Journal of the Chemical Society, Chemical Communications, 1990
Abstract Syntheses of the 4-methyl (2 and 3) and [3.2. 1] bicyclo (4 and 5) analogues of the glyc... more Abstract Syntheses of the 4-methyl (2 and 3) and [3.2. 1] bicyclo (4 and 5) analogues of the glycine/N-methyl-D-aspartate (NMDA) antagonist 3-amino-1-hydroxypyrrolidin-2-one (HA-966, 1) provide evidence that glycine receptor recogntion requires the energetically less ...
1. Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carbo... more 1. Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carboxamide is a potent class I histone deacetylase (HDAC) inhibitor that demonstrated good antiproliferative activity against human tumour cell lines of different origin. 2. This compound showed high in vivo clearance in rats (160 ml min −1 kg −1) due to metabolism. The main metabolite detected in urine after intravenous dosing was characterized as a dihydrohydroxy S-mercapturic acid conjugate. Following oral dosing, however, the mercapturic acid derivative was no longer the main metabolite but the major metabolites were mono-and di-glucuronide conjugates of oxidized species having a mass shift of +34 m/z with respect to the parent. 3. Comparison of plasma concentration after intra-arterial infusion and intravenous infusion and incubation with microsomes from different tissues (liver, kidney, small intestine and lung) in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH) indicated that the compound was highly cleared by the lung. 4. Oxidation of the naphthalene moiety was demonstrated to be the cause of the high in vivo clearance of compound A and the potential for bioactivation of this group was flagged.
Glycine anion (4) and glycine cation (8) synthons are used in efficient syntheses of 4-substitute... more Glycine anion (4) and glycine cation (8) synthons are used in efficient syntheses of 4-substituted analogues of HA-966 (1). A stereospecific route: to cis derivatives involves hydrogenation of enamines such as 16. Introduction of a chiral arlxiliary leads to an enantioselective synthesis of 2a (L-687,414).
The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral... more The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant t...
ASA ϭ American Society of Anesthesiologists BMI ϭ body mass index EBL ϭ estimated blood loss HALS... more ASA ϭ American Society of Anesthesiologists BMI ϭ body mass index EBL ϭ estimated blood loss HALS ϭ hand assisted laparoscopic surgery LDN ϭ laparoscopic donor nephrectomy LNU ϭ laparoscopic nephroureterectomy LP ϭ laparoscopic pyeloplasty LPN ϭ laparoscopic partial nephrectomy LRN ϭ laparoscopic radical nephrectomy LSN ϭ laparoscopic simple nephrectomy SL ϭ standard laparoscopy
A new synthetic approach to tricyclic pyridones bearing a fused seven-membered ring is described.... more A new synthetic approach to tricyclic pyridones bearing a fused seven-membered ring is described. These compounds exhibit atropisomerism and exist in enantiomeric forms. Chiral HPLC separation of the enantiomers has allowed the rates of racemization to be measured and hence the free energy barrier for flipping the seven-membered ring to be deduced. Introduction of a further element of planar chirality leads to diastereomeric atropisomerism. The rate of interconversion of the diastereomers has been quantified by 2D EXSY NMR spectroscopy allowing a full description of the conformational dynamics of the system.
3'-(Arylmethyl)- and 3'4 Aryloxy)-3-phenyl-4-hydroxyquinolin-2( lH)-ones: Orall... more 3'-(Arylmethyl)- and 3'4 Aryloxy)-3-phenyl-4-hydroxyquinolin-2( lH)-ones: Orally Active Antagonists of the Glycine Site on the NMDA Receptor ... Janusz J. Kulagowski, Raymond Baker, Neil R. Curtis Paul D. Leeson,' Ian M. Mawer, Angela M. Moseley, Mark P. Ridgill, Michael ...
Table of Contents Experimental section and synthetic schemes 1 HDAC Enzyme Assays 14 Protocols fo... more Table of Contents Experimental section and synthetic schemes 1 HDAC Enzyme Assays 14 Protocols for cell proliferation assays 17 Protocol for plasma stability 18 Rat PK studies 19 Protocol for tumor xenograft studies 20 Experimental section and synthetic schemes General Experimental Protocols Solvents and reagents were obtained from commercial suppliers and were used without further purification. Flash chromatography purifications were performed on Merck silica gel (200-400 mesh) as the stationary phase or were conducted using prepacked cartridges on a Biotage system, eluting with petroleum ether/ethyl acetate mixtures. HPLC-MS and UPLC-MS analyses were performed on either a Waters Alliance 2795 apparatus, equipped with a diode array and a ZQ mass spectrometer using an X-Terra C18 column (5μM, 4.6x50mm) or on a Acquity UPLC TM
Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has... more Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here a novel class of allosteric inhibitor of NS5B that shows potent affinity for the NS5B enzyme and effective inhibition of subgenomic HCV RNA replication in HUH-7 cells. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.
