ABSTRACT
Compound 5 was identified from a high-throughput screening campaign as a small molecule pharmacological chaperone of glucocerebrocidase (GCase), a lysosomal hydrolase encoded by the GBA1 gene, variants of which are associated with Gaucher disease and Parkinson's disease. Further investigations revealed that compound 5 was slowly transformed into a regio-isomeric compound (6) in PBS buffer, plausibly via a ring-opening at hemiaminal moiety accompanied by subsequent intramolecular CC bond formation. Utilising this unexpected skeletal rearrangement reaction, a series of compound 6 analogues was synthesized which yielded multiple potent GCase pharmacological chaperones with sub-micromolar EC50 values as exemplified by compound 38 (EC50 = 0.14 µM).
Subject(s)
Gaucher Disease , Parkinson Disease , Humans , Glucosylceramidase/genetics , Mutation , Gaucher Disease/drug therapy , Molecular ChaperonesABSTRACT
Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of â¼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified in this work provide a novel starting point for further investigation and optimization.
Subject(s)
Mitophagy , Parkinson Disease , Humans , Mitophagy/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Ubiquitination/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Mutation , Parkinson Disease/drug therapy , Parkinson Disease/genetics , Parkinson Disease/metabolism , Thiolester Hydrolases/genetics , Thiolester Hydrolases/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolismABSTRACT
Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.
Subject(s)
Mitochondrial Proteins/metabolism , Mitophagy , Parkinson Disease/metabolism , Protein Kinases/metabolism , Thiolester Hydrolases/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line , Fibroblasts , HumansABSTRACT
We previously reported a series of 8-methyl-2-aryl-5-alkylaminoquinolines as a novel class of corticotropin-releasing factor-1 (CRF(1)) receptor antagonists. A critical issue encountered for this series of compounds was low aqueous solubility at physiological pH (pH 7.4). To address this issue, derivatization at key sites (R(2), R(3), R(5), R(5'), and R(8)) was performed and the relationships between structure and solubility were examined. As a result, it was revealed that introduction of a methoxy substituent at the C(8) position had a positive impact on the solubility of the derivatives. Consequently, through in vivo and in vitro biological studies, compound 21d was identified as a potent, orally active CRF(1) receptor antagonist with improved physicochemical properties.
Subject(s)
Aminoquinolines/chemistry , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Administration, Oral , Aminoquinolines/chemical synthesis , Aminoquinolines/pharmacokinetics , Animals , Behavior, Animal/drug effects , Drug Design , Half-Life , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Rats , Receptors, Corticotropin-Releasing Hormone/metabolism , Solubility , Structure-Activity RelationshipABSTRACT
This paper describes the design, synthesis, and structure-activity relationships of a novel series of 7-dialkylamino-3-phenyl-6-methoxy pyrazolo[5,1-b]thiazole derivatives for use as selective antagonists of the corticotropin-releasing factor 1 (CRF(1)) receptor. The most promising compound, N-butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]thiazole-7-amine (6t), showed high affinity (IC(50) = 70 nM) and functional antagonism (IC(50) = 7.1 nM) for the human CRF(1) receptor as well as dose-dependent inhibition of the CRF-induced increase in the plasma adrenocorticotropic hormone (ACTH) concentration at a dose of 30 mg/kg (po). Further, in the light/dark test in mice, the compound 6t showed anxiolytic activity at a dose of 30 mg/kg (po).
Subject(s)
Anti-Anxiety Agents/chemical synthesis , Pyrazoles/chemical synthesis , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Thiazoles/chemical synthesis , Administration, Oral , Adrenocorticotropic Hormone/blood , Animals , Anti-Anxiety Agents/chemistry , Anti-Anxiety Agents/pharmacology , Cell Line, Tumor , Corticotropin-Releasing Hormone/pharmacology , Cyclic AMP/metabolism , Defecation/drug effects , Drug Design , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Pyrazoles/chemistry , Pyrazoles/pharmacology , Radioligand Assay , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
We designed and synthesized a series of 2-Ar-8-methyl-5-alkylaminolquinolines as potent corticotropin-releasing factor 1 (CRF(1)) receptor antagonists. The structure-activity relationships of substituents at each position (R(3), R(5), R(5'), and R(8)) was investigated. By derivatization, three compounds (6, 14b, and 14c) were identified as orally active CRF(1) receptor antagonists.
Subject(s)
Quinolines/chemistry , Quinolines/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Administration, Oral , Animals , Depression/drug therapy , Drug Design , HEK293 Cells , Humans , Models, Molecular , Quinolines/administration & dosage , Quinolines/chemical synthesis , Rats , Receptors, Corticotropin-Releasing Hormone/metabolism , Structure-Activity RelationshipABSTRACT
A series of 5-alkylaminolquinolines was designed and synthesized as potential novel CRF(1) receptor antagonists. The structure-activity relationships (SARs) of the substituents on each position (R(2), R(3), R(5) and R(5')) were investigated.