catalyze homologous DNA strand exchange. Single-stranded DNA oligonucleotides complementary to either the short or long strand of a partially duplex DNA substrate affected the rate and extent of unwinding catalyzed by either the full-length HCV NS3 protein fused to a portion of HCV NS4A, or a truncated NS3 protein lacking the protease domain. The oligonucleotides did not, however, sequester HCV helicase and prevent it from separating the original duplex after a single binding cycle. Furthermore, when DNA oligonucleotides were pre-incubated with HCV helicase, they did not prevent subsequent duplex separation, indicating that the enzyme catalyzes strand exchange. The protease portion of NS3 was not needed for this strand exchange. Fluorescent DNA substrates were further used to directly monitor both this ssDNA assisted unwinding and homologous strand exchange, and the effect of a protein trap (poly(U) RNA)...
Current issues in molecular biology, 2007
The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three dom... more The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.