Puromycin activity

Puromycin. Puromycin (19), elaborated by S. alboniger (1—4), inhibits protein synthesis by replacing aminoacyl-tRNA at the A-site of peptidyltransferase (48,49). Photosensitive analogues of (19) have been used to label the A-site proteins of peptidyltransferase and tRNA (30). Compound (19), and its carbocycHc analogue have been used to study the accumulation of glycoprotein-derived free sialooligosaccharides, accumulation of mRNA, methylase activity, enzyme transport, rat embryo development, the acceptor site of human placental 80S ribosomes, and gene expression in mammalian cells (51—60). 

GatCAB amidotransferase.This natural product mimics the charged 3 -terminus of aa-tRNA and has been used as a tool for the study of protein biosynthesis. The parent compound 22 is a very weak inhibitor of AdT. The amino acid chain is related to tyrosine and differs from the glutamic and aspartic side chains transformed in the kinase or the transamidase steps. Replacement of the methoxyphenyl moiety of puromycin by carboxylic acid derivatives (23-26) improved the ability to inhibit this AdT. Stable analogues of the transition state in the last step of the transamidation process (27-29) where the carbonyl to be attacked by NH3 is replaced by tetrahedral sulfur or phosphorus atom with a methyl group mimicking ammonia exhibited the highest activity. 

Although demethylation, which occurs in the liver, is normally considered to be a catabolic process, it may result in conversion of an inactive form of a drug to the active form. Thus 6-(methylthio)purine (XXXIX) is demethylated by the rat to 6-mercaptopurine [205]. This demethylation occurs in the liver micro-somes and is an oxidative process which converts the methyl group to formaldehyde [204, 207]. The 1-methyl derivative of 4-aminopyrazolo[3,4-d] pyrimidine (XLI) is demethylated slowly, but 6-mercapto-9-methylpurine (XLII) not at all [208]. The A -demethylation of puromycin (XLlIl) [209, 210], its aminonucleoside (XLIV) [211], and a number of related compounds, including V-methyladenine and V,V-dimethyladenine, occurs in the liver microsomes of rodents [212]. In the guinea-pig the rate-limiting step in the metabolism of the aminonucleoside appears to be the demethylation of the monomethyl compound, which is the major urinary metabolite [213]. The relationship of lipid solubility to microsomal metabolism [214], and the induction of these demethylases in rats by pre-treatment with various drugs have been studied [215]. 

Although inferior to pyrimethamine plus sulphonamides, both puromycin and the aminonucleoside are active against Toxoplasma gondii in vivo [403]. These drugs are also effective against Endamoeba histolytica in vitro [404], and puromycin is active against the infection in man [405]. 

The mechanism of inhibition of these protozoal infections by the most active drugs, puromycin and the aminonucleoside, is not known. Puromycin and nucleocidin both interfere with protein synthesis, but the aminonucleoside does not. It is known to be demethylated to 3 -amino-3-deoxyadenosine, which is phosphorylated and interferes with nucleic acid metabolism (see above). Whether puromycin must be converted to the aminonucleoside before it can inhibit protozoa has not been established. Some purine analogues known to interfere with nucleic acid metabolism, however, are less effective as antiprotozoal agents, even in vitro, perhaps because their effects are primarily on the de novo pathway which many, if not all, protozoa do not use [406]. 

Puromycin is active against a number of vimses in cell culture. In chick embryo cells it delayed the replication of western equine encephalitis [415] and inhibits Venezulian equine encephalitis [416]. It interferes with the replication... 

There are several antitumor antibiotics resembling the synthetic nucleoside antimetabolites discussed on p. 160. They are adenine derivatives with unusual furanose compounds attached to the 9-position. Psicofuranine (54) is an example. The rather more complex example puromycin (55) is also active against trypanosomes and Gram-positive bacteria. Tubercidin (56) and two related antibiotics have an altered purine system. 

Mechanism and Genetics of Induction in Mammals. Many different mechanisms may be involved in CYP induction. These include increased transcription of DNA, increased mRNA translation to protein, mRNA stabilization, and protein stabilization. Induction can only occur in intact cells and cannot be achieved by the addition of inducers directly to cell fractions such as microsomes. It has been known for some time that in most cases of increase in monooxygenase activity there is a true induction involving synthesis of new enzyme, and not the activation of enzyme already synthesized, since induction is generally prevented by inhibitors of protein synthesis. For example, the protein synthesis inhibitors such as puromycin, ethionine, and cyclo-heximide inhibit aryl hydrocarbon hydroxylase activity. A simplified scheme for gene expression and protein synthesis is shown in Figure 9.7. 

The reactions of phase 2 relate to the attachment of the bridge-carbohydrate residues to the polypeptide chain. There is evidence showing that this addition occurs while the polypeptide chain is still attached to, or perhaps still being synthesized on, the ribosomes.101-103 Thus, 14C-labeled 2-amino-2-deoxy-D-glucose, injected into the circulatory system of the rat, was incorporated into protein in the ribosomes of the rough endoplasmic-reticulum of the liver. Administration of puromycin caused release of the 14C-labeled glycoprotein, which could be isolated by acid-precipitation methods. Examination of the radioactivity data revealed that the subcellular structures most actively involved in glycoprotein synthesis were the ribosomes bound to the membrane, and not free polysomes. 

Sparsomydn inhibits protein synthesis in all organisms and consequently is not used as an antibiotic. However, it has been tested extensively for its antitumor activity [49]. Sparsomycin contains a pseudouracil base with a conjugated link to a sulfur containing tail (Fig. 4.11). This chemical structure has been compared with that of puromycin, and it has been su ested that sparsomycin binds to the A-site much like puromycin [50]. Sparsomycin does not bind to the ribosome unless a P-site substrate is also present. 

Nucleocidin (379) which contains the first fluorosugar to be obtained from a natural source is a more potent inhibitor of protein synthesis in vivo than puromycin but has similar activity in vitro (66JBC(24)l09l). 

Proteosome component C2 Proteosome component Rpt2 Proteosome o6 subunit Proteosome o4 subunit Peptidyl prolylisomerase Prolj4 oligopeptidase Puromycin sensitive aminopeptidase Ubiquitin activating enzyme 1 Ubiquitin carboxy hydrolase... 

The peptidyltransferase activity of ribosomes can be segregated from other translation reactions by the so-called puromycin reaction [129] which monitors the formation of [acetyl-aminoacyl]-puromycin or [peptidyl]-puromycin from puromycin and either [acetyl-aminoacyl]-tRNA or [peptidyl]-tRNA. In its simplest form (termed uncoupled or 30S-subunit-independent peptidyltransferase) the reaction requires large ribosomal subunits and an organic solvent (ethanol or methanol) which is presumably needed to promote the binding of tRNA and puromycin to the 50S subunit. In the absence of organic solvents, however, the reaction (then termed coupled or 30S-subunit-dependent... 

Altamura et al. [178] and Londei et al. [179] investigated the ability of SOS and 30S subunits from phylogenetically disparate archaea to form synthetically active hybrid ribosomes with subunits from bacteria and eucarya, in the presence of Mg " concentrations (lS-18mM) which are optimal for polyphenylalanine synthesis. With poly(U) as the template and Phe-tRNA (or [jV-acetyl-Phe]-puromycin) as the substrate, SOS and 30S subunits from Euryarchaeota (M vannielii) and Crenarchaeota (5. solfataricus) could be assembled into hybrid active monosomes in all reciprocal combinations surprisingly, however, both reciprocal combinations of archaeal (S. solfataricus, M. vannielii) and eucaryal S. cerevisiae) ribosomal subunits gave rise... 

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