Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin‐like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup‐based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I‐OMe‐Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast‐reversible, non‐ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD‐Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds. The Mycobacterium tuberculosis (Mtb) prokaryotic ubiquitin‐like (pup) proteasome system is an attractive target for new drug development. In this study a screen was performed identifying Tyrphostins as low micromolar inhibitors of the Mtb protease Dop. To gain insight in the important functional aspects of these inhibitors, 25 new analogues were prepared and validated, in vitro. Several new compounds were able to inhibit both the depupylating activity of Dop as well as the pupylating activity of de pup ligase PafA.