Isolation and identification of eukaryotic initiation factor 4A as a molecular target for the marine natural product Pateamine A.
Natural products continue to demonstrate their utility both as therapeutics and as molecular probes for the discovery and mechanistic deconvolution of various cellular processes. However, this utility is dampened by the inherent difficulties involved in isolating and characterizing new bioactive natural products, in obtaining sufficient quantities of purified compound for further biological studies, and in developing bioactive probes. Key to characterizing the biological activity of natural products is the identification of the molecular target(s) within the cell. The marine sponge-derived natural product Pateamine A (PatA) has been found to be an inhibitor of eukaryotic translation initiation. Herein, we describe the methods utilized for identification of the eukaryotic translation initiation factor 4A (eIF4A) as one of the primary protein targets of PatA. We begin by describing the synthesis of an active biotin conjugate of PatA (B-PatA), made possible by total synthesis, followed by its use for affinity purification of PatA binding proteins from cellular lysates. We have attempted to present the methodology as a general technique for the identification of protein targets for small molecules including natural products.
Low
Woon-Kai
WK
Department of Pharmacology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
Dang
Yongjun
Y
Schneider-Poetsch
Tilman
T
Shi
Zonggao
Z
Choi
Nam Song
NS
Rzasa
Robert M
RM
Shea
Helene A
HA
Li
Shukun
S
Park
Kaapjoo
K
Ma
Gil
G
Romo
Daniel
D
Liu
Jun O
JO
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D003514:Cyclohexylamines / Q000737:chemistry; D015195:Drug Design; D004852:Epoxy Compounds / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D039601:Eukaryotic Initiation Factor-4A / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D006801:Humans; D018942:Macrolides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D008954:Models, Biological; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013844:Thiazoles / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*
C405347:4,4'-methylenebiscyclohexamine; D001426:Bacterial Proteins; D003514:Cyclohexylamines; D004852:Epoxy Compounds; D018942:Macrolides; D013844:Thiazoles; C421324:pateamine A; C049538:streptavidin-agarose; D001710:Biotin; D012685:Sepharose; D039601:Eukaryotic Initiation Factor-4A