that a benzolactam of type A (Figure 2), in which X and Y are adjusted so that the 3-proton is ac... more that a benzolactam of type A (Figure 2), in which X and Y are adjusted so that the 3-proton is acidic, may bind, but greater delocalization of the negative charge over the 2-carbonyl, X, and Y, could lead to improved brain penetration. In this paper we describe the synthesis of several variants of this type of compound, their binding affinity and antagonist potency and show that, indeed, i n vivo properties can be greatly improved.
It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D recept... more It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.
Incidence and Clinical Characteristics Schizophrenia is a devastating psychiatric illness that af... more Incidence and Clinical Characteristics Schizophrenia is a devastating psychiatric illness that affects approximately 1% of the world population irrespective of ethnic, economical, or cultural boundaries: features that suggest the disorder has no simple, single causative factor. With current drug therapy, approximately 25% of patients recover to some extent within 5 years of starting treatment and about 65% of patients have recurring problems over many years. The remaining 10-15% of patients develop long-term incapacity and around 15% commit suicide. There are substantial costs, both direct and indirect, incurred by this disorder including those of drug treatment, residential accommodation, physician and other healthcare services, and loss of productivity in the workplace. Clinical symptoms are apparent relatively early in life, generally occurring between the ages of 15 and 45. They are characterized by the presence of positive symptoms, for example auditory hallucinations, disorganized thoughts, delusions, and irrational fears, and negative symptoms, including social withdrawal, diminished affect, poverty of speech, lack of energy, and the inability to experience pleasure. In addition, schizophrenic patients may suffer cognitive deficits including impaired attention, verbal fluency, memory recall, and executive function.
5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening... more 5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with > 500-fold selectivity over hD2 and > 200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.
The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a k... more The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3-and 2.4-Å resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small ␣-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.
Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA ... more Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA interference (RNAi) machinery. Three factors that mediate this mechanism have been identified: 1) the RNA-dependent RNA polymerase complex RdRC, 2) the Argonaute-containing RITS (RNA-induced initiation of transcriptional silencing) complex, and 3) the endoribonuclease Dicer ortholog Dcr1. S. pombe mutants lacking a new factor described here, Ers1, are completely defective in RNAi-dependent silencing of centromeric regions but, importantly, not in RNAiindependent silencing at the mat3M or tel2R loci. ers1⌬ cells likewise fail to convert centromeric pre-small interfering RNA transcripts into small interfering RNAs, are defective in histone H3 Lys 9 methylation, and are unable to recruit the RITS complex to centromeric sequences. Surprisingly, Ers1 lacks obvious orthologs outside of the genus Schizosaccharomyces. Within this group, it is diverging rapidly, raising the possibility that it is coevolving with target RNA elements.
Diabetic patients develop a cardiomyopathy that consists of ventricular hypertrophy and diastolic... more Diabetic patients develop a cardiomyopathy that consists of ventricular hypertrophy and diastolic dysfunction. Although the pathogenesis of this condition is poorly understood, previous studies implicated abnormal G-protein activation. In this work, mice with cardiac overexpression of the transcription factor peroxisome proliferator–activated receptor-α (PPAR-α) were examined as a model of diabetic cardiomyopathy. PPAR-α transgenic mice develop spontaneous cardiac hypertrophy, contractile dysfunction, and “fetal” gene induction. We examined the role of abnormal G-protein activation in the pathogenesis of cardiac dysfunction by crossing PPAR-α mice with transgenic mice with cardiac-specific overexpression of regulator of G-protein signaling subtype 4 (RGS4), a GTPase activating protein for Gq and Gi. Generation of compound transgenic mice demonstrated that cardiac RGS4 overexpression ameliorated the cardiomyopathic phenotype that occurred as a result of PPAR-α overexpression without ...
Bioorganic & Medicinal Chemistry Letters, 1999
The syntheses of a number of different N-linked heterocyclic pyrazole replacements based on the s... more The syntheses of a number of different N-linked heterocyclic pyrazole replacements based on the structure 1 are described (compounds 3-12) as hD4 ligands. After further optimisation the best compound identified was 13 which has high affinity for him (5.2 nM) and >300-fold selectivity for hD4 receptors over hD2 and hD3 receptors.
