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Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-impos...

The Plasmodium proteasome (Pf20S) emerged as a target for antimalarials. Pf20S inhibitors are active at multiple stages of the parasite life cycle and synergize with artemisinins, suggesting that Pf20S inhibitors have potential to be prophylactic, therapeutic, and transmission blocking as well as are useful for combination therapy. We recently reported asparagine ethylenediamines (AsnEDAs) as immunoproteasome inhibitors and modified AsnEDAs as selective Pf20S inhibitors. Here, we report further a structure-activity relationship study of AsnEDAs for selective inhibition of Pf20S over human proteasomes. Additionally, we show new mutation that conferred resistance to AsnEDAs and collateral sensitivity to an inhibitor of the Pf20S β2 subunit, the same as previously identified resistant mutation. This resistance could be overcome through the use of the structure-guided inhibitor design. Collateral sensitivity to inhibitors among respective proteasome subunits underscores the potential value of treating malaria with combinations of inhibitors of different proteasome subunits to minimize the emergence of drug resistance.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) are phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill non-replicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target, because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The co-crystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose-dependently led to accumulation of Pup-tagged GFP that is degradable but resistant to depupylation., and death of non-replicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Co-evolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders non-replicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure-activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.