Identification of a Pyrrole Intermediate Which Undergoes C-Glycosidation and Autoxidation to Yield the Final Product in Showdomycin Biosynthesis.

Showdomycin is a C-nucleoside bearing an electrophilic maleimide base. Herein, the biosynthetic pathway of showdomycin is presented. The initial stages of the pathway involve non-ribosomal peptide synthetase (NRPS) mediated assembly of a 2-amino-1H-pyrrole-5-carboxylic acid intermediate. This intermediate is prone to air oxidation whereupon it undergoes oxidative decarboxylation to yield an imine of maleimide, which in turn yields the maleimide upon acidification. It is also shown that this pyrrole intermediate serves as the substrate for the C-glycosidase SdmA in the pathway. After coupling with ribose 5-phosphate, the resulting C-nucleoside undergoes a similar sequence of oxidation, decarboxylation and deamination to afford showdomcyin after exposure to air. These results suggest that showdomycin could be an artifact due to aerobic isolation; however, the autoxidation may also serve to convert an otherwise inert product of the biosynthetic pathway to an electrophilic C-nucleotide thereby endowing showdomycin with its observed bioactivities.

The role of the maleimide ring system on the structure-activity relationship of showdomycin.

Showdomycin produced by Streptomyces showdoensis ATCC 15227 is a C-nucleoside microbial natural product with antimicrobial and cytotoxic properties. The unique feature of showdomycin in comparison to other nucleosides is its maleimide base moiety, which has the distinct ability to alkylate nucleophilic thiol groups by a Michael addition reaction. In order to understand structure-activity relationships of showdomycin, we synthesized a series of derivatives with modifications in the maleimide ring at the site of alkylation to moderate its reactivity. The showdomycin congeners were designed to retain the planarity of the base ring system to allow Watson-Crick base pairing and preserve the nucleosidic character of the compounds. Consequently, we synthesized triphosphates of showdomycin derivatives and tested their activity against RNA polymerases. Bromo, methylthio, and ethylthio derivatives of showdomycin were incorporated into RNA by bacterial and mitochondrial RNA polymerases and somewhat less efficiently by the eukaryotic RNA polymerase II. Showdomycin derivatives acted as uridine mimics and delayed further extension of the RNA chain by multi-subunit, but not mitochondrial RNA polymerases. Bioactivity profiling indicated that the mechanism of action of ethylthioshowdomycin was altered, with approximately 4-fold reduction in both cytotoxicity against human embryonic kidney cells and antibacterial activity against Escherichia coli. In addition, the ethylthio derivative was not inactivated by medium components or influenced by addition of uridine in contrast to showdomycin. The results explain how both the maleimide ring and the nucleoside nature contribute to the bioactivity of showdomycin and demonstrates for the first time that the two activities can be separated.

Showdomycin as a versatile chemical tool for the detection of pathogenesis-associated enzymes in bacteria.

Showdomycin is a potent nucleoside antibiotic that displays a high structural similarity to uridine and pseudouridine. No detailed target analysis of this very unusual electrophilic natural product has been carried out so far. To unravel its biological function, we synthesized a showdomycin probe that can be appended with a fluorophor or a biotin marker via click chemistry and identified diverse enzymes which were important for either the viability or virulence of pathogenic bacteria. Our results indicate that the antibiotic effect of showdomycin against Staphylococcus aureus may be due to the inhibition of various essential enzymes, especially MurA1 and MurA2, which are required for cell wall biosynthesis. Although real-time polymerase chain reaction revealed that the MurA2 gene was expressed equally in four S. aureus strains, our probe studies showed that MurA2 was activated in only one multiresistant S. aureus strain, and only this strain was resistant to elevated concentrations of the MurA inhibitor fosfomycin, suggesting its potential role as an antibiotic bypass mechanism in the case of MurA1 inhibition. Moreover, we utilized this tool to compare enzyme profiles of different pathogenic strains, which provided unique insights in regulatory differences as well as strain-specific signatures.

Differential competition with cytotoxic agents: an approach to selectivity in cancer chemotherapy.

