Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway

Macrocyclic peptides
are sought-after molecular scaffolds for drug
discovery, and new methods to access diverse libraries are of increasing
interest. Here, we report the enzymatic synthesis of pyridine-based
macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides
are a recently described class of ribosomally synthesized and post-translationally
modified peptides (RiPPs) and are related to the long-known thiopeptide
natural products. RiPP precursors typically contain an N-terminal
leader region that is physically engaged by the biosynthetic proteins
that catalyze modification of the C-terminal core region of the precursor
peptide. We demonstrate that pyritide-forming enzymes recognize both
the leader region and a C-terminal tripeptide motif, with each contributing
to site-selective substrate modification. Substitutions in the core
region were well-tolerated and facilitated the generation of a wide
range of pyritide analogues, with variations in macrocycle sequence
and size. A combination of the pyritide biosynthetic pathway with
azole-forming enzymes was utilized to generate a thiazole-containing
pyritide (historically known as a thiopeptide) with no similarity
in sequence and macrocycle size to the naturally encoded pyritides.
The broad substrate scope of the pyritide biosynthetic enzymes serves
as a future platform for macrocyclic peptide lead discovery and optimization.