Ανάπτυξη Και Χαρακτηρισμός Νέων Αντιβιοτικών - Αναστολέων Της Πρωτεϊνικής Σύνθεσης

Abstract

Ribosomes translate the genetic code into proteins in all living cells with extremely high efficiency, owing to their inherent flexibility and to their spectacular architecture. These large ribonucleoprotein particles synthesize proteins in all cells, using messenger RNA as the template, confirming that the ribosome is indeed a ribozyme, and aminoacyl-transfer RNAs as substrates (Steitz and Moore, 2003; Simonović and Steitz, 2008; Schmeing and Ramakrishnan, 2009). As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. New studies indicate that in some cases, the tunnel plays a more active role. Accumulated evidence had definitely concluded that ribosomal tunnel is not only a passive conduit for the nascent chains but a rather functionally important compartment, where specific peptide sequences establish direct interactions with the tunnel, talking back to the ribosome in order to regulate peptide synthesis, leading to translational stalling (Wilson and Beckmann, 2011; Ramu et al., 2011; Vazquez-Laslop et al., 2008; Seidelt et al., 2009; Wei et al., 2012; Bhushan et al., 2010).In this study, we have investigated functional interactions between distinct locations of the ribosomal tunnel and specific residues of the peptidyltransferase, using new macrolides, which were kindly provided by the pharmaceutical company Κosan Biosciences Inc. These compounds represent the newest generation of macrolide antibiotics, known as ketolides, carrying also fluorine attached to the lactone ring either directly or indirectly. According to our results, the new antibiotics exhibited better antimicrobial activity in vivo against Gram-positive and negative bacteria and strongly inhibited the translational apparatus and could be useful tools in the future for treatment of bacterial infections. Binding studies with radiolabeled erythromycin revealed that these drugs bound competitively with erythromycin at the large ribosomal subunit, with extremely low dissociation constants. In parallel, RNA footprinting indicated that the new ketolides occupy the main macrolide binding site in the ribosome, which is located at the entrance of the exit tunnel adjacent to the peptidyltransferase center (Bulkley et al., 2010; Dunkle et al., 2010; Tu et al., 2005). These drugs interact with A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central A2058, A2059 and A2062 in the central loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, loop of domain V 23S rRNA, as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended as well their extended heteroaromatic alkylalkyl alkylalkyl-aryl side chain penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, penetrates deeper in the tunnel protecting A752 of Helix 35, interacting with basepair A752-U2609.To explore further this interaction, we have synthesized new peptidyl conjugates of chloramphenicol, which resemble nascent peptidyl-tRNA chains bound to the A-site of the24ribosome, with their peptide sequence located deeper within tunnel, adopting an extended configuration. According to our data, these compounds did indeed bind to the peptidyl transferase center, competing with radiolabelled- chloramphenicol for binding to the ribosome. The binding of each analog, as well as its inhibitory activity of ribosomal function, is absolutely idiosyncratic, depending on the sequence of amino acid of peptide moiety. Studying the footprinting pattern of these compounds on the ribosome, we confirmed that while the chloramphenicol base maintains its position on the A-site, the peptide moiety penetrates deeper in the entrance of the exit tunnel, interacting with nucleotide A752. Therefore, we believe that these chloramphenicol peptides could be useful tools for probing nascent polypeptide chain interaction with the ribosome.