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Background: The majority of scientists, physicians, and healthcare professionals were trained with the paradigm: “antibiotics are for bacteria only !”, because they misunderstood the definition of the ribosome targeting antibiotics. In the context of the current worldwide COVID-19 pandemic, it might be useful to recall as precisely as possible the definition of the word antibiotic and provide evidence that some classes of antibiotics could offer excellent means to counteract viral infections via specific mechanisms. Methods: Molecular modeling and docking studies were used, as well as the tRNAox labeling reaction of the ribosomal protein eL42 in situ on human 80S ribosomes to demonstrate that cycloheximide and its thiosemicarbazone analogues bind to the catalytic Lys-53 residue of the human large subunit ribosomal protein eL42. Results: Comparison of the binding sites for Cycloheximide (CHX) and Sparsomycin (SPS) on the evolutionarily conserved E. coli bL12 and S. cerevisiae eL42 by means of molecular modeling and docking studies showed that: (i) SPS binds in proximity to the catalytic Lys-65 residue of the GANK motif of rp bL12 and to the catalytic Lys-55 residue of the GGQTKP motif of rp eL42; (ii) CHX failed to bind to the GANK motif, while the glutarimide moiety of SPS and CHX was found to make contact with Lys-55 of the GGQTKP motif of rp eL42. Conclusion: In this report, we demonstrate that cycloheximide and its thiosemicarbazone analogues are capable of inhibiting the human 80S ribosomes selectively through their binding to the ε-amino group of the side chain of Lys-53. As a consequence, these small-molecule inhibitors of translation are susceptible to exhibit antiviral activities by preventing the human ribosomes of the SARS-CoV-2 infected cells from synthesizing the viral proteins and enzymes.

Objectives Sparsomycin, which exhibits rare broad-spectrum antibiotic and antitumour activity against bacteria, Archaea, Eucarya and various cancer cell lines, has been widely used as a powerful tool to study protein synthesis. Here we report the draft genome sequence of Streptomyces sparsogenes ATCC 25498 from the American Type Culture Collection (ATCC), which has become an organism of interest owing to its ability to produce sparsomycin. Methods The whole-genome sequence of S. sparsogenes ATCC 25498 was determined using a high-throughput Illumina HiSeq 2000 platform and genome assembly was performed using the SOAPdenovo method. Results Whole-genome sequencing analysis revealed a genome size of 10.0 Mb. A total of 41 secondary metabolite biosynthetic gene clusters were identified. The gene cluster for the biosynthesis of sparsomycin was localised on scaffold 9. Conclusions The genome sequence of S. sparsogenes ATCC 25498 will not only aid in understanding the regulatory mechanism of sparsomycin biosynthesis but will also reveal the ability of the isolate to produce novel bioactive secondary metabolites.

Hundred and twenty microbial isolates could be isolated from different soil samples collected from different localities in Egypt. One of the bacterial cultures AZ-SH-B8 was found to produce a wide spectrum antimicrobial agent when cultivated on plastic wastes. The bacterial culture AZ-SH-B8 could be isolated from a soil sample collected from Sharkia governorate, Egypt. From the taxonomic features, the bacterial isolate AZ-SH-B8 matches with Pseudomonas aeurginosa in the morphological, physiological and biochemical characters and confirmation by using API20E system. Thus, it was given the suggested name P. aeurginosa , AZ-SH-B8. The parameters controlling the biosynthetic process of antimicrobial agent formation includes: innoculum size, different pH values, different temperatures, different incubation period, different carbon and nitrogen sources and different mineral salts concentrations (KNO 3 , K 2 HPO 4 , MgSO 4 ·7H 2 O and KCl) were fully investigated. The active metabolite was extracted using n-butanol (1:1, v/v) at pH 7.0. The separation of the active ingredient and its purification was performed using both thin layer chromatography (TLC) and column chromatography (CC) techniques. The physico-chemical characteristics of the purified antibiotic viz. color, melting point, solubility, elemental analysis, spectroscopic characteristics and chemical reactions have been investigated. This analysis indicates a suggested empirical formula of C 13 H 21 N 3 O 6 S 2 . The minimum inhibition concentrations “MICs” of the purified antimicrobial agent were also determined. The purified antimicrobial agent was suggestive of belonging to Sparsomycin antibiotic produced by P. aeurginosa , AZ-SH-B8.