An approach to increasing the selectivity of cancer chemotherapeutic agents is presented in which noncytotoxic competitive substrates are used to discern the differences in structural requirements for transport of cytotoxic agents between tumor cells and a sensitive host tissue, the hematopoietic precursor cells of the bone marrow. Examples are given for two such systems, one responsible for the transport of nucleosides and another for the transport of amino acids. Cytidine is twice as effective in reducing the toxicity of showdomycin for murine bone marrow cells in culture as it is for murine L1210 leukemia cella. Conversely, homoleucine is twice as effective in reducing the toxicity of melphalan for L1210 cells as it is for bone marrow cells. These observations can serve as a basis for the development of bone marrow protective agents and for the design of cytotoxic agents that may be preferentially transported into tumor cells.

Conformational analysis (geometry optimization) of nucleosidic antitumor antibiotic showdomycin by Arguslab 4 software.

Showdomycin is a naturally maleimide antitumor antibiotic of the C-nucleoside, it inhibits the nucleic acid synthesis in bacteria. Conformational analysis and geometry optimization of showdomycin was performed according to the Hartree-Fock (HF) calculation method by ArgusLab 4.0.1 software. The minimum potential energy is calculated by geometry convergence function by ArgusLab software. The most feasible position for the drug to interact with the receptor was found to be 0.269696 K.cal/mole.

Discovery of the Showdomycin Gene Cluster from Streptomyces showdoensis ATCC 15227 Yields Insight into the Biosynthetic Logic of C-Nucleoside Antibiotics.

Nucleoside antibiotics are a large class of pharmaceutically relevant chemical entities, which exhibit a broad spectrum of biological activities. Most nucleosides belong to the canonical N-nucleoside family, where the heterocyclic unit is connected to the carbohydrate through a carbon-nitrogen bond. However, atypical C-nucleosides were isolated from Streptomyces bacteria over 50 years ago, but the molecular basis for formation of these metabolites has been unknown. Here, we have sequenced the genome of S. showdoensis ATCC 15227 and identified the gene cluster responsible for showdomycin production. Key to the detection was the presence of sdmA, encoding an enzyme of the pseudouridine monophosphate glycosidase family, which could catalyze formation of the C-glycosidic bond. Sequence analysis revealed an unusual combination of biosynthetic genes, while inactivation and subsequent complementation of sdmA confirmed the involvement of the locus in showdomycin formation. The study provides the first steps toward generation of novel C-nucleosides by pathway engineering.

Conformational analysis of nucleoside and nucleotide antibiotics prossessing five-membered base rings. A comparison of the glycosyl barrier for purine, pyrimidine and imidazole rings.

The preferred conformations of the nucleosides comprising five-membered base rings viz., ribavirin, tetrazole, showdomycin and pyrazomycin and their 5'-nucleoside monophosphates have been explored by semiempirical potential energy calculations. The nature of the substituents on the five-membered base ring has been found to influence significantly the favoured glycosyl conformation and the hydrogen bonding between the base and the sugar. In the absence of any such hydrogen bonding, the imidazole nucleosides expectedly exhibit nearly 'free' rotation around the glycosyl bond for the commonly found C(3') endo and C(2') endo sugar ring conformations, thus increasing the probability of anti in equilibrium high-anti in equilibrium syn conformational interconversions as compared to common purine and pyrimidine nucleosides. However, the C(2') exo pucker imparts a high-energy barrier to glycosyl rotation, locking the base in the high-anti region. The conformation behaviour is significantly modified in the nucleoside 5'-monophosphates. The additional attractive interactions between the carboxamide and the phosphate groups favour the syn glycosyl conformation in the 5'-monophosphates of ribavirin as well as pyrazomycin smaller to guanosine 5'-monophosphate whereas the anti conformation is strongly favoured for the 5'-nucleotides of tetrazole and showdomycin similar to other common purine and pyrimidine nucleotides. The conformational properties of five-membered base ring nucleosides have a strong resemblance to purine nucleosides. Molecular modelling studies reveal that only the anti or high anti-glycosyl conformations of ribavirin can mimic the inosine base. It is suggested that experiments on the configurational isomers of ribavirin, viz., aravirin and cycloaravirin would conclusively establish the conformational specificity and biological function of ribavirin and analogues.

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin.

Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations.

Effect of inhibiting dolichol phosphate-mannose formation on the biosynthesis of the N-acetylglucosaminylpyrophosphoryl polyprenols by the retina.

