ABSTRACT
Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.
Subject(s)
Disease Models, Animal , Membrane Proteins/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridazines/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/therapeutic use , Usher Syndromes/drug therapy , Animals , High-Throughput Screening Assays , Humans , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrazoles/therapeutic use , Pyridazines/chemical synthesis , Pyridazines/chemistry , Pyridazines/therapeutic use , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Usher Syndromes/geneticsABSTRACT
Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIß (PI4KIIIß), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIß in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIß carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model.
Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Enterovirus/drug effects , Enterovirus/metabolism , Animals , Enterovirus/pathogenicity , Enzyme Activation/drug effects , Fluorescent Antibody Technique , HeLa Cells , Humans , Male , Mice , Molecular Structure , Pancreatitis/drug therapy , Pancreatitis/metabolism , Virus Replication/drug effectsABSTRACT
Rhinovirus (genus enterovirus) infections are responsible for many of the severe exacerbations of asthma and chronic obstructive pulmonary disease. Other members of the genus can cause life-threatening acute neurological infections. There is currently no antiviral drug approved for the treatment of such infections. We have identified a series of potent, broad-spectrum antiviral compounds that inhibit the replication of the human rhinovirus, Coxsackie virus, poliovirus, and enterovirus-71. The mechanism of action of the compounds has been established as inhibition of a lipid kinase, PI4KIIIß. Inhibition of hepatitis C replication in a replicon assay correlated with enterovirus inhibition.
ABSTRACT
Scaffold hopping from a non-basic series of 5-HT(2A) receptor antagonists developed in-house that possessed reduced activity in vivo enabled the discovery of a novel series of diaryl sulfones that gave excellent occupancy on oral dosing. Not only does this work further demonstrate that oral bioavailability of a given series can be enhanced by improving physicochemical parameters such as log P, but it corroborates the growing evidence that a protonated amine is not essential for affinity at aminergic GPCRs.
Subject(s)
Serotonin 5-HT2 Receptor Antagonists , Sulfones/chemical synthesis , Administration, Oral , Amines , Animals , Biological Availability , Drug Discovery , Humans , Ligands , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Serotonin Receptor Agonists , Sleep Wake Disorders/drug therapy , Sulfones/pharmacology , Sulfones/therapeutic useABSTRACT
Amalgamation of the structure-activity relationship of two series of GlyT1 inhibitors developed at Merck led to the discovery of a clinical candidate, compound 16 (DCCCyB), which demonstrated excellent in vivo occupancy of GlyT1 transporters in rhesus monkey as determined by displacement of a PET tracer ligand.
ABSTRACT
3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.
Subject(s)
GABA Agonists/pharmacology , GABA-A Receptor Agonists , Isoxazoles/pharmacology , Triazines/pharmacology , Animals , Anxiety/psychology , Behavior, Animal/drug effects , Convulsants/pharmacology , Dogs , Dose-Response Relationship, Drug , Electric Stimulation , Electrophysiology , Excitatory Postsynaptic Potentials/drug effects , Fibroblasts , Flumazenil/metabolism , GABA Agonists/metabolism , GABA Agonists/pharmacokinetics , GABA Modulators/metabolism , Hepatocytes/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Macaca mulatta , Male , Maze Learning/drug effects , Mice , Patch-Clamp Techniques , Postural Balance/drug effects , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Young AdultABSTRACT
This report describes the effect of replacing the central basic amine present in many known 5-HT(2A) ligands with an aromatic residue. We targeted the isomeric phenethylpyridines 2 and 3 and these compounds proved to be excellent leads, possessing good 5-HT(2A) receptor binding affinity and selectivity over the 5-HT(2C) subtype. Optimization of one isomer led to the identification of 25, a compound with sub-nanomolar 5-HT(2A) affinity and selectivity over 5-HT(2C) of greater than 4600-fold.
Subject(s)
Chemistry, Pharmaceutical/methods , Pyridines/chemistry , Pyridines/chemical synthesis , Receptor, Serotonin, 5-HT2A/chemistry , Serotonin Antagonists/chemistry , Serotonin Antagonists/chemical synthesis , Animals , Drug Design , Humans , Kinetics , Ligands , Models, Chemical , Molecular Conformation , Pyridines/pharmacology , Rats , Serotonin Antagonists/pharmacology , Sulfones/chemistryABSTRACT
Based on an existing series of 5-HT2A receptor ligands containing a basic nitrogen, we designed a non-basic lead that had reduced affinity for both the 5-HT2A receptor and the IKr potassium channel. The present paper describes the development of this lead to a novel series of non-basic piperidine sulfonamides and amides that have high affinity for the 5-HT2A receptor, whilst maintaining excellent selectivity over off target activities such as the IKr channel. This work has shown that the proposed pharmacophore model for the 5-HT2A receptor which suggests that a basic nitrogen is required for the binding of ligands is questionable.
