Puromycin Analogues Research Articles

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.

Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

The Role of Ribosomal Protein Deficiency in T-MDS Pathogenesis

Therapy-related myelodysplastic syndrome (t-MDS) is a lethal complication of cytotoxic cancer treatment and a leading cause of non-relapse mortality among patients undergoing autologous hematopoietic cell transplantation (aHCT) for lymphoma. The molecular abnormalities leading to t-MDS, however, are poorly understood. We are studying a prospective, longitudinal cohort of patients undergoing aHCT for lymphoma at our center with serial sample collection and banking. Using this approach we have identified impaired hematopoietic function and altered gene expression in CD34+ progenitor cells from pre-aHCT samples from patients who later developed t-MDS (cases) as compared to patients who did not develop t-MDS (controls) (Cancer Cell 2011, 20:591). These results indicate that genetic programs associated with t-MDS are perturbed long before disease onset. The alterations seen include down-regulation of genes related to ribosome biogenesis and translation, highlighting the potential contribution of altered ribosome function to disease development. Interestingly, a group of inherited ribosomal disorders including Diamond-Blackfan Anemia (DBA), called ribosomopathies, are characterized by cytopenias and a propensity for MDS development, features that closely resemble those of t-MDS patients. We evaluated the expression level of 79 ribosomal genes and a selected group of ribosome associated genes in cases and controls by multiplex Q-RT-PCR. Expression of DBA associated genes RPL10, RPL11, and RPL27 was significantly reduced in cases compared to controls. Each of these genes is reported to be associated with DBA. In addition, expression of p53, p21, c-Myc, and hdm2 were significantly upregulated in t-MDS cases. Altered expression of RPL11 is hypothesized to contribute to hematopoietic dysfunction by modulating the p53 ubiquitin ligase, HDM2, and activating p53-dependent apoptosis. We therefore characterized the effect of reduced RPL11 expression on hematopoietic function and determined the role of p53 activation in mediating the effects of RPL11 knockdown. We generated lentiviral vectors expressing shRNAs against RPL11 to reduce its expression in hematopoietic cell lines and CD34+ stem cell populations. Cells were cultured for 48 to 96 hours post-transduction followed by apoptosis analysis using Annexin-V staining, and proliferation by dye dilution assays. We confirmed >90% knockdown of RPL11 in shRNA expressing cells (MOLM13) by Q-RT-PCR and Western blot analysis. RPL11 knockdown resulted in 5-fold increase in apoptosis (Figure 1a) and 2-fold inhibition of proliferation of MOLM13 hematopoietic cells that express wild type p53, but did not affect survival and proliferation of p53 mutated HL60 cells. This suggests that cellular responses to RPL11 deficiency may depend at least in part on intact p53 signaling. This was confirmed in experiments showing that concomitant shRNA mediated knockdown of p53 and RPL11 (Figure 1a) resulted in a 2-fold reduction of apoptosis compared to RPL11 knockdown alone. Furthermore, RPL11 knockdown in CD34+ stem/progenitor cells resulted in 40% increase in apoptosis, 30% inhibition of proliferation and 90% decrease in colony forming cell (CFC) frequency in methylcellulose progenitor assays. We measured protein translation by measuring the intensity of an alkyne analog of puromycin (OPP) integrated into newly translated proteins, ligation to an azide dye, and flow cytometry analysis. We observed a 5-fold drop in global protein translation with RPL11 knockdown using the Click-iT Plus Alexa Fluor Protein Synthesis Assay (Figure 1b). Our results suggest that ribosomal abnormalities occur early in the course of t-MDS development and may lead to enhanced cell death and impaired proliferation. These results support a potential role of aberrant RPL11 expression in altered cell survival, proliferation, and differentiation, which may contribute to hematopoietic dysfunction seen early in the course of t-MDS development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Imaging of protein synthesis with puromycin

With great interest, we read the work by Liu et al. (1), which described the development of an alkyne analog of puromycin for labeling and imaging of newly synthesized peptides. Available techniques for this type of measurement are very limited, and the work by Liu et al. (1) claimed that an alternative method, which is based on antipuromycin immunohistochemistry (SUnSET) (2), results in a subcellular localization of nascent peptides that is not consistent with the expected location of newly synthesized proteins [e.g., “antipuromycin staining shows the strongest signal at the plasma membrane” (1)]. For the following reasons, we have serious concerns with this potentially misleading claim. First, the claim is largely based on studies in which azidohomoalanine (Aha)/homopropargylglycine (Hpg) analogs of methionine were used to identify the localization of newly synthesized proteins. However, there is currently … [↵][1]1To whom correspondence should be addressed. E-mail: pierre{at}ciml.univ-mrs.fr. [1]: #xref-corresp-1-1

Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin

Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation, or death. Methods that allow imaging and identification of nascent proteins are critical for dissecting regulation of translation, both spatially and temporally, particularly in whole organisms. We introduce a simple and robust chemical method to image and affinity-purify nascent proteins in cells and in animals, based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-puro). OP-puro forms covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized or captured by copper(I)-catalyzed azide-alkyne cycloaddition. Unlike methionine analogs, OP-puro does not require methionine-free conditions and, uniquely, can be used to label and assay nascent proteins in whole organisms. This strategy should have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

The Shortest Synthetic Route to Puromycin Analogues Using a Modified Robins Approach

We are reporting on the utility of commercial vinyl isocyanate for a practical synthetic route from adenosine to N(6)-bis-demethylpuromycin in seven steps and 65% overall yield. A clean one-pot conversion of 3'-bromo-2'-carbamoyl derivative 8 to 3'-amino-3'-deoxyadenosine derivative 10 is the main feature of this synthetic pathway. This synthesis is the shortest synthetic route toward 3'-(aminoacylamido)deoxyadenosines to date.

ChemInform Abstract: Analogues of the Antibiotic Puromycin as Potential Prodrugs of 3′- Amino-3′-deoxythymidine.

Condensation of L- and D-3′-amino-2′,3′-dideoxynucleosides 2–5 with N-BOC-protected aminoacids 6 and 13 using dicyclohexylcarbodiimide and N-hydroxysuccinimide in DMF is reported to give the N-BOC-protected acylamino aminonucleosides 7–9 and 14 in 51–81% yield. After deprotection using trifluoroacetic acid the corresponding unprotected new analogues of puromucin 10–12 and 15 were obtained in 43–56% yield. These compounds did not show any significant antiviral activity using HIV (stain HTLV-III B)-infected MT-4 cells as target system.

Total Syntheses of a Conformationally Locked North‐Type Methanocarba Puromycin Analogue and a Dinucleotide Derivative

An original synthetic approach for the first synthesis of an enantiopure methanocarba puromycin (3'-alpha-aminoacylamino-3'-deoxyadenosine) analogue and its cytidine dinucleotide derivative is described. Each compound is conformationally locked in a North-type pucker and exhibits both a pseudoaxial hydroxy group and a pseudoequatorial aminoacyl group. The syntheses were accomplished from D-ribose in 18 and 19 steps, respectively, with key steps being a ring-closing metathesis, a Luche reduction, a Simmons-Smith cyclopropanation, a Mitsunobu coupling, a Mattocks bromoacetylation, a regioselective and a stereoselective nucleophilic substitution, a chemoselective phosphoramidite coupling and a Staudinger-Vilarrasa coupling. Both molecules are being tested for peptidyl transfer efficiency in ribosomes for comparison with the peptidyl transfer kinetics of natural puromycin and other natural and synthetic ribosomal A site substrates.

Synthesis of Conformationally Locked Versions of Puromycin Analogues

Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North (2 and 3) and South (6 and 7) 3'-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine. North azides 2 and 3 were reported earlier. The 3'-azido intermediates 6 and 7 that are necessary for the synthesis of the South puromycin analogues are described herein for the first time.

Total syntheses of a North methanocarba Puromycin analog and its dinucleotide derivative

North methanocarba Puromycin analog 5 and its di-nucleotide derivative 6 were synthesized from D-ribose in respectively 18 and 19 steps, in order to be tested for peptidyl transfer efficiency in ribosomes.

Structural and functional prerequisites for ribosomal nascentpeptide acceptors: Attempts to decipher the nature of the ribosome's catalysis of peptide bond formation.

Aminoacyl ribonucleoside analogues that are capable of binding to the acceptor site of ribosomes and taking over the nascent peptide bear, if properly designed, the potential of antibiotic and cytostatic activity. Here we present a study on the intrinsic conformations of natural and synthetic peptide acceptors and the basicities of their peptide accepting amino groups. The conformations and thermodynamic parameters of several synthetic puromycin analogues have been elucidated through ab initio calculations as well as temperature and pH dependent (1)H NMR experiments. The intrinsic basicities of their peptide accepting amino groups were determined through (1)H NMR and compared to the effective basicities of the peptide accepting amino groups of aminoacyl transfer RNAs with the same amino acid side chains, as estimated from the pH dependent kinetics of mRNA programmed ribosomal peptidyl transfer.

