Structure‐Activity Relationships for Binding and Inactivation of Rabbit Reticulocyte Ribosomes by Quassinoid Antineoplastic Agents

Abstract

A series of quassinoid compounds was investigated to determine the structure-activity relationships for the binding and inactivation steps associated with the inhibition of protein synthesis by these compounds in a rabbit reticulocyte system. The binding of these compounds to rabbit reticulocyte run-off ribosomes was measured in a competition-binding assay using ['4C]trichodermin as the competing ligand. Previous studies have shown that trichodermin and quassinoids compete for a single binding site on yeast ribosomes. Our experiments confirmed this behavior with rabbit reticulocyte ribosomes and indicated that binding equilibrium could be reached in 2.5 min at 30 "C. The comparative binding data indicated the following: (a) The nature of the side-chain at position 15 has very little effect on the association of these compounds with rabbit reticulocyte ribosomes. There is one exception to this general rule, however. Saturation of the double bond in brusatol (compound 11) to give dihydrobrusatol (compound IV) increases the Kd from 1.6 pM to 8.7 pM. (b) Easy access to the A ring is required for optimal binding. The addition of any bulky hydrophobic group to the A ring increased the Kd significantly. However, the double bond in the A ring does not appear to be required for binding since the Kd values for dihydrobrusatol (compound IV) and tetrahydrobrusatol (compound VI) are virtually identical. (c) The lactone ring plays only a very minor role in binding, since conversion of the lactone (compound VI) to the corresponding lactol (compound X) has only a minor effect on Kd. In order to determine whether inactivation of rabbit reticulocyte ribosomes had any structural requirements which differed from those required for simple binding, we also compared the concentrations of these compounds required to give 50 inhibition of protein synthesis (IDso) in rabbit reticulocyte lysates. Previous studies had indicated that binding of quassinoids to the peptidyl transferase center and inhibition of peptide bond formation was probably the only mode of action of these compounds. Our experiments indicated that the IDso values for several of these compounds were the same in whole reticulocytes and reticulocyte lysates, suggesting that transport and/or metabolism of these compounds was probably not limiting for their inhibitory effects. The inhibition studies indicated that (a) the nature of the side-chain at position 15 had no detectable effect on the inhibition; (b) the double bonds in the A ring and lactone ring are probably involved in the inactivation of rabbit reticulocyte ribosomes.

Eurycomalactone Inhibits Expression of Endothelial Adhesion Molecules at a Post-Transcriptional Level

“…Quassinoids were reported in the 1970s and early 1980s to bind to the peptidyl transferase center of ribosomes inhibiting peptide bond formation in eukaryotes, thus acting as elongation inhibitors. 21 , 22 Actively synthesizing ribosomes will continue protein synthesis and need to terminate before quassinoids bind. 23 − 25 Silva et al quite recently reported that the quassinoid isobrucein B isolated from the Amazonian medicinal plant Picrolemma sprucei exerted in vivo and in vitro anti-inflammatory activity.…”

“…This mechanism may at least contribute to the inhibition of luciferase gene expression as observed in our previous publication and reduced endothelial adhesion molecules as shown in Figure . Quassinoids were reported in the 1970s and early 1980s to bind to the peptidyl transferase center of ribosomes inhibiting peptide bond formation in eukaryotes, thus acting as elongation inhibitors. , Actively synthesizing ribosomes will continue protein synthesis and need to terminate before quassinoids bind. − Silva et al quite recently reported that the quassinoid isobrucein B isolated from the Amazonian medicinal plant Picrolemma sprucei exerted in vivo and in vitro anti-inflammatory activity . They showed that isobrucein B inhibits the release of pro-inflammatory cytokines in LPS-activated primary murine peritoneal macrophages in a concentration-dependent manner within a similar concentration range to that used in our study (0.1–10 μM).…”

“…The differences in the described effects of some investigated quassinoids might also be a result of the structural heterogeneity within this compound class, which is currently subdivided into C-18, C-19, C-20, C-22, and C-25 types . Unfortunately larger SAR studies were only carried out using C-20-type quassinoids ,, and are missing to our knowledge for the other subtypes. Therefore, the impact of changes in the total number of carbons and shape of the basic skeletons remains rather unclear and seems to be even more complex since Kupchan et al showed that the antileukemic activity of the bruceolide derivatives already varies widely with differences in the ester substituent …”

Ailanthus Quassinoids and Their Biological Activity

“…NOE and HMBC correlation, together with other spectral data, suggested that 49 was the 1,7-epoxy and 11,12-derivative of picrasane, which was confirmed by single-crystal X-ray diffraction analysis. Biological activity: Activity of quassinoids are influenced by (i) the nature of the C-15 side chain, (ii) the nature of A ring modifications, (iii) the presence or absence of a sugar moiety, and (iv) the presence of an epoxymethano bridge [80,[82][83][84][85]88]. Some of the quassinoids for which biological activity has been reported are listed in Table 4.…”