Interaction of the antibiotic sparsomycin with the ribosome

Abstract

Phenol-alanine-sparsomycin, a derivative of sparsomycin carrying a p-hydroxy-benzyl function easily labeled by iodination, has been used to study the interaction of this drug with the ribosome. Our study indicated that the binding of the drug to the ribosome is sensitive to trichloroacetic acid and is equally affected by disintegration of the particle after RNase and protease treatments. The ribosome is not irreversibly inactivated, and the chemical structure of the drug is not affected by interaction with the particle. These data are not compatible with the proposed covalent association of sparsomycin with the ribosome by G. A. Flynn and R. J. Ash (Biochem. Biophys. Res. Commun. 114:1-7, 1983); therefore, the antibiotic must inhibit protein synthesis through a reversible interaction with the ribosome.Sparsomycin is a strong inhibitor of protein synthesis in eucaryotic and procaryotic cells, including highly antibioticresistant organisms such as the archaebacterium Sulfolobus solfataricus (1), suggesting that the drug must interact at a very critical and conserved region of the ribosome. Sparsomycin blocks peptide bond formation by interfering with the interaction of the acceptor tRNA molecule with the A site of the peptidyl transferase center while at the same time strongly stimulating the binding of the donor tRNA to the P site (see Ottenheijm et al. [10] for a review).Interest in the drug as an antitumor agent diminished after a reported sparsomycin-related retinopathy was detected in early clinical studies, although the drug has been used extensively as a tool in studies of ribosome function (10). The recent synthesis of a number of derivatives having up to 20-fold-higher inhibitory activity than the original drug (16, 17) has rekindled interest in sparsomycin as an antitumor antibiotic, and some of these derivatives are currently at the level of clinical testing (19).In this new wave of interest in sparsomycin, a very interesting model has been proposed for the mode of action of the drug; this model implies the occurrence of a chemical reaction involving the sulfoxide moiety of the sparsomycin molecule (4, 5). According to this model, a Pummerer process can be initiated by an imidazole-activated intermediate of the peptide bond-forming reaction, resulting in covalent binding of the drug through its sulfoxide sulfur atom to either the nascent peptide or a ribosomal component.This model is compatible with the analogous stereochemical confirmation of sparsomycin and the 3' aminoacyl adenosine of the aminoacyl-tRNA (4, 5) and provides a plausible explanation for the stimulation of sparsomycin inhibitory activity through preincubation of ribosomes with the drug prior to the initiation of protein synthesis (2). According to the model, this "preincubation effect" would facilitate chemical reaction of the drug at the interaction site. However, experimental evidence supporting the model is based exclusively on the binding of radioactive sparsomycin to ribosomes on sucrose gradients, which does not necessarily deman...

On the specificity of antibiotics targeting the large ribosomal subunit

“…This is understandable since the conjugated tail of SPAR overlaps with the aminoacyl‐moiety of an A‐tRNA (Fig. 4B) and thus would prevent binding not only of A‐tRNA, but also CAM and lincomycin in agreement with previous reports 34,39 . The binding site of SPAR in the D50S structures in contrast spans across the P site and does not encroach on the A site (Fig.…”

“…Sparsomycin (SPAR), a nucleoside analog of uracil (Fig. 4A) produced by Streptomyces sparsogenes , is a potent inhibitor of peptidyltransferase activity in bacteria, archaea, and eukaryotes (reviewed by Lazaro et al 34 ). SPAR has been crystallized together with tRNA mimics in complex with both archaeal (H50S) 9,35 and bacterial (D50S) 36 ribosomes (Table 1).…”

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

“…We stored sparoxomycin and its analogues (3)(4)(5)(6)(7) in the dark after their recrystallization from MeOH, because the trans double bond tended to easily isomerize to the cis-isomer in a solution.…”

“…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.…”

Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin

“…The biological activity of sparsomycin is the result of its ability to inhibit the peptide bond-forming step of protein biosynthesis by interacting with the large ribosomal subunit in a poorly understood, complex manner (16)(17)(18)30). Although the clinical use of sparsomycin is precluded due to drug-related retinopathy, a number of more potent analogs have been prepared which may prove useful in cancer chemotherapy (16). One of the novel components of the sparsomycin molecule is (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid (UCA).…”