Biochemical and kinetic characteristics of the interaction of the antitumor antibiotic sparsomycin with prokaryotic and eukaryotic ribosomes

Abstract

Using 125I-labeled phenol-alanine sparsomycin, an analogue of sparsomycin having higher biological activity than the unmodified antibiotic, we studied the requirements and the characteristics of its interaction with the ribosome. The drug does not bind to either isolated ribosomal subunits or reconstituted whole ribosomes. For sparsomycin binding to 70S and 80S ribosomes, the occupation of the peptidyltransferase P-site by an N-blocked aminoacyl-tRNA is a definitive requirement. The sparsomycin analogue binds to bacterial and yeast ribosomes with Ka values of around 10(6) M-1 and 0.6 x 10(6) M-1, respectively, but its affinity is probably affected by the character of the peptidyl-tRNA bound to the P-site. Chloramphenicol, lincomycin, and 16-atom ring macrolides compete with sparsomycin for binding to bacterial ribosomes, but streptogramins and 14-atom ring macrolides do not. Considering the reported low affinity of puromycin for bacterial ribosomes, this antibiotic is also a surprisingly good competitor of sparsomycin binding to these particles. In the case of yeast ribosomes, blasticidin is a relatively good competitor of sparsomycin interaction, but anisomycin, trichodermin, and narciclasin are not. As expected, puromycin is a poor competitor of the binding in this case. The results from competition studies carried out with different sparsomycin analogues reveal, in some cases, a discrepancy between the drug ribosomal affinity and its biological effects. This suggests that some intermediate step, perhaps a ribosomal conformational change, is required for the inhibition to take place.

Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes

“…To demonstrate unambiguously that sparsomycin was crosslinked to the A2602 region of 23S rRNA, the phenol-alanine derivative of sparsomycin was radioactively labeled, by iodination at the phenol group, to produce [ 125 I]phenol-alanine-sparsomycin (Fig. 1), which retains biological activity (6,19). Proc.…”

“…It is also special in that it binds very weakly, if at all, to ribosomes lacking an N-blocked aminoacyltRNA (6). This effect is reciprocated in that sparsomycin stimulates PЈ-site binding of N-blocked tRNA fragments containing the 3Ј-terminal-CCA-end (4, 7).…”

“…Phenol-alanine-sparsomycin (named according to ref. 9) was dissolved in 20 l 50% DMSO at 7 mM and was iodinated with [ 125 I]NaI (100 Ci, Amersham Pharmacia) by using a mild procedure (6,19). [ 125 I]Phenol-alanine-sparsomycin (50 M) was subsequently complexed to 70S ribosomes (40 pmol) in the presence of poly(U) (1 g͞pmol ribosome) and N-Ac-[ 14 C]Phe-tRNA (1.3 mol͞mol ribosomes) in 80 l of 20 mM Tris⅐HCl (pH 7.5), 50 mM NH 4 Cl, and 10 mM MgCl 2 for 20 min at 37°C, and half of the sample was irradiated at 365 nm as described above.…”

Synthesis and Activity of Pyrimidinylpropenamide Antibiotics: The Alkyl Analogues of Sparsomycin

“…The biological activity resulted from its ability to inhibit the peptide bond-forming step of protein biosynthesis by interacting with the large ribosomal subunit. [7][8][9][10] These potentially important biological activities have made 3 an attractive target for a potential antineoplastic compound. 11) Since thê rst synthetic study of sparsomycin in 1976, 12) synthetic studies of sparsomycin, [13][14][15] total synthesis, [16][17][18] biosynthesis, 19,20) and structure-activity relationship studies [21][22][23][24][25][26][27] have been widely reported.…”

“…A retrosynthetic analysis was carried out based on Helquist's protocol 18) as summarized in Scheme 1. The structure of sparsomycin might enable it to be produced from 6-methyluracylacryllic acid (8) and the hydroxy-protected amino dithioacetal mono-S-oxide part (III) and to couple these two building blocks by amide formation. The alkyl sulfenyl group of III was prepared by sulfenylation of the methyl sulfoxide (II) with alkyl disulˆde.…”