Subject(s)
Serotonin 5-HT2 Receptor Antagonists , Humans , Molecular Structure , Receptor, Serotonin, 5-HT2A/metabolism , Structure-Activity RelationshipABSTRACT
(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (1) was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors and enhances performance in animal models of cognition. The routes of metabolism of this compound in vivo in rat have been well characterised, the identities of the major metabolites are confirmed by synthesis and their biological profiles were evaluated. An unusual oxidation of the pyrazolo[1,5-d][1,2,4]triazine core to the corresponding pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one scaffold by aldehyde oxidase has been observed.
Subject(s)
GABA Agonists/metabolism , GABA Agonists/pharmacokinetics , GABA-A Receptor Agonists , Isoxazoles/metabolism , Isoxazoles/pharmacokinetics , Protein Subunits/agonists , Triazines/metabolism , Triazines/pharmacokinetics , Animals , Dogs , Dose-Response Relationship, Drug , GABA Agonists/chemical synthesis , Hydrolysis , Isoxazoles/chemical synthesis , Macaca mulatta , Mice , Molecular Conformation , Rats , Stereoisomerism , Structure-Activity Relationship , Tissue Distribution , Triazines/chemical synthesisABSTRACT
Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.
Subject(s)
Pyridazines/chemical synthesis , Receptors, GABA-A/drug effects , Animals , Cell Line , Humans , In Vitro Techniques , Ligands , Male , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Patch-Clamp Techniques , Protein Subunits/physiology , Pyridazines/pharmacokinetics , Pyridazines/pharmacology , Radioligand Assay , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/physiology , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Incorporation of fluorine at the 4-position of an existing series of sulfonyl piperidine 5-HT2A antagonists gave compounds with increased selectivity over the IKr potassium channel. This work led to the identification of 3b, a compound that gave no increase in QTc in the anesthetized dog up to plasma levels as high as 148 microM. Furthermore, 3b has been shown to increase slow-wave sleep bout duration and to decrease the number of awakenings in rats, indicating the potential utility of 5-HT2A antagonists in the treatment of insomnia.
Subject(s)
Piperidines/pharmacology , Piperidines/therapeutic use , Serotonin 5-HT2 Receptor Antagonists , Sleep Initiation and Maintenance Disorders/drug therapy , Animals , Dogs , Drug Evaluation, Preclinical , Ligands , Molecular Structure , Piperidines/chemical synthesis , Rats , Structure-Activity Relationship , Time FactorsABSTRACT
(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors. 13 is orally bioavailable, readily penetrates the CNS, and enhances performance in animal models of cognition. It does not exhibit the convulsant, proconvulsant, or anxiogenic activity associated with nonselective GABA(A) inverse agonists.
Subject(s)
Cognition/drug effects , GABA-A Receptor Agonists , Isoxazoles/chemical synthesis , Nootropic Agents/chemical synthesis , Triazines/chemical synthesis , Administration, Oral , Animals , Binding Sites , Biological Availability , Dogs , Isoxazoles/adverse effects , Isoxazoles/pharmacology , Maze Learning/drug effects , Mice , Nootropic Agents/adverse effects , Nootropic Agents/pharmacology , Patch-Clamp Techniques , Protein Subunits , Radioligand Assay , Rats , Receptors, GABA-A/metabolism , Triazines/adverse effects , Triazines/pharmacologyABSTRACT
The identification of a novel series of 7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazines as GABA(A)alpha5 inverse agonists, which have both binding and functional (efficacy) selectivity for the benzodiazepine binding site of alpha5- over alpha1-, alpha2-, and alpha3-containing GABA(A) receptor subtypes, is described. Binding selectivity was determined to a large part by the degree of planarity of the fused ring system whereas functional selectivity was dependent on the nature of the heterocycle at the 3-position of the triazolopyridazine ring. 3-Furan and 5-methylisoxazole were shown to be optimal for GABA(A)alpha5 functional selectvity. 3-(5-Methylisoxazol-3-yl)-6-(2-pyridyl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine (43) was identified as a full inverse agonist at the GABA(A)alpha5 subtype with functional selectivity over the other GABA(A) receptor subtypes and good oral bioavailability.
Subject(s)
GABA-A Receptor Agonists , Isoxazoles/chemical synthesis , Phthalazines/chemical synthesis , Triazoles/chemical synthesis , Administration, Oral , Animals , Binding Sites , Biological Availability , Cell Line , Female , Humans , Isoxazoles/chemistry , Isoxazoles/pharmacology , Male , Maze Learning/drug effects , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Phthalazines/chemistry , Phthalazines/pharmacokinetics , Phthalazines/pharmacology , Protein Subunits/agonists , Radioligand Assay , Rats , Structure-Activity Relationship , Triazoles/pharmacokinetics , Triazoles/pharmacology , Xenopus laevisABSTRACT
A series of substituted 3,4-dihydronaphthalen-1(2H)-ones with high binding affinity for the benzodiazepine site of GABAA receptors containing the alpha5-subunit has been identified. These compounds have consistently higher binding affinity for the GABAA alpha5 receptor subtype over the other benzodiazepine-sensitive GABAA receptor subtypes (alpha1, alpha2 and alpha3). Compounds with a range of efficacies for the benzodiazepine site of alpha5-containing GABAA receptors were identified, including the alpha5 inverse agonist 3,3-dimethyl-8-methylthio-5-(pyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 22 and the alpha5 agonist 8-ethylthio-3-methyl-5-(1-oxidopyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 19.