The syntheses of compounds 14–19, 6-methyluracilacryloyl derivatives, and 27–29, photoreactive aryl azide analogs of the antitumor antibiotic sparsomycin (1) are described. All of the modifications in the 6-methyluracilacryloyl part of 1 were achieved by selective reduction or by selective N- and O-methylation of 6 (Scheme 2). The key step in the synthetic scheme to the photo-affinity analogs is the reaction of the chloromethyl sulfoxides 20 and 21 with the properly protected cystamine 22 in liquid ammonia, to give the sparsomycin N-protected amines 23 and 24, respectively (Scheme 4). Subsequently, the photoreactive groups, viz. a p-azidosalicylyl and a 4-azido-2-nitrophenyl group, were coupled with these amines. For an evaluation of the biological activity of the compounds prepared, inhibition of protein biosynthesis in two cell-free assays, cell-growth inhibition of E. coli in liquid medium and inhibition of murine leukemia L1210 colony formation in soft agar were studied. All variations introduced in the “western A” fragment of 1 render the drug biologically inactive. Apparently, the uracil ring plays an important role in the interaction of sparsomycin with the ribosomal peptidyl transferase and its unmodified presence is crucial for displaying activity. The photoprobes, carrying modifications in the “eastern C” fragment of 1, do interact with the ribosome, but less effectively than sparsomycin itself.

Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA Pro leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA Pro by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA Pro to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNA Pro from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression.

In Escherichia coli , interactions between the nascent TnaC-tRNA Pro peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA Pro cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA Pro cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA Pro peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.

To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is approximately 2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Psi2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.

Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA Pro , the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNA Pro peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding/competition assays. The ribosome-TnaC-tRNA Pro complexes analyzed were formed in vitro ; they contained either wild-type TnaC-tRNA Pro or its nonfunctional substitute, TnaC(W12R)-tRNA Pro . Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNA Pro complexes but not of ribosome-TnaC(W12R)-tRNA Pro complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNA Pro ; it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNA Pro mutant complexes. These findings establish that Trp-12 of TnaC-tRNA Pro is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center.

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0 A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H. marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of K m for initiator tRNA and a 100-fold decrease in V max in puromycin reaction. Comparison of the K i values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.

The large ribosomal subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-Å resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the Haloarcula marismortui 50S ribosomal subunit. A P site substrate, CCA-Phe-caproic acid–biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The α-NH 2 group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2′ OH of the A-76 ribose in the P site or with the 2′ OH of A2486 (2451). These interactions position the α amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack.

The peptidyltransferase center is the ribosomal site where peptide bond formation occurs and the site where many antibiotics of diverse structures act. By definition, the peptidyltransferase cavity must be able to accommodate any of the diverse hydrophobic and hydrophilic side chains of the amino acids, which suggests that it exhibits a high degree of structural complexity and/or conformational flexibility. An early model for the mechanisms of antibiotic inhibition at the peptidyltransferase center was based on molecular mimicry occurring between the antibiotics and the 3' termini of either aminoacylor peptidyl-tRNAs. Many lines of evidence, including mutational studies and rRNA footprinting and cross-linking studies with antibiotics and tRNAs, strongly suggest that the peptidyltransferase loop of 23S rRNA constitutes the main component of the peptidyltransferase cavity. Some of the cross-linked nucleotides may lie in intermediate states that are occupied while entering or leaving the P' site of the peptidyltransferase center on (otherwise) free ribosomes. Several peptidyltransferase antibiotics, including puromycin, reduced the yield of the sparsomycinribosomal cross-link; the only exceptions were erythromycin and a streptogramin B, both of which belong to the MLSB ntibiotics, which do not primarily act on peptide bond formation. Consideration of the synergistic effects observed with streptogramin A and B antibiotics, which potentially represent the two classes of drugs described in this chapter, may yield further insight into their inhibitory mechanisms.

