Transition state analogues rescue ribosomes from saporin-L1 ribosome inactivating protein.
Ribosome inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves, and it demonstrated robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic, and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (K(i)*) of 2.3-8.7 nM under physiological conditions and bind up to 40000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1-linked immunotoxins.
Sturm
Matthew B
MB
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Tyler
Peter C
PC
Evans
Gary B
GB
Schramm
Vern L
VL
D000818:Animals; D016923:Cell Death; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008958:Models, Molecular; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000502:physiology; D054788:Ribosome Inactivating Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects*; D029750:Saponaria / Q000737:chemistry; D000076985:Saporins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011480:Protease Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D054788:Ribosome Inactivating Proteins; D000076985:Saporins