A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane

Recent work has resulted in the development of potent inhibitors of oligosa ccharyl transferase (OT), the enzyme that catalyzes the cotranslational gly cosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Sec. 118, 7636-7637; Kellenberger, C., H endrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environmen t have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH 2 was previously shown to exhibit nanomolar inhibition (K-i = 37 nM) throug h slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M. , and Imperiali, B. (1996) J. Anl. Chem. Sec. 118, 7636-7637]. Included her ein is the redesign of this prototype inhibitor fur achieving both passive and active translocation into model membrane systems representing the endop lasmic reticulum (ER). The strategy for passive transport involved the inco rporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membran es [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechn ol. 16, 370-375]. Assessment of function in intact ER membranes revealed th at the inhibitor targeted toward passive diffusion demonstrated concentrati on-dependent inhibition of two different glycosylation substrates. Thus, th is modified inhibitor achieved potent inhibition of glycosylation after bei ng successfully transported through the ER membrane. In the active transloc ation approach, the lead OT inhibitor and a corresponding substrate were re designed to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient trans port and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modifie d substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless o f the extensions to the carboxy-terminus of the root inhibitor. The above f amily of redesigned inhibitors provides a template for generating a transce llular pathway and represents the first step toward OT inhibition in intact cells.