It was recently demonstrated that peptide bond formation can occur using an Escherichia coli naked 23 S ribosomal RNA without any of the ribosomal proteins. Here, the six domains of the 23 S ribosomal RNA were individually synthesized and shown to be capable, when complexed together, of stimulating the reaction. Omission and addition experiments indicated that the activity could be reconstituted solely by domain V at a concentration 10 times higher than that of the intact 23 S ribosomal RNA, whereas domain VI could enhance the activity in trans . These findings suggest that fragments of an RNA molecule have the ability to associate into a functional whole.

The effects of two peptidyl-transferase inhibitors, anisomycin and sparsomycin, on ribosomal frameshifting efficiencies and the propagation of yeast double-stranded RNA viruses were examined. At sublethal doses in yeast cells these drugs specifically alter the efficiency of −1, but not of +1, ribosomal frameshifting. These compounds promote loss of the yeast L-A double-stranded RNA virus, which uses a programmed −1 ribosomal frameshift to produce its Gag-Pol fusion protein. Both of these drugs also change the efficiency of −1 ribosomal frameshifting in yeast and mammalian in vitro translation systems, suggesting that they may have applications to control the propagation of viruses of higher eukaryotes, which also use this translational regulatory mechanism. Our results offer a new set of antiviral agents that may potentially have a broad range of applications in the clinical, veterinary, and agricultural fields.

Ethyldeshydroxy-sparsomycin (EdSm) is a ribosomal protein synthesis inhibitor which synergistically enhances the antitumor activity of cisplatin against L1210 leukemia in vivo. Because cellular glutathione (GSH) and glutathione S-transferases (GST) are reported to interfere with the antitumor activity of cisplatin, we analyzed the effect of EdSm and cisplatin on GSH and GST activity in selected tumor cells. For this purpose we used three murine leukemia tumors with different sensitivities towards EdSm and cisplatin: L1210-WT, sensitive to both drugs, L1210-Sm, resistant to EdSm, and L1210-CDDP, resistant to cisplatin. No significant differences were detectable between these three cell lines regarding the population doubling time, the cell size, and the cellular level of protein and glutathione. Neither of the resistant L1210 subclones showed P-glycoprotein expression. Drug exposure, however, changed the intracellular dynamics. Exposure to EdSm strongly decreased the amount of cellular protein, decreased the overall GST activity and led to GSH depletion, whereas exposure to cisplatin induced a rise in the amount of protein, in GSH, and in the total GST activity. These effects are dose-dependent and correlate well with the sensitivity of the tumor cells for EdSm or cisplatin. In addition, exposure to EdSm lowered the V(max) of GST in L1210-WT and L1210-Sm; however, in L1210-CDDP both the V(max) and the K(m) were increased. That this was not a direct effect of EdSm on GST was shown in a cell-free system, where EdSm did not influence the GST activity nor could it act as a substrate for GST. Our results suggest that the synergistic combination of EdSm and cisplatin might be explained by EdSm switching off the cellular detoxification mechanism for cisplatin, i.e. by inhibition of de novo synthesis and subsequent depletion of GSH and GST.

The structures of {[(S)-(4,5-dihydro-2-phenyloxazol-4-yl)methyl]-(S)-sulfinyl}octane, C18H27NO2S, (2), {[(S)-(4,5-dihydro-2-phenyloxazol-4­yl) methyl]-(R)-sulfinyl}octane, C18H27NO2S, (3) and 4-{[(S)-(4,5-dihydro- 2-phenyloxazol-4­yl)methyl]-(S)-sulfinyl}phenol, C16H15NO3S, (5), have been determined. The known chirality (S) at the C10 atom in all three compounds enabled assignment of the absolute configuration at the S13 atom. The C10 chirality was subsequently confirmed by refinement of the Flack parameter. The absolute configuration at S13 for compounds (2), (3) and (5) is R, S and S, respectively. Comparison with the stereochemistry of sparsomycin enables proper choice of diastereomers for analogue development.