Previously (Kean, E.L. (1982) J. Biol. Chem. 257, 7952-7954), it was shown that dolichol phosphate-mannose stimulated the formation of the N-acetylglucosamine-containing mono-, di- and trisaccharide intermediates of the dolichol pathway. Consistent with this activating role, the present report demonstrates that inhibition of the formation of dolichol phosphate-mannose by the antibiotics, showdomycin and diumycin, also blocked the stimulatory phenomenon.

Showdomycin, a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase.

Showdomycin [2-(beta-D-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na+ + K+)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01-mol- minus 1-min- minus 1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K+, 100 MUM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na+ + K+)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase and inhibiton is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.

Effect of maleimide derivatives, sulfhydryl reagents, on stimulation of neutrophil superoxide anion generation with concanavalin A.

Using maleimide derivatives with relatively rare side reactions, their effect on stimulation of O-2 production by guinea-pig neutrophils with Con A was studied. Showdomycin, a very slowly penetrating agent, did not affect O-2 production whereas NEM, a rapidly penetrating agent, markedly inhibited O-2 production without interference with binding of Con A to cells. Particulate fractions from neutrophils stimulated with Con A showed markedly increased NADPH-dependent O-2 production compared with fractions from unstimulated cells. Treatment of neutrophils with NEM before exposure to Con A inhibited the enhancement of NADPH-dependent O-2 production of particulate fractions by Con A. However, particulate fractions from Con A-stimulated and unstimulated cells hardly exhibited reduced NADPH oxidase activity after direct exposure to NEM. Treatment of neutrophils with NEM after activation by Con A had no effect on NADPH-dependent O-2 production of particulate fractions. These results indicate that NEM inhibits the activation process of the O-2-generating enzyme, probably NADPH oxidase with Con A.

Showdomycin analogues: synthesis and antitumor evaluation.

The synthesis of N-beta-D-ribofuranosyl derivatives of maleimide, 3-methylmaleimide, and 3-chloromaleimide was accomplished in three steps from ribosylamine. The synthetic ribosides can be considered N-nucleoside analogues of showdomycin, which is an antitumor antibiotic of the C-nucleoside type. Although the three analogues were cytotoxic to cultured L1210 cells, no in vivo antitumor activity was found with the murine P388 leukemia test system. Drug transport studies were done in an attempt to trace the biological fate of the analogues.

Degradation and intracellular phosphorylation of showdomycin.

The anti-tumor agent showdomycin is degraded in the presence of 0.05 M NH4HCO3 (pH 8.0) to a compound which carries a negatively charged group. Together with previous data (Darnall et al., 1967) it is suggested that ammonia causes a basic hydrolysis of the maleimide moiety under formation of a maleic acid-like compound. During this modification the C-glycosidic bond is not split. Intracellularly, in mouse lymphoma cells (L5178y), showdomycin is phosphorylated to showdomycintriphosphate, via the mono- and diphosphate stage.

Organ secificity of rat sodium- and potassium-activated adenosine triphosphatase.

We compared several Na,K-ATPase preparations from various organs of the rat. The brain Na,K-ATPase differed from the enzymes of other organs in its pH dependence and responses to ouabain and N-ethylmaleimide in spite of similarities in the kinetic parameters of activation by Na+, K+, Mg2+, and ATP. The optimum pH of the brain MaI-enzyme was at 7.4 to 7.5 at 37 degrees D. The Lubrol extract of this brain enzyme preparation showed a lower optimum oH of 6.6. When the Lubrol extract of the brain was fractionated wtih (NH4)2SO4, the activity of the precipitate in the neutral pH region was restored. On the other hand, the optimum pH of the kidney NaI-enzyme was slightly affected by Lubrol and ammonium sulfate treatments (pH 7.5 leads to 7.3). The brain enzyme (K 1/2 = 0.9 microM) showed about 100-fold higher sensitivity to ouabain than the enzymes from other organs (I 1/2 = 100 microM) in the presence of 120 mM Na+ and 10 mM K+. In a Hill plot of the ouabain inhibition, the former failed to give a linear relationship, while the latter gave a straight line with a Hill coefficient of 1.0. The effect of K4 on the brain enzyme-ouabain interaction led us to consider that the brain enzyme might have two components as regards ouabain affinity, high and low affinity components. The time course of N-ethylmaleimide inhibition of the brain enzyme was rapid and biphasic, while the kidney enzyme showed only a slow phase following pseudo-first order kinetics. ATP protected the kidney enzyme activity completely agai,st N-ethylmaleimide inhibition, but the protection of the brain enzyme activity by ATP was only partial. We divided rat Na,K-ATPases into two groups, the brain type, which is restricted to the central nervous system, and the kidney type, which is found in most organs.

