Molecular Therapy - Methods & Clinical Development, 2019
Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (S... more Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector adeno-associated virus (AAV)rh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vector genomes [vg]/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion, and neuroinflammation at 12 weeks and 25 weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0E+12 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.
Proceedings of the National Academy of Sciences, 2016
Significance Laquinimod is an oral drug currently being evaluated for the treatment of relapsing,... more Significance Laquinimod is an oral drug currently being evaluated for the treatment of relapsing, remitting, and primary progressive multiple sclerosis as well as Huntington’s disease. It is thought that laquinimod has a primary effect on the peripheral innate immune system and also acts directly on resident cells within the CNS. However, the exact mechanism of action of laquinimod has not been fully elucidated. We investigated gene expression in laquinimod-treated mice and show induction of genes downstream to activation of the aryl hydrocarbon receptor (AhR). In this paper, we examine the role of the AhR in laquinimod treatment of experimental autoimmune encephalomyelitis and demonstrate that AhR is the molecular target of laquinimod in this model.
Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by... more Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by secondary endpoints in clinical trials. Originally described as a dopamine stabilizer, this mechanism is insufficient to explain the clinical and preclinical effects of pridopidine. This study therefore explored pridopidine's potential mechanisms of action. The effect of pridopidine versus sham treatment on genome-wide expression profiling in the rat striatum was analysed and compared to the pathological expression profile in Q175 knock-in (Q175 KI) vs Q25 WT mouse models. A broad, unbiased pathway analysis was conducted, followed by testing the enrichment of relevant pathways. Pridopidine upregulated the BDNF pathway (P ¼ 1.73E-10), and its effect on BDNF secretion was sigma 1 receptor (S1R) dependent. Many of the same genes were independently found to be downregulated in Q175 KI mice compared to WT (5.2e-7 < P < 0.04). In addition, pridopidine treatment upregulated the glucocorticoid receptor (GR) response, D1R-associated genes and the AKT/PI3K pathway (P ¼ 1E-10, P ¼ 0.001, P ¼ 0.004, respectively). Pridopidine upregulates expression of BDNF, D1R, GR and AKT/PI3K pathways, known to promote neuronal plasticity and survival, as well as reported to demonstrate therapeutic benefit in HD animal models. Activation of S1R, necessary for its effect on the BDNF pathway, represents a core component of the mode of action of pridopidine. Since the newly identified pathways are downregulated in neurodegenerative diseases, including HD, these findings suggest that pridopidine may exert neuroprotective effects beyond its role in alleviating some symptoms of HD.
Recombinant biotin-binding phages were affinity-selected from a random peptide library expressed ... more Recombinant biotin-binding phages were affinity-selected from a random peptide library expressed on the surface of filamentous phage. Phage binding to biotinylated proteins was half-maximally inhibited by micromolar concentrations of a monobiotinylated molecule. Sequencing of the peptide inserts of selected phages led to the identification of a previously unknown biotin-binding motif, CXWXPPF(K or R)XXC. A synthetic peptide containing this sequence motif inhibited streptavidin binding to biotinylated BSA with an IC50 of 50 microM. This compound represents the shortest non-avidin biotin-binding peptide identified to date. Our results illustrate that phage display technology can be used to identify novel ligands for a small non-proteinaceous molecule.
Liver microsomal preparations are routinely used to predict drug interactions that can occur in v... more Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent K m and V max values of 2.1 vs. 2.8 lM and 0.74 nmolAEmin)1 per mg microsomal protein vs. 0.11 nmolAEmin)1 per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC 50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC 50 in hepatocytes than in microsomes. The cellassociated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.
Proceedings of the National Academy of Sciences, 1996
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containin... more Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance bindi...
Supporting Information 4,5-Dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidine carob... more Supporting Information 4,5-Dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidine caroboxamides are potent, selective HIV-1 integrase inhibitors with good pharmacokinetic profiles in preclinical species.
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which nov... more Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.
A strategy to obtain a fully orthogonal estrogen-receptorbased gene switch responsive to molecule... more A strategy to obtain a fully orthogonal estrogen-receptorbased gene switch responsive to molecules with acceptable pharmacological properties is presented. From a series of tetrahydrofluorenones active on the wild-type estrogen receptor (ER) an inactive analogue is chosen as a new lead compound. Coevolution of receptor mutants and ligands leads to an ER-based gene switch suitable for studies in animal models.
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which nov... more Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired offtarget activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC 50) 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.
Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzyme... more Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIVintegrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide 38, with a CIC 95 of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent.