Ricin A-chain was used as a test protein to study the effects of deletion of codons on the ribosomal synthesis, release and chaperone-mediated folding of the proteins. Synthesis of wild-type ricin and five mutant proteins was carried out in an Escherichia coli cell-free coupled transcription/translation system from otherwise identical non-linearized plasmids. The deletions involved small numbers of contiguous amino acid residues at different points from the N terminus to the C terminus of the wild-type protein. Deletion of the N-terminal 20 amino acid residues caused a 45% reduction in total protein synthesis whereas deletion of the next three amino acid residues caused a 1.5-fold increase in synthesis compared with wild-type with an accumulation of full-length polypeptides as peptidyl-tRNA in the ribosomal P site. Intermediate levels of synthesis and release were seen with the other three mutants. Enzymatic activity was detected only with wild-type protein and a mutant lacking the C-terminal five amino acid residues. These were the only ricin species in which chaperone-dependent reactions could be detected by fluorescence from coumarin incorporated with methionine at the N terminus of the proteins. By using sparsomycin to block termination of full-length peptidyl-tRNA, it was demonstrated that the chaperone-mediated reactions detected by fluorescence occur on the ribosomes and involve folding of the nascent protein as peptidyl-tRNA. The results presented provide a direct demonstration of two points of fundamental importance: folding of nascent proteins involving chaperone-mediated reactions can occur on ribosomes and is directly related to the conformation of the native enzyme. Deletion of amino acid residues at different points from the N terminus to the C terminus affects the reactions of elongation, chaperone-mediated folding and release of full-length protein.

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

Fluorescently labeled rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system. A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide. Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in accumulation of enzymatically inactive full-length rhodanese polypeptides on the ribosomes. These can be activated and released in the presence of the added chaperones, DnaJ, DnaK, GrpE, GroEL, GroES and ATP. Fluorescence parameters (quantum yield, anisotropy and the emission maximum) of ribosome-bound coumarin-labeled rhodanese are affected differentially by addition of the chaperones individually or sequentially. Rhodanese released from the ribosomes in the presence of all chaperones (enzymatically active) differs in fluorescence properties from rhodanese released by GroES or DnaK only or by puromycin (enzymatically inactive) indicating a difference in conformation. Using sparsomycin, an inhibitor of the peptidyl transferase reaction, full-length rhodanese can be trapped on the ribosomes. A ribosome-bound intermediate formed by DnaJ or DnaJ plus DnaK was demonstrated by the effect of these chaperones on fluorescence spectra resulting from binding of anticoumarin antibodies to the N terminus of newly synthesized rhodanese. The results support the hypothesis that folding of nascent proteins can take place on the ribosome.

Precursor incorporation experiments have ruled out the involvement of N′-formylkynurenine in the biosynthesis of the uracil acrylic acid moiety of sparsomycin. An enzyme has been partially purified from the sparsomycin-producer that catalyzes the synthesis of the uracil acrylic acid from a pyrimidine acrylic acid previously shown to be a sparsomycin precursor. The enzymatic synthesis of the uracil acrylic acid requires NAD + , a result which indicates that the mechanism of the uracil acrylic acid synthase is similar to that of inosine-5′-monophosphate dehydrogenase. Analog studies demonstrate that the enzyme lacks rigid substrate specificity.

The modulating effect of ethyldeshydroxy-sparsomycin (EDSM), an inhibitor of ribosomal protein synthesis, on cytostatic agents was studied on cultured B16 melanoma cells using the microculture tetrazolium test (MTT). The data were analyzed for true synergism using the combination index and the median effect principle. The extent of cytotoxic drug interaction was influenced by the duration of drug exposure, the dose ratio, as well as the treatment schedule. When drug ratios were used, synergism was observed upon pre- and post-treatment in combination with cisplatin, cytosine arabinoside (Ara-C), methotrexate (MTX) and 5-fluorouracil (5-FU). The combination of a fixed dose of EDSM was synergistic with cisplatin, Ara-C, vincristine (VCR) and MTX, in the order MTX > Ara-C > VCR > cisplatin, while the combinations with doxorubicin, 5-FU and etoposide (VP-16) were shown to be antagonistic. These results suggest that only certain drugs and treatment schedules might be worthwhile for combination studies with EDSM in vivo and indicate a role for EDSM as modifier of antitumor responses in cancer chemotherapy.

