ABSTRACT
Dopamine (DA)-producing neurons are critically involved in the production of motor behaviors in multiple circuits that are conserved from basal vertebrates to mammals. Although there is increasing evidence that DA neurons in the hypothalamus play a locomotor role, their precise contributions to behavior and the circuit mechanisms by which they are achieved remain unclear. Here, we demonstrate that tyrosine-hydroxylase-2-expressing (th2+) DA neurons in the zebrafish hypothalamus fire phasic bursts of activity to acutely promote swimming and modulate audiomotor behaviors on fast timescales. Their anatomy and physiology reveal two distinct functional DA modules within the hypothalamus. The first comprises an interconnected set of cerebrospinal-fluid-contacting DA nuclei surrounding the 3rd ventricle, which lack distal projections outside of the hypothalamus and influence locomotion through unknown means. The second includes neurons in the preoptic nucleus, which send long-range projections to targets throughout the brain, including the mid- and hindbrain, where they activate premotor circuits involved in swimming and sensorimotor integration. These data suggest a broad regulation of motor behavior by DA neurons within multiple hypothalamic nuclei and elucidate a novel functional mechanism for the preoptic DA neurons in the initiation of movement.
Subject(s)
Brain Stem/physiology , Dopaminergic Neurons/metabolism , Preoptic Area/physiology , Swimming/physiology , Animals , Brain Stem/cytology , Evoked Potentials, Motor/physiology , Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , Intravital Microscopy/methods , Male , Microscopy, Fluorescence, Multiphoton , Models, Animal , Nerve Net/physiology , Optogenetics , Preoptic Area/cytology , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , Video Recording , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 µg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.
Subject(s)
ADAM Proteins/antagonists & inhibitors , ADAM Proteins/metabolism , Biphenyl Compounds/pharmacology , Procollagen N-Endopeptidase/antagonists & inhibitors , Procollagen N-Endopeptidase/metabolism , Protease Inhibitors/pharmacology , Sulfonamides/pharmacology , ADAMTS4 Protein , Animals , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacokinetics , Drug Design , Humans , Male , Matrix Metalloproteinase 13/metabolism , Models, Molecular , Osteoarthritis/drug therapy , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , Proteoglycans/metabolism , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.
Subject(s)
ADAM Proteins/antagonists & inhibitors , ADAM Proteins/metabolism , Chemistry, Pharmaceutical/methods , Osteoarthritis/drug therapy , Procollagen N-Endopeptidase/antagonists & inhibitors , Procollagen N-Endopeptidase/metabolism , Sulfonamides/chemical synthesis , ADAMTS4 Protein , Cartilage/drug effects , Cartilage/metabolism , Drug Design , Humans , Inhibitory Concentration 50 , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 2/metabolism , Models, Chemical , Molecular Conformation , Proteoglycans/chemistry , Sulfonamides/pharmacologyABSTRACT
N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-microM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.
Subject(s)
ADAM Proteins/antagonists & inhibitors , ADAM Proteins/chemistry , Acetamides/chemical synthesis , Acetamides/pharmacology , ADAM Proteins/metabolism , ADAMTS4 Protein , ADAMTS5 Protein , Animals , Chemistry, Pharmaceutical/methods , Cytochrome P-450 CYP3A , Cytochrome P-450 CYP3A Inhibitors , Cytochrome P-450 Enzyme Inhibitors , Drug Design , Humans , Inhibitory Concentration 50 , Microsomes, Liver/drug effects , Models, Chemical , Osteoarthritis/drug therapy , Procollagen N-Endopeptidase/metabolism , RatsABSTRACT
Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.
Subject(s)
ADAM Proteins/chemistry , Procollagen N-Endopeptidase/chemistry , ADAMTS4 Protein , ADAMTS5 Protein , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Protein ConformationABSTRACT
5'-Phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. Selected compounds 23, 33-35 show sub-micromolar ADAMTS-5 potency and strong SAR trends with selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP12, and MMP13. This series of compounds represents progress toward a selective ADAMTS-5 inhibitor as a disease modifying osteoarthritis drug.
Subject(s)
Endopeptidases/metabolism , Indoles/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Thiadiazoles/chemistry , Molecular Structure , Protease Inhibitors/chemical synthesis , Spiro Compounds/chemical synthesis , Structure-Activity Relationship , Thiadiazoles/chemical synthesis , Thiadiazoles/pharmacologyABSTRACT
5-Benzylidene-2-thioxo-thiazolidin-4-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. The identified compounds show micromolar ADAMTS-5 potency and demonstrate SAR trends for both the aryl group and thioxothiazolidinone zinc chelator. This series of compounds represents steps toward a metalloprotease inhibitor as a disease-modifying osteoarthritis drug.
Subject(s)
ADAM Proteins/antagonists & inhibitors , Thiazolidinediones/chemical synthesis , Thiazolidinediones/pharmacology , ADAMTS5 Protein , Chelating Agents , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Metalloproteases/antagonists & inhibitors , Osteoarthritis/drug therapy , Structure-Activity Relationship , ZincABSTRACT
A series of 5-((1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one inhibitors of ADAMTS-5 (aggrecanase-2) is described. These compounds show microM functional inhibition of ADAMTS-5, and represent a new class of agents with the potential of inhibiting degradation of aggrecan, a major component of cartilage which is lost in osteoporosis. Compound 12 is noteworthy in that it has an ADAMTS-5 IC50: 1.1 microM and shows >40-fold functional selectivity over ADAMTS-4 (aggrecanase-1).
Subject(s)
ADAM Proteins/antagonists & inhibitors , Thiazolidinediones/chemical synthesis , ADAMTS4 Protein , ADAMTS5 Protein , Aggrecans/drug effects , Aggrecans/metabolism , Cartilage , Humans , Inhibitory Concentration 50 , Osteoporosis/drug therapy , Procollagen N-Endopeptidase , Structure-Activity Relationship , Thiazolidinediones/pharmacologyABSTRACT
Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of biphenylsulfonamidocarboxylic acid inhibitors of aggrecanase-1 are reported. Compound 24 demonstrated 89% inhibition of proteoglycan degradation at 10 microg/mL and has an oral bioavailability in rat of 35%.