"Slim" nucleosides and nucleotides. I. Synthetic, structural and biophysical investigations of showdomycins.

A new, convenient, and short synthesis of 2'-deoxyshowdomycin, along with an improved procedure for the preparation of showdomycin, have been presented. A single-crystal X-ray structure of 1-benzyl-2'-deoxyshowdomycin (9) has been reported. Conformational studies using C.D. indicated that showdomycin exists predominantly in an anti conformation in aqueous solution. Molecular mechanics calculations using AMBER point to comparable binding energy of showdomycin-adenosine pair with the natural uridine-adenosine pair, but with a significant base-ribose conformational deviation from the natural array in the former. Implications of such a conformational deviation on tumor and viral replications have been discussed. Base-pairing studies employing high resolution NMR spectroscopy indicate that both showdomycin and epishowdomycin base-pair with adenosine-5'-monophosphate (AMP); however, while showdomycin also shows evidence of stacking, that was absent in epishowdomycin. Molecular modeling studies using QUANTA/CHARMm show that showdomycin is capable of forming a homopolymer duplex by base-pairing with poly(A), but with a considerably broader and deeper major groove. A heteropolymer duplex with a single insert of showdomycin exhibits tighter coiling at the point of insertion. A ten-picosecond dynamics simulation of the above heteroduplex revealed relaxation of the helix with disruption of H-bonding for two base pairs on either side of the insertion point, forming a large central cavity.

Synthesis of pseudo-C-nucleosides.

Whereas the reaction of 1,2-O-isopropylidene-alpha-D-xylo-5-hexulofuranuronamide (1) with the Wittig reagent ethoxycarbonylmethylenetriphenylphosphorane gave 3-(1,2-O-isopropylidene-beta-L-threofuranos-4-yl)maleimide (2, 15%) and ethyl 5-carbamoyl-5,6-dideoxy-1,2-O-isopropylidene-alpha-D-xylo-hept-5-enof uranuronate (3, 76%), a similar reaction of 3-O-benzyl-1,2-O-isopropylidene-alpha-D-ribo-5-hexulofuranuronamide++ + (4) gave only 3-(3-O-benzyl-1,2-O-isopropylidene-alpha-D-erythrofuranos-4-yl)mal eimide (5), 80%), and that of 3-O-benzyl-1,2-O-isopropylidene-alpha-D-xylo-5-hexulofuranuronamide++ + (6) gave only ethyl 3-O-benzyl-5-carbamoyl-5,6-dideoxy-1,2-O-isopropylidene- alpha-D-xylo-hept-5-enofuranuronate (7, 85%). The formation of the maleimide ring depended on the orientation and substitution of HO-3'. Compounds 2 and 5 are analogous of showdomycin.

The disturbance of oxidative phosphorylation by N-acetoxy-N-acetyl-2-aminofluorene, a model ultimate carcinogen.

Currently N-acetoxy-N-acetyl-2-aminofluorene is favored by many investigators to be a model of the ultimate electrophilic carcinogenic agent derived metabolically from the carcinogen N-acetyl-2-aminofluprene. The model induced in vitro a delayed ATP energized increase in mitochondrial volume as indicated by the decrease in absorbancy at 520 nm. The ATP energized decrease in absorbancy was inhibited by rutamycin, 2,4-dinitrophenol and a high level of antimycin known to induce ATPase activity. The known to inhibit respiration without inducing ATPase activity. Malate or potassium ion did not affect the phenomenon, however, sulfate ion which has been implicated in liver carcinogenesis shortened the induction period. Showdomycin stimulated the phenomenon. N-Acetoxy-N-acetyl-2-aminofluorene interacts with the machinery of oxidative phosphorylation. N-Acetoxy-N-acetyl-2-aminofluorene was enzymically converted by the mitochondria to N-hydroxy-N-acetyl-2-aminofluorene. These findings extend the experimental confluence of oxidative phosphorylation with carcinogenesis.