Sparsomycin is an antibiotic that targets the peptidyl transferase center of the ribosome and has the ability to promote ribosomal translocation in the absence of EF-G and GTP. Here we show that changes in the configurations at the two chiral centers of sparsomycin, especially at the chiral carbon, can greatly affect its capability to promote ribosomal translocation. More importantly, the incorporation of the pseudo-uracil moiety of sparsomycin into linezolid through a covalent linkage conferred on linezolid derivatives the ability to promote translocation, thus indicating the importance of interactions between this pseudo-uracil moiety, rRNA, and tRNA for promoting translocation. In addition, these translocation promoters can also effectively inhibit spontaneous reverse translocation; this suggests that they might promote forward translocation by trapping the ribosome in the post-translocation state and shifting the equilibrium between the pre- and post-translocation ribosome in the forward direction.

Four hybrid antibiotics combining structural features of chloramphenicol (1a), sparsomycin (2b), lincomycin (5c), and puromycin (6d)--lincophenicol (1c), chloramlincomycin (5a), sparsolincomycin (5b), and sparsopuromycin (6b)--were synthesized. They were investigated as inhibitors of several partial reactions of procaryotic and eucaryotic protein synthesis as well as potential antimicrobial agents. Lincophenicol (1c) was active as inhibitor of Escherichia coli ribosomal peptidyltransferase-catalyzed puromycin reaction. Both lincophenicol (1c) and sparsophenicol (1b) inhibited the binding of the iodophenol analogue of sparsomycin to E. coli ribosomes. The results are discussed in terms of a retro-inverso hypothesis advanced earlier for interpretation of biological activity of chloramphenicol (1a) and sparsophenicol (1b). Chloramlincomycin (5a) suppressed the growth of Streptococcus pyogenes with MIC 6.25 micrograms/mL.

The biosynthesis of the antitumor antibiotic sparsomycin (1) has been investigated by administration of isotopically labeled precursors to Streptomyces sparsogenes var. sparsogenes. These studies indicated that the dithioacetal moiety (2) of sparsomycin is derived from L-cysteine via the intermediacy of 5-methylcysteine and S-(methylthiomethyl)cysteine, with reduction of the carboxyl group of 5-(methylthiomethyl)cysteine preceding attachment of the uracil moiety (3). Investigations of the origin of the uracil moiety (3) of sparsomycin revealed that it is derived from L-tryptophan by a process that involves loss of the side chain and oxidative ring opening of both rings of the amino acid. The intermediacy of yV-formylanthranilic acid in this process was ruled out. Investigations of the terminal stage in the biosynthesis of the uracil moiety (3) demonstrated that it resembles the conversion of inosine-5'-monophosphate into xanthosine-5'-monophosphate.

The syntheses of compounds 14–19, 6-methyluracilacryloyl derivatives, and 27–29, photoreactive aryl azide analogs of the antitumor antibiotic sparsomycin (1) are described. All of the modifications in the 6-methyluracilacryloyl part of 1 were achieved by selective reduction or by selective N- and O-methylation of 6 (Scheme 2). The key step in the synthetic scheme to the photo-affinity analogs is the reaction of the chloromethyl sulfoxides 20 and 21 with the properly protected cystamine 22 in liquid ammonia, to give the sparsomycin N-protected amines 23 and 24, respectively (Scheme 4). Subsequently, the photoreactive groups, viz. a p-azidosalicylyl and a 4-azido-2-nitrophenyl group, were coupled with these amines. For an evaluation of the biological activity of the compounds prepared, inhibition of protein biosynthesis in two cell-free assays, cell-growth inhibition of E. coli in liquid medium and inhibition of murine leukemia L1210 colony formation in soft agar were studied. All variations introduced in the “western A” fragment of 1 render the drug biologically inactive. Apparently, the uracil ring plays an important role in the interaction of sparsomycin with the ribosomal peptidyl transferase and its unmodified presence is crucial for displaying activity. The photoprobes, carrying modifications in the “eastern C” fragment of 1, do interact with the ribosome, but less effectively than sparsomycin itself.