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containin... more Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific ␣-receptor subunit (CNTFR) and the signal-transducing -subunits gp130 and leukemia inhibitory factor receptor- (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in nonneuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either ␣or -receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.
A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT s... more A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [ 14 C]UDPGA, and [ 14 C]-labelled glucuronidation products are separated from the unreacted nucleotide sugar by solid phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics was studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of 5 compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone, UGT1A6 and UGT1A8 for 1-naphthol, UGT2B7 for naloxone, UGT1A3 and UGT2B7 for ketoprofen, and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional HPLC method coupled to mass spectrometric detection. The new assay should prove to be valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, for identifying high turnover compounds during drug discovery, and for reaction phenotyping studies.
Human HIV integrase inhibitors are a novel class of antiretroviral drugs that act by blocking inc... more Human HIV integrase inhibitors are a novel class of antiretroviral drugs that act by blocking incorporation of the proviral DNA into the host cell genome, a crucial step in the life cycle of HIV. In the present work, quantitative methods for prediction of human pharmacokinetics were used to guide the selection of development candidates from a series of dihydroxypyrimidine and N-methylpyrimidinone carboxamide inhibitors of HIV integrase, which are cleared mainly by O-glucuronidation. The pharmacokinetics of 10 drugs from this series were determined in several preclinical species, including rats, dogs, rhesus monkeys and rabbits, and the in vitro turnover, plasma protein binding and blood to plasma partition ratio was studied using preparations from both preclinical species and humans. Two clearance prediction methods, based on physiologically based scaling or allometric scaling normalized for differences in microsomal turnover, were used to extrapolate human clearance. For three clinical candidates, including the novel AIDS drug raltegravir, oral drug exposure was predicted and compared to that observed in healthy human volunteers. Both scaling methods gave a reasonable correspondence between predicted and observed oral exposure. Prediction errors for the physiologically based method were less than 1.7-fold for 2 drugs, including raltegravir, and less than 3.5fold for one drug. The exposures predicted using normalized allometric scaling were within 1.1-to 1.5-fold of observed values for all 3 compounds. The accuracy of prediction by normalized allometric scaling was similar when using data from either 4 preclinical species, or from rats and dogs only. The prediction methods used may be applicable to other drugs cleared predominantly by glucuronidation.
Molecular Therapy - Methods & Clinical Development, 2019
Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (S... more Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector adeno-associated virus (AAV)rh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vector genomes [vg]/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion, and neuroinflammation at 12 weeks and 25 weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0E+12 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.
Proceedings of the National Academy of Sciences, 2016
Significance Laquinimod is an oral drug currently being evaluated for the treatment of relapsing,... more Significance Laquinimod is an oral drug currently being evaluated for the treatment of relapsing, remitting, and primary progressive multiple sclerosis as well as Huntington’s disease. It is thought that laquinimod has a primary effect on the peripheral innate immune system and also acts directly on resident cells within the CNS. However, the exact mechanism of action of laquinimod has not been fully elucidated. We investigated gene expression in laquinimod-treated mice and show induction of genes downstream to activation of the aryl hydrocarbon receptor (AhR). In this paper, we examine the role of the AhR in laquinimod treatment of experimental autoimmune encephalomyelitis and demonstrate that AhR is the molecular target of laquinimod in this model.
Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by... more Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by secondary endpoints in clinical trials. Originally described as a dopamine stabilizer, this mechanism is insufficient to explain the clinical and preclinical effects of pridopidine. This study therefore explored pridopidine's potential mechanisms of action. The effect of pridopidine versus sham treatment on genome-wide expression profiling in the rat striatum was analysed and compared to the pathological expression profile in Q175 knock-in (Q175 KI) vs Q25 WT mouse models. A broad, unbiased pathway analysis was conducted, followed by testing the enrichment of relevant pathways. Pridopidine upregulated the BDNF pathway (P ¼ 1.73E-10), and its effect on BDNF secretion was sigma 1 receptor (S1R) dependent. Many of the same genes were independently found to be downregulated in Q175 KI mice compared to WT (5.2e-7 < P < 0.04). In addition, pridopidine treatment upregulated the glucocorticoid receptor (GR) response, D1R-associated genes and the AKT/PI3K pathway (P ¼ 1E-10, P ¼ 0.001, P ¼ 0.004, respectively). Pridopidine upregulates expression of BDNF, D1R, GR and AKT/PI3K pathways, known to promote neuronal plasticity and survival, as well as reported to demonstrate therapeutic benefit in HD animal models. Activation of S1R, necessary for its effect on the BDNF pathway, represents a core component of the mode of action of pridopidine. Since the newly identified pathways are downregulated in neurodegenerative diseases, including HD, these findings suggest that pridopidine may exert neuroprotective effects beyond its role in alleviating some symptoms of HD.
