Exploiting ligand conformation in selective inhibition of non-ribosomal peptide synthetase amino acid adenylation with designed macrocyclic small molecules

Abstract

Macrocyclic aminoacyl-AMP analogs have been developed to inhibit non-ribosomal peptide synthetase amino acid adenylation domains selectively by mimicking a cisoid ligand binding conformation observed in crystal structures. In contrast, these macrocycles do not inhibit aminoacyl-tRNA synthetases, which are mechanistically closely related but bind their ligands in a distinct transoid conformation. The macrocycles contain a two- or three-carbon linker between Cβ of the amino acid moiety and C8 of the adenine ring and a sulfamate in place of the phosphate group. These compounds are potent inhibitors of the cysteine adenylation domain activity of the yersiniabactin siderophore synthetase HMWP2 and, unlike the corresponding linear aminoacyl-AMP analogs, do not inhibit protein translation in vitro. Selective small molecule inhibitors of non-ribosomal peptide synthesis should provide a powerful means to study the biological functions of non-ribosomal peptide natural products and a potential avenue to develop novel antibiotics.

Figure 1

(a) Reactions catalyzed by amino acid adenylation domains during NRP biosynthesis (PCP-SH nucleophile) and by aminoacyl-tRNA synthetases during protein translation (tRNA-OH nucleophile). PCP = peptidyl carrier protein. (b) Cisoid conformation of phenylalanine and AMP ligands in a phenylalanine adenylation domain active site (PheA; PDB 1AMU). (c) Transoid conformation of a Phe-AMP analog in a phenylalanyl-tRNA synthetase active site (PheRS; PDB 1B7Y). Adenine-C8-to-phenylalanine-Cβ distances are indicated (gray).

Figure 2

Structures of adenylation domain inhibitors and general synthetic approach to macrocycles. (R¹, R² = Boc or H; Y = NHBoc or H).