ABSTRACT
Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.
ABSTRACT
The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.
Subject(s)
Huntington Disease , Animals , Brain/metabolism , Disease Models, Animal , Huntingtin Protein/metabolism , Huntington Disease/drug therapy , Mice , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , rho-Associated KinasesABSTRACT
Using an iterative structure-activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in vivo. Given the lack of understanding of endogenous class IIa HDAC substrates, we developed a surrogate readout to measure compound effects in vivo, by exploiting the >100-fold selectivity compound 12 exhibits over class I/IIb HDACs. We achieved adequate brain exposure with compound 12 in mice to estimate a class I/IIb deacetylation EC50, using class I substrate H4K12 acetylation and global acetylation levels as a pharmacodynamic readout. We observed excellent correlation between the compound 12 in vivo pharmacodynamic response and in vitro class I/IIb cellular activity. Applying the same relationship to class IIa HDAC inhibition, we estimated the compound 12 dose required to inhibit class IIa HDAC activity, for use in preclinical models of Huntington's disease.
ABSTRACT
We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and â¼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for â¼8â¯h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.
Subject(s)
Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Animals , Dose-Response Relationship, Drug , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Mice , Molecular Structure , Structure-Activity RelationshipABSTRACT
Synthesis of (S)-2-methyl-3-fluorophenyl cyclopentanone methyl ester (1S)-1 has been achieved by both inter- and intramolecular alkylation reactions on multigram scale, using chiral pool reagents. The intramolecular variant is a novel example of a chiral bis-electrophile reacting with a carbon nucleophile to form an enantiomerically pure all-carbon quaternary center.
ABSTRACT
Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington's disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a "closed-loop" form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.
Subject(s)
Drug Design , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Huntington Disease/drug therapy , Animals , Dose-Response Relationship, Drug , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacokinetics , Histone Deacetylases/classification , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Histone deacetylase (HDAC) inhibitors have received considerable attention as potential therapeutics for a variety of cancers and neurological disorders. Recent publications on a class of pimelic diphenylamide HDAC inhibitors have highlighted their promise in the treatment of the neurodegenerative diseases Friedreich's ataxia and Huntington's disease, based on efficacy in cell and mouse models. These studies' authors have proposed that the unique action of these compounds compared to hydroxamic acid-based HDAC inhibitors results from their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3, resulting in a distinctive pharmacological profile and reduced toxicity. Here, we evaluate the HDAC subtype selectivity, cellular activity, absorption, distribution, metabolism and excretion (ADME) properties, as well as the central pharmacodynamic profile of one such compound, HDACi 4b, previously described to show efficacy in vivo in the R6/2 mouse model of Huntington's disease. Based on our data reported here, we conclude that while the in vitro selectivity and binding mode are largely in agreement with previous reports, the physicochemical properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi 4b render this compound suboptimal to investigate central Class I HDAC inhibition in vivo in mouse per oral administration. A drug administration regimen using HDACi 4b dissolved in drinking water was used in the previous proof of concept study, casting doubt on the validation of CNS HDAC3 inhibition as a target for the treatment of Huntington's disease. We highlight physicochemical stability and metabolic issues with 4b that are likely intrinsic liabilities of the benzamide chemotype in general.
Subject(s)
Central Nervous System/metabolism , Friedreich Ataxia/drug therapy , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Huntington Disease/drug therapy , Pimelic Acids/pharmacology , Administration, Oral , Animals , Caco-2 Cells , Chromatography, High Pressure Liquid , Dogs , Friedreich Ataxia/enzymology , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/pharmacokinetics , Histone Deacetylase Inhibitors/therapeutic use , Humans , Huntington Disease/enzymology , Madin Darby Canine Kidney Cells , Mice , Microsomes, Liver/metabolism , Pimelic Acids/administration & dosage , Pimelic Acids/chemical synthesis , Pimelic Acids/pharmacokinetics , Pimelic Acids/therapeutic use , Tandem Mass SpectrometryABSTRACT
Until the recent advent of genetically engineered drugs, small molecules constituted the predominant method of treatment for autoimmune diseases. Both modalities have advantages and disadvantages; while protein-based therapeutics interfere very selectively with the function of their biological targets, they have to be administered subcutaneously or intravenously. Small molecules have the potential for oral administration. Due to their cell permeability, they can interact with extra- and intracellular targets, thus opening opportunities for interfering with novel biochemical pathways. We herein describe the preclinical stages of typical small-molecule research programmes and outline hurdles that may have to be overcome. A few examples of small molecules that are currently under clinical evaluation and arose from diverse discovery pathways will be discussed.
