NMR Structures of Thiostrepton Derivatives for Characterization of the Ribosomal Binding Site

The synthesis of complex, biologically active molecules by catalyst-controlled, selective functionalization of complex molecules is an emerging capability. We describe the application of Rh-catalyzed conjugate arylation to the modification of thiostrepton, a complex molecule with potent antibacterial properties for which few analogs are known. By this approach, we achieve the site- and stereoselective functionalization of one subterminal dehydroalanine residue (Dha16) present in thiostrepton. The broad scope of this method enabled the preparation and isolation of 24 new analogs of thiostrepton, the biological testing of which revealed that the antimicrobial activity of thiostrepton tolerates the alternation of Dha16 to a range of amino acids. Further analysis of this Rh-catalyzed process revealed that use of sodium or potassium salts was crucial for achieving high stereoselectivity. The catalyst system was studied further by application to the synthesis of amino esters and amides from dehydroalanine monomers, a process which was found to occur with up to 93 : 7 er under conditions milder than those previously reported for analogous reactions. Furthermore, the addition of the same sodium and potassium salts as applied in the case of thiostrepton lead to a nearly full reversal of the enantioselectivity of the reaction. As such, this study of site-selective catalysis in a complex molecular setting also delivered synergistic insights in the arena of enantioselective catalysis. In addition, these studies greatly expand the number of known thiostrepton analogs obtained by any method and reveal a high level of functional group tolerance for metal-catalyzed, site-selective modifications of highly complex natural products.

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“…2a). 9 Using either KOH or Et 3 N as base, the major product observed is compound 3 ( 37–43% conversion by UPLC, Fig. 2b, entries 1–2).…”

Section: Resultsmentioning

confidence: 99%

Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-Catalyzed Conjugate Arylation: New Analogues and Collateral Enantioselective Synthesis of Amino Acids

Key

Miller

2017

J. Am. Chem. Soc.

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“…(44) The C-terminal tail, on the other hand, does sample a variety of conformations in solution and could access a position poised for nucleophilic attack of the Dha16 or Dha17 side chain by the Cys4 thiol. (45) It is also worth noting that Lan or MeLan formation could precede the installation of one or both macrocycles of the native thiostrepton architecture. To examine the possibilities for the location of the thioether bridge, one- and two-dimensional NMR studies were performed with thiostreptons Ala4Cys F1 and F2 (Figures S4–S10, S12–S18, and Tables S1 and S2).…”

Section: Resultsmentioning

confidence: 99%

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Zhang

Kelly

2015

ACS Chem. Biol.

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Thiopeptides are posttranslationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery’s tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogs. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched, hydrophobic residues, are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogs were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

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“…It is not yet clear whether TS binds iron or simply mimics an iron-bound conformation, as it has multiple hydroxyls positioned in a manner that could coordinate metals ( Fig 1A ). Its structure has been solved both by X-ray crystallography and NMR, but no bound metals were reported (47, 48). The FpvA receptor is also exploited by S-pyocins, 40-80 kDa peptide antibiotics produced by competing P. aeruginosa strains, showing that it is a key promiscuous entry point for diverse molecules in addition to its usual ligand, pyoverdine (33, 34, 49).…”

Section: Discussionmentioning

confidence: 99%

Thiostrepton hijacks pyoverdine receptors to inhibit growth ofPseudomonas aeruginosa

Ranieri

Chan

Yaeger

et al. 2019

Preprint

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17Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and intrinsically resistant to many 18 antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered 19 that the thiopeptide antibiotic, thiostrepton (TS) -considered inactive against Gram-negative 20 bacteria -stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is 21 characteristic of exposure to antimicrobial compounds at sub-inhibitory concentrations, suggesting that 22 TS was active against P. aeruginosa. Supporting this observation, TS inhibited growth of a panel of 96 23 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low micromolar concentrations. TS also had 24 activity against Acinetobacter baumannii clinical isolates. Expression of Tsr -a 23S rRNA-modifying 25 methyltransferase -in trans conferred TS resistance, confirming that the drug acted via its canonical 26 mode of action, inhibition of ribosome function. Deletion of oligopeptide permease systems used by 27 other peptide antibiotics for uptake failed confer TS resistance. TS susceptibility was inversely 28 proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is 29 increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A.30 baumannii was potentiated by FDA-approved iron chelators deferiprone and deferasirox. Screening of P. 31 aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to 32 cross the outer membrane. Our data show that the biofilm stimulation phenotype can reveal cryptic 33 sub-inhibitory antibiotic activity, and that TS has activity against select multidrug resistant Gram-34 negative pathogens under iron-limited growth conditions, similar to those encountered at sites of 35 infection.

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NMR Structures of Thiostrepton Derivatives for Characterization of the Ribosomal Binding Site

Cited by 33 publications

References 45 publications

Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-Catalyzed Conjugate Arylation: New Analogues and Collateral Enantioselective Synthesis of Amino Acids

Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-Catalyzed Conjugate Arylation: New Analogues and Collateral Enantioselective Synthesis of Amino Acids

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Thiostrepton hijacks pyoverdine receptors to inhibit growth ofPseudomonas aeruginosa

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