Recombinant biotin-binding phages were affinity-selected from a random peptide library expressed ... more Recombinant biotin-binding phages were affinity-selected from a random peptide library expressed on the surface of filamentous phage. Phage binding to biotinylated proteins was half-maximally inhibited by micromolar concentrations of a monobiotinylated molecule. Sequencing of the peptide inserts of selected phages led to the identification of a previously unknown biotin-binding motif, CXWXPPF(K or R)XXC. A synthetic peptide containing this sequence motif inhibited streptavidin binding to biotinylated BSA with an IC50 of 50 microM. This compound represents the shortest non-avidin biotin-binding peptide identified to date. Our results illustrate that phage display technology can be used to identify novel ligands for a small non-proteinaceous molecule.
Liver microsomal preparations are routinely used to predict drug interactions that can occur in v... more Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent K m and V max values of 2.1 vs. 2.8 lM and 0.74 nmolAEmin)1 per mg microsomal protein vs. 0.11 nmolAEmin)1 per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC 50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC 50 in hepatocytes than in microsomes. The cellassociated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.
Proceedings of the National Academy of Sciences, 1996
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containin... more Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance bindi...
Supporting Information 4,5-Dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidine carob... more Supporting Information 4,5-Dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidine caroboxamides are potent, selective HIV-1 integrase inhibitors with good pharmacokinetic profiles in preclinical species.
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which nov... more Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.
A strategy to obtain a fully orthogonal estrogen-receptorbased gene switch responsive to molecule... more A strategy to obtain a fully orthogonal estrogen-receptorbased gene switch responsive to molecules with acceptable pharmacological properties is presented. From a series of tetrahydrofluorenones active on the wild-type estrogen receptor (ER) an inactive analogue is chosen as a new lead compound. Coevolution of receptor mutants and ligands leads to an ER-based gene switch suitable for studies in animal models.
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which nov... more Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired offtarget activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC 50) 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.
Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzyme... more Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIVintegrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide 38, with a CIC 95 of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent.
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containin... more Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific ␣-receptor subunit (CNTFR) and the signal-transducing -subunits gp130 and leukemia inhibitory factor receptor- (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in nonneuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either ␣or -receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.
A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT s... more A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [ 14 C]UDPGA, and [ 14 C]-labelled glucuronidation products are separated from the unreacted nucleotide sugar by solid phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics was studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of 5 compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone, UGT1A6 and UGT1A8 for 1-naphthol, UGT2B7 for naloxone, UGT1A3 and UGT2B7 for ketoprofen, and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional HPLC method coupled to mass spectrometric detection. The new assay should prove to be valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, for identifying high turnover compounds during drug discovery, and for reaction phenotyping studies.
Human HIV integrase inhibitors are a novel class of antiretroviral drugs that act by blocking inc... more Human HIV integrase inhibitors are a novel class of antiretroviral drugs that act by blocking incorporation of the proviral DNA into the host cell genome, a crucial step in the life cycle of HIV. In the present work, quantitative methods for prediction of human pharmacokinetics were used to guide the selection of development candidates from a series of dihydroxypyrimidine and N-methylpyrimidinone carboxamide inhibitors of HIV integrase, which are cleared mainly by O-glucuronidation. The pharmacokinetics of 10 drugs from this series were determined in several preclinical species, including rats, dogs, rhesus monkeys and rabbits, and the in vitro turnover, plasma protein binding and blood to plasma partition ratio was studied using preparations from both preclinical species and humans. Two clearance prediction methods, based on physiologically based scaling or allometric scaling normalized for differences in microsomal turnover, were used to extrapolate human clearance. For three clinical candidates, including the novel AIDS drug raltegravir, oral drug exposure was predicted and compared to that observed in healthy human volunteers. Both scaling methods gave a reasonable correspondence between predicted and observed oral exposure. Prediction errors for the physiologically based method were less than 1.7-fold for 2 drugs, including raltegravir, and less than 3.5fold for one drug. The exposures predicted using normalized allometric scaling were within 1.1-to 1.5-fold of observed values for all 3 compounds. The accuracy of prediction by normalized allometric scaling was similar when using data from either 4 preclinical species, or from rats and dogs only. The prediction methods used may be applicable to other drugs cleared predominantly by glucuronidation.
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Papers by Ralph Laufer