ABSTRACT
Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase 1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation,sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.
Subject(s)
Azepines/pharmacology , Nerve Growth Factor/pharmacology , Receptor, trkA/agonists , Animals , Azepines/chemistry , Binding Sites , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Hippocampus/cytology , Hippocampus/metabolism , Humans , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Weight , NIH 3T3 Cells , Nerve Growth Factor/genetics , Nerve Growth Factor/metabolism , PC12 Cells , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/metabolism , Rats , Receptor, trkA/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.
Subject(s)
Azepines/pharmacology , Nerve Growth Factor/pharmacology , Receptor, trkA/agonists , Animals , Azepines/chemistry , Binding Sites , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Hippocampus/cytology , Hippocampus/metabolism , Humans , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Weight , NIH 3T3 Cells , Nerve Growth Factor/genetics , Nerve Growth Factor/metabolism , PC12 Cells , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/metabolism , Rats , Receptor, trkA/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
This study explored the effectiveness of biologic markers in alcoholism where correct diagnosis does not result from relatively simple inspection. After review of the use of individual biologic markers and their limitations, data is presented from a multivariate analysis of 351 young healthy male alcoholics and 339 nonalcoholic male patients in which an overall accuracy rate of 84.3% was obtained. A discussion of what biologic markers may be measuring, issues in sample selection, statistical issues and problems with laboratory variability in the use of biologic markers is presented.
