ABSTRACT
Eight monosaccharide analogs of the potent antitumor OSW saponins (2-9) were synthesized. One analog, 2-O-acetyl-alpha-l-arabinopyranoside 3, showed antiproliferative activity against the Jurkat cells (IC(50)=0.078microM) comparable to that of the disaccharide derivative (1).
Subject(s)
Antineoplastic Agents, Phytogenic/chemical synthesis , Antineoplastic Agents, Phytogenic/pharmacology , Carbohydrates/chemistry , Cholestenones/chemical synthesis , Cholestenones/pharmacology , Ornithogalum/chemistry , Saponins/chemical synthesis , Saponins/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , HeLa Cells , Humans , Indicators and Reagents , Jurkat Cells , Thymidine/metabolismABSTRACT
PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.
