A series of
brusatol, bisbrusatol, and
bruceantin esters were examined for their ability to inhibit
protein synthesis in P-388
lymphocytic leukemia cells. Compounds which produced high T/C % values (170-272) resulted in ID50 of 5.4-15.5 microM for inhibition of whole cell
protein synthesis, ID50 of 1.3-13 microM for inhibition of endogenous
protein synthesis in cell homogenates, and ID50 of 1.9-6 microM for inhibition of polyuridine directed
polyphenylalanine synthesis using "runoff" ribosomes and a "pH 5"
enzyme preparation. The polyuridine directed
polyphenylalanine synthesis requires neither initiation nor termination factors, suggesting that
quassinoids are exclusively elongation inhibitors.
Bruceantin,
brusatol, and
bisbrusatolyl malonate allowed a runoff of the polyribosomes to 80S free ribosomes. However, formation of the ternary complex and 80S initiation complex were not inhibited by the
quassinoids. Thus, these agents do not affect the individual steps leading to the formation of a stable 80S initiation complex in P-388 cells.
Brusatol,
bruceantin, and
bisbrusatolyl malonate inhibited the formation of the first
peptide bond between
puromycin and [3H]methionyl-
transfer RNA bound to the initiation complex, indicating
peptidyl transferase activity is inhibited by the
quassinoids in P-388 cells. These studies also suggest that the free 80S ribosome is the site of binding by the
quassinoid. Ribosomes actively conducting
protein synthesis will continue
protein synthesis and terminate before the
quassinoids bind. This proves
quassinoids are elongation inhibitors of
tumor cells. A strong correlation was observed between potent antileukemic activity and the ability to inhibit
protein synthesis in P-388
lymphocytic leukemia cells.