Thiostrepton Variants Containing a Contracted Quinaldic Acid Macrocycle Result from Mutagenesis of the Second Residue

Abstract

The thiopeptides are a family of ribosomally synthesized and posttranslationally modified peptide metabolites, and the vast majority of thiopeptides characterized to date possess one highly modified macrocycle. A few members, including thiostrepton A, harbor a second macrocycle that incorporates a quinaldic acid moiety and the four N-terminal residues of the peptide. The antibacterial properties of thiostrepton A are well established, and its recently discovered ability to inhibit the proteasome has additional implications for the development of antimalarial and anticancer therapeutics. We have conducted the saturation mutagenesis of Ala2 in the precursor peptide, TsrA, to examine which variants can be transformed into a mature thiostrepton analogue. Although the thiostrepton biosynthetic system is somewhat restrictive towards substitutions at the second residue, eight thiostrepton Ala2 analogues were isolated. The TsrA Ala2Ile and Ala2Val variants were largely channeled through an alternate processing pathway wherein the first residue of the core peptide, Ile1, is removed and the resulting thiostrepton analogues bear quinaldic acid macrocycles abridged by one residue. This is the first report revealing that quinaldic acid loop size is amenable to alteration during the course of thiostrepton biosynthesis. Both the antibacterial and proteasome inhibitory properties of the thiostrepton Ala2 analogues were examined. While the identity of the residue at the second position of the core peptide influences thiostrepton biosynthesis, our report suggests it may not be crucial for antibacterial and proteasome inhibitory properties of the full-length variants. In contrast, the contracted quinaldic acid loop can, to differing degrees, affect both types of biological activity.

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…271, 277, 305 Other studies have also sought to increase the water solubility of thiostrepton without reducing target potency by introducing polar residues at positions 2 and 4. These positions were chosen because they point toward bulk solvent in the co-crystal structure of thiostrepton bound to the 50S ribosome and are less likely to affect binding potency.…”

“…Despite these lower potencies against live bacteria, in vitro translation inhibition was approximately identical for most of the substitutions, indicating the lowering in whole cell activity comes from other sources such as uptake, export, or stability. 277, 305 …”

“…277, 306 The quinaldic acid ring can also be contracted if Val or Ile are substituted at position 2 because the leader protease appears to cleave the peptide just before Val/Ile so the traditional position 1 will be removed when position 2 is Val/Ile. 277 Additionally, a fourth ring has been formed by introducing a Cys into the side ring that can then adventitiously react with a dehydroalanine in the tail region. 305 However, both of these modifications likely disrupt the structure of the quinaldic acid ring, preventing it from interacting with the 23S ribosomal subunit properly, and result in dramatic loss of antibacterial activity.…”

Efficient methylation of C2 in l-tryptophan by the cobalamin-dependent radical S-adenosylmethionine methylase TsrM requires an unmodified N1 amine

“…Although it is currently used in topical treatments in veterinary medicine, its clinical use in humans is hampered by its poor solubility and poor gastric absorption (6). Therefore, efforts are under way to alter its pharmacokinetic properties through both chemical and biosynthetic processes (7,8).…”

Natural product scaffolds as inspiration for the design and synthesis of 20S human proteasome inhibitors

“…115 Zhang et al employed the use of an S. laurentii tsrA mutant organism to produce Ala2 thiostrepton analogs and additionally evaluated them for their inhibitory activity towards the 20S proteasome. 116 Several macrocyclic peptide scaffolds that have been identified as inhibitors of the proteasome have yet to be further evaluated through SAR studies (Charts 8 and 9). Among these natural products are the phepropeptins.…”