Inhibition of Helicobacter pylori Aminoacyl-tRNA Amidotransferase by Puromycin Analogues

Recent genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and archaea. The survival of microorganisms missing one or both of these essential enzymes implies an alternative pathway for the formation of Gln-tRNAGln and Asn-tRNAAsn. This indirect pathway involves misacylation of tRNAGln with Glu (or tRNAAsn with Asp) by a nondiscriminating synthetase and subsequent transamidation of the misacylated aa-tRNA by an amidotransferase. Analogues of the natural product puromycin have been developed to inhibit this class of enzymes which has been only recently investigated at the mechanistic and structural levels. The widespread use of the indirect transamidation pathway among prominent human pathogens and its absence in the cytosol of mammalian cells identify aminoacyl-tRNA amidotransferases as an interesting target for the development of antimicrobial agents with a novel mode of action. These mechanism-based inhibitors will provide useful probes for further mechanistic investigations and ligands for X-ray crystallography.

Facile and Rapid Access to Inosine Puromycin Analogues through the Use of Adenylate Deaminase

To study the ribosomal peptidyl transfer, puromycin analogues are of interest in which adenine has been replaced by hypoxanthine. We synthesized inosine puromycin analogues from 3'-azidodeoxyadenosine derivatives using adenylate deaminase for the quantitative transformation of the N-heterocycle. The amino acid coupling was carried out under Staudinger-Vilarrasa conditions in 94% yield starting from the protected and in 82% using the unprotected azide, thus, in the presence of two hydroxyls and a lactam function.

Shorter puromycin analog synthesis by means of an efficient Staudinger–Vilarrasa coupling

An efficient Staudinger–Vilarrasa coupling generates amides from azides and 1-hydroxybenzotriazole esters of amino- or hydroxy acid derivatives in very high isolated yields and purity. New puromycin analogs, mostly putative biosynthetic intermediates, were synthesized in nine steps from adenosine.

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1P198 ピューロマイシン類似体を用いたトロンボポエチンのC末端蛍光標識と1分子イメージングへの応用(接着・運動・骨格・伝達・膜)))

High-yield immobilization of a puromycin analogue for the solid support synthesis of aminoacyl-tRNA fragments.

[reaction: see text] An efficient procedure for the immobilization of 3'-deoxy-3'-(O-methyltyrosyl)aminoadenosine was developed. A poly(ethylene glycol)-derived diacid linker/spacer was attached to aminomethyl polystyrene. Coupling of the 2'-hydroxy instead of the 2'-O-succinylated ribonucleoside resulted in high immobilization yields (over 80%) and allowed for the recovery of valuable unreacted material. This specific procedure should be applicable to other ribonucleosides containing a bulky modification at the 3'-position and can be used for the stepwise construction of 3'-aminoacyl- or 3'-peptidyl-RNA conjugates.

The puromycin route to assess stereo- and regiochemical constraints on peptide bond formation in eukaryotic ribosomes.

We synthesized a series of puromycin analogues to probe the chemical specificity of the ribosome in an intact eukaryotic translation system. These studies reveal that both d-enantiomers and beta-amino acid analogues can be incorporated into protein, and provide a quantitative means to rank natural and unnatural residues. Modeling of a d-amino acid analogue into the 50S ribosomal subunit indicates that steric clash may provide part of the chiral discrimination. The data presented provide one metric of the chiral and regiospecificity of mammalian ribosomes.

Next-generation protein-handling method: puromycin analogue technology

Puromycin is a well-known antibiotic that inhibits protein synthesis by competitive incorporation against an aminoacyl tRNA on the ribosome A site. Novel technology using this property of puromycin has been developed for convenient handling methods in protein research. Puromycin modified with another molecule is incorporated into a protein at the C-terminus, thus linking the desired molecule to the protein. Combination of in vitro translation with puromycin analogues has resulted in novel technologies such as display technology for screening, fluorescence labeling, affinity purification, and protein chip for proteomics.

A practical route to 3'-amino-3'-deoxyadenosine derivatives and puromycin analogues.

3'-aminoacylamino-3'-deoxyadenosines, analogues of the antibiotic puromycin, have been synthesized from adenosine. They key 3'-azido derivative 10 was obtained through a 3'-oxidation/reduction/substitution procedure. A modified purification protocol on a larger scale was developed for the oxidation step using the Garegg reagent. The coupling reaction between an Fmoc-l-amino acid and the fully protected form of 3'-amino-3'-deoxyadenosine 11 furnished the aminoacylated compounds 12 in high yields. The puromycin analogues were obtained in 10 steps and up to 23% (14c) overall yield.

Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.