Carboxylic acids and derivatives thereof and pharmaceutical compositions containing them
A composition and drug technology, applied in the direction of drug combination, anhydride/acid/halide active ingredients, medical preparations containing active ingredients, etc.
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Embodiment 1
[0100] Long-chain acyl-CoAs are ligands for HNF-4α
[0101] Acyl-CoAs of various chain lengths and degrees of saturation were found to bind specifically to HNF-4α.
[0102] Binds to the HNF-4α ligand-binding domain incorporated into glutathione-S-transferase (GST-HNF-4α (LBD)) or to the full-length HNF-4α protein tagged with 6 histidines (His-HNF-4α )as an example. Palmitoyl(16:0)-CoA bound to the ligand-binding domain or full-length HNF-4α protein is saturable with a Kd of 2.6 μM and approaches the saturation of 1 mole of fatty acyl-CoA / mole of HNF-4α ( Figure 1A). Binding is specific for acyl-CoA and has no activity against free fatty acids or free CoA. Binding of acyl-CoAs of various chain lengths and degrees of saturation was demonstrated by competition with radiolabeled palmitoyl(16:0)-CoA bound to recombinant GST-HNF-4α (LBD) or His-HNF-4α (attached Figure 1B). No binding was observed for saturated fatty acyl-CoAs with chain lengths shorter than C12. However, the b...
Embodiment 2
[0105] Regulation of HNF-4α activity by long-chain acyl-CoAs
[0106]HNF-4α activity as a function of long-chain acyl-CoA binding by studying the association of HNF-4α with its apo CIII promoter sequence (-87 / -66)6 C3P elements at various chain lengths with or without addition, saturation, Binding in the presence of acyl-CoA and degree of substitution were evaluated.
[0107] Binding was confirmed by gel mobility shift analysis. As shown in Figure 2, as the concentration of His-HNF-4α increases, the binding of C3P to HNF-4α also increases and is activated by natural saturated fatty acyl-CoA with a chain length of C12-C16. Activation was concentration dependent and was maximal in the presence of myristoyl(14:0)-CoA in the concentration range required for its binding to HNF-4α. Furthermore, some fatty acyl-CoAs as well as heterologous acyl-CoAs were found to act as true HNF-4α antagonists, ie inhibit their internal binding to their cognate enhancers.
[0108] Thus, incubation...
Embodiment 3
[0110] Regulation of HNF-4α-induced transcription by HNF-4α agonists and antagonists
[0111] The effects of agonistic or antagonistic HNF-4α-ligands were further analyzed by analysis catalyzed by addition of HeLa nuclear extracts, induced by recombinant HNF-4α, initiated by HSV thymidine kinase and chicken ovalbumin promoter, and induced by apo The in vitro transcription rate of the test template consisting of a 377 bp G-less cassette enhanced by three copies of the CIII gene promoter was evaluated. Transcriptional activation by HNF-4α was assessed in the presence or absence of added representative long-chain fatty acyl-CoAs. Template transcription consisting of a 200 bp G-less cassette driven by the adenoviral major late major (AdML) promoter and lacking the HNF-4α enhancer was used as an internal control template. As shown in Figure 3, the in vitro transcription of the test template increased as a function of HNF-4α, and was close to saturation when the concentration of HN...
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