Journal of the Chemical Society, Chemical Communications, 1990
Abstract Syntheses of the 4-methyl (2 and 3) and [3.2. 1] bicyclo (4 and 5) analogues of the glyc... more Abstract Syntheses of the 4-methyl (2 and 3) and [3.2. 1] bicyclo (4 and 5) analogues of the glycine/N-methyl-D-aspartate (NMDA) antagonist 3-amino-1-hydroxypyrrolidin-2-one (HA-966, 1) provide evidence that glycine receptor recogntion requires the energetically less ...
1. Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carbo... more 1. Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carboxamide is a potent class I histone deacetylase (HDAC) inhibitor that demonstrated good antiproliferative activity against human tumour cell lines of different origin. 2. This compound showed high in vivo clearance in rats (160 ml min −1 kg −1) due to metabolism. The main metabolite detected in urine after intravenous dosing was characterized as a dihydrohydroxy S-mercapturic acid conjugate. Following oral dosing, however, the mercapturic acid derivative was no longer the main metabolite but the major metabolites were mono-and di-glucuronide conjugates of oxidized species having a mass shift of +34 m/z with respect to the parent. 3. Comparison of plasma concentration after intra-arterial infusion and intravenous infusion and incubation with microsomes from different tissues (liver, kidney, small intestine and lung) in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH) indicated that the compound was highly cleared by the lung. 4. Oxidation of the naphthalene moiety was demonstrated to be the cause of the high in vivo clearance of compound A and the potential for bioactivation of this group was flagged.
Glycine anion (4) and glycine cation (8) synthons are used in efficient syntheses of 4-substitute... more Glycine anion (4) and glycine cation (8) synthons are used in efficient syntheses of 4-substituted analogues of HA-966 (1). A stereospecific route: to cis derivatives involves hydrogenation of enamines such as 16. Introduction of a chiral arlxiliary leads to an enantioselective synthesis of 2a (L-687,414).
The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral... more The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant t...
ASA ϭ American Society of Anesthesiologists BMI ϭ body mass index EBL ϭ estimated blood loss HALS... more ASA ϭ American Society of Anesthesiologists BMI ϭ body mass index EBL ϭ estimated blood loss HALS ϭ hand assisted laparoscopic surgery LDN ϭ laparoscopic donor nephrectomy LNU ϭ laparoscopic nephroureterectomy LP ϭ laparoscopic pyeloplasty LPN ϭ laparoscopic partial nephrectomy LRN ϭ laparoscopic radical nephrectomy LSN ϭ laparoscopic simple nephrectomy SL ϭ standard laparoscopy
A new synthetic approach to tricyclic pyridones bearing a fused seven-membered ring is described.... more A new synthetic approach to tricyclic pyridones bearing a fused seven-membered ring is described. These compounds exhibit atropisomerism and exist in enantiomeric forms. Chiral HPLC separation of the enantiomers has allowed the rates of racemization to be measured and hence the free energy barrier for flipping the seven-membered ring to be deduced. Introduction of a further element of planar chirality leads to diastereomeric atropisomerism. The rate of interconversion of the diastereomers has been quantified by 2D EXSY NMR spectroscopy allowing a full description of the conformational dynamics of the system.
3'-(Arylmethyl)- and 3'4 Aryloxy)-3-phenyl-4-hydroxyquinolin-2( lH)-ones: Orall... more 3'-(Arylmethyl)- and 3'4 Aryloxy)-3-phenyl-4-hydroxyquinolin-2( lH)-ones: Orally Active Antagonists of the Glycine Site on the NMDA Receptor ... Janusz J. Kulagowski, Raymond Baker, Neil R. Curtis Paul D. Leeson,' Ian M. Mawer, Angela M. Moseley, Mark P. Ridgill, Michael ...
Table of Contents Experimental section and synthetic schemes 1 HDAC Enzyme Assays 14 Protocols fo... more Table of Contents Experimental section and synthetic schemes 1 HDAC Enzyme Assays 14 Protocols for cell proliferation assays 17 Protocol for plasma stability 18 Rat PK studies 19 Protocol for tumor xenograft studies 20 Experimental section and synthetic schemes General Experimental Protocols Solvents and reagents were obtained from commercial suppliers and were used without further purification. Flash chromatography purifications were performed on Merck silica gel (200-400 mesh) as the stationary phase or were conducted using prepacked cartridges on a Biotage system, eluting with petroleum ether/ethyl acetate mixtures. HPLC-MS and UPLC-MS analyses were performed on either a Waters Alliance 2795 apparatus, equipped with a diode array and a ZQ mass spectrometer using an X-Terra C18 column (5μM, 4.6x50mm) or on a Acquity UPLC TM
Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has... more Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here a novel class of allosteric inhibitor of NS5B that shows potent affinity for the NS5B enzyme and effective inhibition of subgenomic HCV RNA replication in HUH-7 cells. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.