Subject(s)
Naphthalenes/pharmacology , Receptors, GABA-A/drug effects , Allosteric Site , Benzodiazepines/antagonists & inhibitors , Binding Sites , GABA-A Receptor Agonists , GABA-A Receptor Antagonists , Humans , Ligands , Naphthalenes/chemical synthesis , Protein Binding , Protein Subunits , Structure-Activity RelationshipABSTRACT
Nonselective inverse agonists at the gamma-aminobutyric acid(A) (GABA-A) benzodiazepine binding site have cognition-enhancing effects in animals but are anxiogenic and can precipitate convulsions. Herein, we describe novel GABA-A alpha5 subtype inverse agonists leading to the identification of 16 as an orally active, functionally selective compound that enhances cognition in animals without anxiogenic or convulsant effects. Compounds of this type may be useful in the symptomatic treatment of memory impairment associated with Alzheimer's disease and related dementias.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , GABA-A Receptor Agonists , Nootropic Agents/chemical synthesis , Phthalazines/chemical synthesis , Triazoles/chemical synthesis , Animals , Binding, Competitive , Biological Availability , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Convulsants/chemical synthesis , Convulsants/chemistry , Convulsants/pharmacology , Dogs , Humans , Macaca mulatta , Mice , Nootropic Agents/chemistry , Nootropic Agents/pharmacology , Oocytes/metabolism , Patch-Clamp Techniques , Phthalazines/chemistry , Phthalazines/pharmacology , Radioligand Assay , Rats , Receptors, GABA-A/physiology , Structure-Activity Relationship , Triazoles/chemistry , Triazoles/pharmacology , Xenopus laevisABSTRACT
In pursuit of a GABA(A) alpha5-subtype-selective inverse agonist to enhance cognition, a series of 6,7-dihydro-2-benzothiophen-4(5H)-ones has been identified as a novel class of GABA(A) receptor ligands. These thiophenes have higher binding affinity for the GABA(A) alpha5 receptor subtype compared to the GABA(A) alpha1, alpha2, and alpha3 subtypes, and several analogues exhibit high GABA(A) alpha5 receptor inverse agonism. 6,6-Dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (43) has been identified as a full inverse agonist at the GABA(A) alpha5 receptor and is functionally selective over the other major GABA(A) receptor subtypes. 43 readily penetrates into the CNS to give selective occupancy of GABA(A) alpha5 receptors. In addition, 43 enhances cognitive performance in rats in the delayed 'matching-to-place' Morris water maze test-a hippocampal-dependent memory task-without the convulsant or proconvulsant activity associated with nonselective, GABA(A) receptor inverse agonists.
Subject(s)
Cognition/drug effects , GABA Agonists/chemical synthesis , Nootropic Agents/chemical synthesis , Receptors, GABA-A/drug effects , Thiazoles/chemical synthesis , Thiophenes/chemical synthesis , Animals , Brain/metabolism , Cell Line , Female , GABA Agonists/adverse effects , GABA Agonists/pharmacology , Hippocampus/physiology , Humans , In Vitro Techniques , Ligands , Male , Maze Learning/drug effects , Memory/drug effects , Mice , Nootropic Agents/adverse effects , Nootropic Agents/pharmacology , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Protein Subunits , Radioligand Assay , Rats , Seizures/chemically induced , Structure-Activity Relationship , Thiazoles/adverse effects , Thiazoles/pharmacology , Thiophenes/adverse effects , Thiophenes/pharmacology , Xenopus laevisABSTRACT
Nonselective inverse agonists at the benzodiazepine binding site on the GABA-A chloride ion channel enhance cognitive performance in animals but cannot be used in the treatment of cognitive disorders because of anxiogenic and convulsant side effects. We have identified a novel series of GABA-A alpha5 receptor ligands during our search for alpha5 receptor inverse agonists as potential cognition enhancers. In particular, 6,6-dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (26) has been identified as a functionally selective GABA-A alpha5 inverse agonist.
Subject(s)
GABA-A Receptor Agonists , Ketones/chemical synthesis , Thiophenes/chemical synthesis , Animals , Cells, Cultured , Cognition , Humans , Ketones/pharmacology , Mice , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Thiophenes/pharmacology , Xenopus laevisABSTRACT
On the basis of a spirocyclic ether screening lead, a series of acyclic sulfones have been identified as high-affinity, selective 5-HT(2A) receptor antagonists. Bioavailability lacking in the parent, 1-(2-(2,4-difluorophenyl)ethyl)-4-(phenylsulfonyl)piperidine (12), was introduced by using stability toward rat liver microsomes as a predictor of bioavailability. By this means, the 4-cyano- and 4-carboxamidophenylsulfonyl derivatives 26 and 31 were identified as orally bioavailable, brain-penetrant analogues suitable for evaluation in animal models. Bioavailability was also attainable by N substitution leading to the N-phenacyl derivative 35. IKr activity detected through counterscreening was reduced to insignificant levels in vivo with the latter compound.