Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium tuberculosis Proteasome

Abstract

The 20S core particle of the proteasome in Mycobacterium tuberculosis (Mtb) is a promising, yet unconventional, drug target. This multimeric peptidase is not essential, yet degrades proteins that have become damaged and toxic via reactions with nitric oxide (and/or the associated reactive nitrogen intermediates) produced during the host immune response. Proteasome inhibitors could render Mtb susceptible to the immune system, but they would only be therapeutically viable if they do not inhibit the essential 20S counterpart in humans. Selective inhibitors of the Mtb 20S were designed and synthesized on the bases of both its unique substrate preferences and the structures of substrate-mimicking covalent inhibitors of eukaryotic proteasomes called syringolins. Unlike the parent syringolins, the designed analogues weakly inhibit the human 20S (Hs 20S) proteasome and preferentially inhibit Mtb 20S over the human counterpart by as much as 74-fold. Moreover, they can penetrate the mycobacterial cell envelope and render Mtb susceptible to nitric oxide-mediated stress. Importantly, they do not inhibit the growth of human cell lines in vitro and thus may be starting points for tuberculosis drug development.

Keywords: host−pathogen interaction; innate immune response; nitric oxide; peptidomimetic; protein homeostasis; syringolins.

Figure 1

Chemical structures of syringolin A (SylA) and syringolin B (SylB) proteasome inhibitors.

Figure 2

Structural model for proteasome substrate mimicry by the syringolins. Substituents at R and R′ correspond to P1 and P3 residues of a peptide substrate, respectively.

Figure 3

Evaluating the cell permeability and activity of selective Mtb 20S inhibitors using M. bovis BCG. Apparent activity reflects proteasome-activity observed in lysates from the treated and untreated cultures. The proteasome activity observed in the untreated sample was set as 100%. The experiments were performed in triplicate. See supporting information for experimental protocols.

Figure 4

Mtb nitric-oxide stress assays for compound 14. 0 μM experiment indicates CFU/mL value of DMSO control experiment. The lightly colored bars show CFU counts in assays lacking NaNO₂. Assays were performed in triplicate. See supporting information for protocol.

Scheme 1

Synthesis of syringolin analogs.^–