The asymmetric synthesis of (R C,SS) and (S C,R S), important intermediates in the synthesis of optically pure Sparsomycin analogs, is discussed. The enantioselective synthesis of both isomers, R and S, of Oxisuran and three of their bioisosters is also described. In both strategies o.p. methanesulfinate esters of DAG were used as starting material.

The structures of {[(S)-(4,5-dihydro-2-phenyloxazol-4-yl)methyl]-(S)-sulfinyl}octane, C18H27NO2S, (2), {[(S)-(4,5-dihydro-2-phenyloxazol-4­yl) methyl]-(R)-sulfinyl}octane, C18H27NO2S, (3) and 4-{[(S)-(4,5-dihydro- 2-phenyloxazol-4­yl)methyl]-(S)-sulfinyl}phenol, C16H15NO3S, (5), have been determined. The known chirality (S) at the C10 atom in all three compounds enabled assignment of the absolute configuration at the S13 atom. The C10 chirality was subsequently confirmed by refinement of the Flack parameter. The absolute configuration at S13 for compounds (2), (3) and (5) is R, S and S, respectively. Comparison with the stereochemistry of sparsomycin enables proper choice of diastereomers for analogue development.

Three pyrimidinylpropanamide antibiotics sparsomycin ( 1 ), sparoxomycins A1, A2 ( 2, 3 ), and also six analogues ( 4–9 ) have been synthesized by employing asymmetric sulfide oxidation conditions as a key step. Sparsomycin ( 1 ) and its alkyl analogues ( 5–7 ) showed higher morphological reversion activities on src ts NMR cells than 2 and 3 .

The standard (absolute) binding free energy of the antibiotic sparsomycin with the 50S bacteria ribosomal subunit is calculated using molecular dynamics (MD) free energy perturbation (FEP) simulations with restraining potentials developed by Wang et al. [Biophys. J. 91:2798–2814 (2006)]. In the simulation protocol, restraining potentials are activated for the orientational and translational movements of the ligand relative to the binding site when it is decoupled from the binding pocket, and then released once the ligand fully interacts with the rest of the system. A reduced system is simulated to decrease the computational cost of the FEP/MD calculations and the effects of the surrounding atoms are incorporated using the generalized solvent boundary potential (GSBP) method. The loss of conformational freedom of the ligand upon binding is characterized using the potential of mean force (PMF) as a function of the root-mean-square deviation (RMSD) relative to the bound conformation. The number of water molecules in the binding pocket is allowed to fluctuate dynamically in response to the ligand during the calculations by combining FEP/MD with grand canonical Monte Carlo (GCMC) simulations. The calculated binding free energy is about −6 kcal/mol, which is in reasonable agreement with the experimental value. The information gleaned from this study provides new insight on the recognition of ribosome by sparsomycin and highlights the challenges in calculations of absolute binding free energies in these systems. Copyright © 2010 John Wiley & Sons, Ltd.

The biosynthesis of the antitumor antibiotic sparsomycin (1) has been investigated by administration of isotopically labeled precursors to Streptomyces sparsogenes var. sparsogenes. These studies indicated that the dithioacetal moiety (2) of sparsomycin is derived from L-cysteine via the intermediacy of 5-methylcysteine and S-(methylthiomethyl)cysteine, with reduction of the carboxyl group of 5-(methylthiomethyl)cysteine preceding attachment of the uracil moiety (3). Investigations of the origin of the uracil moiety (3) of sparsomycin revealed that it is derived from L-tryptophan by a process that involves loss of the side chain and oxidative ring opening of both rings of the amino acid. The intermediacy of yV-formylanthranilic acid in this process was ruled out. Investigations of the terminal stage in the biosynthesis of the uracil moiety (3) demonstrated that it resembles the conversion of inosine-5'-monophosphate into xanthosine-5'-monophosphate.