that a benzolactam of type A (Figure 2), in which X and Y are adjusted so that the 3-proton is ac... more that a benzolactam of type A (Figure 2), in which X and Y are adjusted so that the 3-proton is acidic, may bind, but greater delocalization of the negative charge over the 2-carbonyl, X, and Y, could lead to improved brain penetration. In this paper we describe the synthesis of several variants of this type of compound, their binding affinity and antagonist potency and show that, indeed, i n vivo properties can be greatly improved.
It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D recept... more It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.
Incidence and Clinical Characteristics Schizophrenia is a devastating psychiatric illness that af... more Incidence and Clinical Characteristics Schizophrenia is a devastating psychiatric illness that affects approximately 1% of the world population irrespective of ethnic, economical, or cultural boundaries: features that suggest the disorder has no simple, single causative factor. With current drug therapy, approximately 25% of patients recover to some extent within 5 years of starting treatment and about 65% of patients have recurring problems over many years. The remaining 10-15% of patients develop long-term incapacity and around 15% commit suicide. There are substantial costs, both direct and indirect, incurred by this disorder including those of drug treatment, residential accommodation, physician and other healthcare services, and loss of productivity in the workplace. Clinical symptoms are apparent relatively early in life, generally occurring between the ages of 15 and 45. They are characterized by the presence of positive symptoms, for example auditory hallucinations, disorganized thoughts, delusions, and irrational fears, and negative symptoms, including social withdrawal, diminished affect, poverty of speech, lack of energy, and the inability to experience pleasure. In addition, schizophrenic patients may suffer cognitive deficits including impaired attention, verbal fluency, memory recall, and executive function.
5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening... more 5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with > 500-fold selectivity over hD2 and > 200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.
The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a k... more The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3-and 2.4-Å resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small ␣-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.
Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA ... more Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA interference (RNAi) machinery. Three factors that mediate this mechanism have been identified: 1) the RNA-dependent RNA polymerase complex RdRC, 2) the Argonaute-containing RITS (RNA-induced initiation of transcriptional silencing) complex, and 3) the endoribonuclease Dicer ortholog Dcr1. S. pombe mutants lacking a new factor described here, Ers1, are completely defective in RNAi-dependent silencing of centromeric regions but, importantly, not in RNAiindependent silencing at the mat3M or tel2R loci. ers1⌬ cells likewise fail to convert centromeric pre-small interfering RNA transcripts into small interfering RNAs, are defective in histone H3 Lys 9 methylation, and are unable to recruit the RITS complex to centromeric sequences. Surprisingly, Ers1 lacks obvious orthologs outside of the genus Schizosaccharomyces. Within this group, it is diverging rapidly, raising the possibility that it is coevolving with target RNA elements.
Diabetic patients develop a cardiomyopathy that consists of ventricular hypertrophy and diastolic... more Diabetic patients develop a cardiomyopathy that consists of ventricular hypertrophy and diastolic dysfunction. Although the pathogenesis of this condition is poorly understood, previous studies implicated abnormal G-protein activation. In this work, mice with cardiac overexpression of the transcription factor peroxisome proliferator–activated receptor-α (PPAR-α) were examined as a model of diabetic cardiomyopathy. PPAR-α transgenic mice develop spontaneous cardiac hypertrophy, contractile dysfunction, and “fetal” gene induction. We examined the role of abnormal G-protein activation in the pathogenesis of cardiac dysfunction by crossing PPAR-α mice with transgenic mice with cardiac-specific overexpression of regulator of G-protein signaling subtype 4 (RGS4), a GTPase activating protein for Gq and Gi. Generation of compound transgenic mice demonstrated that cardiac RGS4 overexpression ameliorated the cardiomyopathic phenotype that occurred as a result of PPAR-α overexpression without ...
Bioorganic & Medicinal Chemistry Letters, 1999
The syntheses of a number of different N-linked heterocyclic pyrazole replacements based on the s... more The syntheses of a number of different N-linked heterocyclic pyrazole replacements based on the structure 1 are described (compounds 3-12) as hD4 ligands. After further optimisation the best compound identified was 13 which has high affinity for him (5.2 nM) and >300-fold selectivity for hD4 receptors over hD2 and hD3 receptors.
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Papers by Michael Rowley