Subject(s)
Autoimmune Diseases/drug therapy , Drug Design , Immunosuppressive Agents/pharmacology , Animals , HumansABSTRACT
The development of a series of novel aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, JNK2 and CDK2, and the in vitro inhibitory value for a c-Jun cellular assay are discussed.
Subject(s)
Enzyme Inhibitors/pharmacology , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Mitogen-Activated Protein Kinase 9/antagonists & inhibitors , Models, Chemical , Structure-Activity RelationshipABSTRACT
The study of non-oxazole containing indole fragments as inhibitors of inosine monophosphate dehydrogenase (IMPDH) is described. The synthesis and in vitro inhibitory values for IMPDH II are discussed.
Subject(s)
Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Indoles/pharmacology , Carbamates/chemistry , Carbamates/pharmacology , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hydrogen Bonding , Indoles/chemical synthesis , Indoles/chemistry , Models, Molecular , Molecular Structure , Molecular Weight , Oxazoles/pharmacology , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Sensitivity and Specificity , Structure-Activity RelationshipABSTRACT
The elaboration of previously reported indole fragments as inhibitors of inosine monophosphate dehydrogenase (IMPDH) is described. The synthesis, in vitro inhibitory values for IMPDH II, PBMC proliferation and physicochemical properties are discussed.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Indoles/chemical synthesis , Indoles/pharmacology , Biological Transport, Active/drug effects , Caco-2 Cells , Cell Proliferation/drug effects , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , In Vitro Techniques , Indoles/chemistry , Leukocytes, Mononuclear/drug effects , Molecular Structure , Molecular Weight , Structure-Activity RelationshipABSTRACT
The synthesis and biological activity of a novel series of 7-methoxy-6-oxazol-5-yl-2,3-dihydro-1H-quinazolin-4-ones are described. Some of these compounds were found to be potent inhibitors of inosine 5'-monophosphate dehydrogenase type II (IMPDH II).
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Quinazolines/chemistry , Quinazolines/pharmacology , Enzyme Inhibitors/chemical synthesis , Humans , Molecular Conformation , Quinazolines/chemical synthesis , Structure-Activity RelationshipABSTRACT
The development of a series of novel quinazolinethiones and quinazolinediones as inhibitors of inosine monophosphate dehydrogenase (IMPDH) is described. The synthesis, in vitro inhibitory values for IMPDH II and in vitro inhibitory value for PBMC proliferation are discussed.
Subject(s)
IMP Dehydrogenase/antagonists & inhibitors , Quinazolines/chemical synthesis , Quinazolines/pharmacology , Blood Cells , Cell Proliferation/drug effects , Enzyme Inhibitors/chemical synthesis , Humans , Inhibitory Concentration 50 , Ketones/chemical synthesis , Ketones/pharmacology , Monocytes/drug effects , Structure-Activity Relationship , Thiones/chemical synthesis , Thiones/pharmacologyABSTRACT
A series of bicyclic heteroaryl ring systems was considered as a replacement for the 3-cyclopentyloxy-4-methoxyphenyl moiety in rolipram resulting in the discovery of 8-methoxyquinoline-5-carboxamides as potent inhibitors of phosphodiesterase type 4 (PDE4).
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Amides/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Phosphodiesterase Inhibitors/therapeutic use , Amides/pharmacology , Anti-Asthmatic Agents/pharmacology , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Phosphodiesterase Inhibitors/pharmacologyABSTRACT
The synthesis and pharmacological profile of a novel series of 2-substituted 8-methoxyquinolines is described. The 2-trifluoromethyl compound was found to be a potent inhibitor of phosphodiesterase type 4 (PDE4).
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Animals , Area Under Curve , Cyclic Nucleotide Phosphodiesterases, Type 4 , Guinea Pigs , Phosphodiesterase Inhibitors/pharmacokinetics , Quinolines/pharmacokinetics , Vomiting/chemically inducedABSTRACT
The syntheses and pharmacological profiles of some 2-trifluoromethyl-8-methoxyquinoline-5-carboxamides are described. SCH351591 is a potent selective inhibitor of phosphodiesterase type 4 (PDE4).
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Cyclic N-Oxides/chemical synthesis , Cyclic N-Oxides/pharmacology , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Animals , Area Under Curve , Cyclic N-Oxides/pharmacokinetics , Cyclic Nucleotide Phosphodiesterases, Type 4 , Guinea Pigs , Phosphodiesterase Inhibitors/pharmacokinetics , Quinolines/pharmacokineticsABSTRACT
The synthesis and pharmacological profile of a novel series of 7-methoxy-furo[2,3-c]pyridine-4-carboxamides is described. Some of these compounds were found to be potent inhibitors of phosphodiesterase type 4 (PDE4).