Before CI isomerizes to C*I, we detect a competitive phase of inhibition (Ki = k5/k4 = 0.05 microM) which eventually, by increasing the concentration of I, becomes linear mixed noncompetitive and involves C*I in place of CI. The equilibration of C and I according to reaction 2 is much slower than the equilibration between C and S in reaction 1 (time-dependent inhibition). The inactivation plots obey reaction 2 and allow us to estimate k6 as equal to 2.2 min-1. The isomerized C*I, free of excess I, can be studied as a mixture with complex C. From the kinetics of the regeneration of C from C*I, in the presence of puromycin, we can estimate k7 to be between 0.22 min-1 and 0.06 min-1. Although the isomerized C*I survives after adsorption on cellulose nitrate filter disks, it does not survive after gel chromatography on a Sepharose CL-4B column but is converted quantitatively to complex C containing D of unchanged reactivity. This result does not support the proposed [Flynn, G. A., & Ash, R. J., (1990) Biochem. Biophys. Res. Commun. 166, 673-680] chemical reaction between D and I toward new products. The isomerized C*I can be obtained not only from the already-made complex C but also de novo from D, R, and M. In the latter case, the reactions which lead to C are represented by the following hypothetical scheme: D + R + M in equilibrium with DRM or C (binding reaction). When C*I is formed de novo, this reaction is coupled to reaction 2 and the ultimate product is a mixture of C and C*I.(ABSTRACT TRUNCATED AT 250 WORDS)

The sensitivity to different antibiotics of in-vitro polyuridylic acid-dependent polypeptide synthesizing system from Bacteroides vulgatus RYC18F6 and two clindamycin-resistant derivatives was studied. The ribosomes from the resistant strains were not affected by concentrations of up to 0.1 mM clindamycin and lincomycin. In contrast, streptogramin B was found to cause strong stimulation of the clindamycin-resistant polymerizing systems. The modified ribosomes from the resistant strains were more sensitive to other antibiotics like sparsomycin and chloramphenicol. The data indicate that resistance in these B. vulgatus mutant strains is due to alteration of the ribosome structure.

The accurate calculation of absolute binding free energy is one of the holy grails of computer-aided drug design. The emerging successes reported in computing the binding free energy of small ligands to proteins using molecular dynamics (MD) simulations indicated that such physics-based approaches hold the promise of expediting the rational drug discovery process. Among numerous receptor-ligand systems, ribosome-antibiotic binding provides an important paradigm for studying the molecular recognition of RNAs by small molecules. The interactions of the 50S bacteria ribosomal subunit with antibiotic sparsomycin and its derivatives have been studied through the calculation of the binding free energy and the characterization of conformational dynamics. The standard binding free energies of the complexes were calculated using free energy perturbation (FEP) method. Restraining potentials affecting the orientational, translational and conformational freedom of the ligand and receptor were applied and then removed during the simulations to enhance the sampling and the convergence. The loss of ligand conformational entropy upon binding was estimated with Umbrella Sampling method by calculating the Potential of Mean Force as a function of the RMSD relative to the reference conformation of the ligand. Due to the large size of the ribosome, the Generalized Solvent Boundary Potential method was used to reduce the computational cost of MD/FEP calculations. For a deeply buried binding pocket in the ribosome, the fluctuation of solvent occupancy during the alchemical free energy calculation was also characterized by combining the MD with Grand Canonical Monte Carlo simulation. This computational study further revealed the mechanism of ribosome-antibiotics interactions and shed light on the design of ribosomal drugs. With the above stated developments, the evaluation of the binding free energies has become computationally more appealing for large systems.

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.