Structure Elucidation, Biosynthetic Gene Cluster Distribution, and Biological Activities of Ketomemicin Analogs in Salinispora

Pseudopeptides are attractive agents for protease inhibition due to their structural similarities to the natural substrates of these enzymes, as well as their enhanced stability and resistance to enzymatic degradation. We report three new ketomemicin pseudopeptides (<b>1</b>-<b>3</b>) from extracts of the marine actinomycete <i>Salinispora pacifica</i> strain CNY-498. Their constitution and relative configuration were elucidated using NMR, mass spectrometry, and quantum chemical calculations. Using GNPS molecular networking and publicly available <i>Salinispora</i> LCMS datasets, five additional ketomemicin analogs (<b>4</b>-<b>8</b>) were identified with ketomemicin production detected broadly across <i>Salinispora</i> species. The ketomemicin biosynthetic gene cluster (<i>ktm</i>) is highly conserved in <i>Salinispora</i>, occurring in 79 of 118 public genome sequences, including eight of the nine named species. Outside <i>Salinispora</i>, <i>ktm</i> homologs were detected in various genera of the phylum Actinomycetota that might encode novel ketomemicin analogs. Ketomemicins <b>1</b>-<b>3</b> were tested against a panel of eleven proteases, with <b>2</b> displaying moderate inhibitory activity. This study describes the first report of ketomemicin production by <i>Salinispora</i> cultures, the distribution of the corresponding biosynthetic gene cluster, and the protease inhibitory activity of new ketomemicin derivatives.