Selective Phenylimidazole-Based Inhibitors of the Mycobacterium tuberculosis Proteasome

Abstract

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-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders nonreplicating 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.

Figure 1.

Chemical structures of the Mtb20S selective inhibitors. Compounds in the class of 1,3,4-oxathiazol-2-ones and analogs of syringolin 14 are irreversible inhibitors, whereas dipeptide DPLG2 is a noncovalent inhibitor.

Figure 2.

Illustration of CyclOps™ operation for iterative structure-activity study of the N,C-capped dipeptides. Reproduced with permission from Journal of Medicinal Chemistry with slight modification. Copyright 2013 American Chemical Society.

Figure 3.

Dose-dependent inhibition of β5 activity of the Mtb20S, hu c-20S and i-20S by N,C-capped dipeptides. Data are representative of at least three independent experiments. Suc-LLVY-AMC was used as substrate for Mtb20S and c-20S at 100 μM and 25 μM final concentrations, respectively. Ac-ANW-AMC (15 μM) was used as substrate for i-20S.

Figure 4.

Electron density maps of proteasome inhibitors in the active site of Mtb20S proteasome core particle. The 2mFo-DFc maps are scaled to 1 σ and are shown in grey mesh. The two neighboring β-subunits are shown in green and cyan and the Thr-1 position is labeled in red. A & C A85 (PDB: 6OCW). B & D, A86 (PDB: 6OCZ). Possible hydrogen bonds between ligands and proteasome are depicted as orange dashed lines. The inhibitors are shown in yellow sticks; the citrates are in grey sticks; water molecules are shown as red spheres.

Figure 5.

Peptidomimetic evolution of N,C-capped dipeptides to phenylimidazoles as selective Mtb20S inhibitors.

Figure 6.

Inhibition of Mtb20S, hu c-20S and i-20S by phenylimidazoles. Data are representative of at least three independent experiments. Suc-LLVY-AMC was used as substrate for Mtb20S and c-20S at 100 μM and 25 μM final concentration, respectively. Ac-ANW-AMC (15 μM) was used as substrate for i-20S.

Figure 7.. Structures of Mtb20SOG with B6 (PDB: 6ODE).

(A) Electron density maps of B6 in the active site of proteasome core particle. The 2mFo-DFc maps are scaled to 1σ and are shown in grey mesh. The two neighboring β-subunits are shown in green and cyan and the Thr-1 position is labeled in red. (B) B6 is shown in yellow sticks. (C) Phenylimidazole moiety of B6 binds to S1 pocket (top); (D) Phenylpyrrolidinyl moiety of B6 binds to S3 pocket. (E) Hydrogen bond network between B6 and the Mtb20S: P1 imidazole ring forms two hydrogen bonds with the backbone carbonyl group of Ser-20 and the backbone NH of Gly-47, respectively.

Figure 8.

Comparison of the S1 binding pockets of Mtb20S, c-20S and i-20S. Structure of B6 and Mtb20S in (A), and human c-20S (5LF3) in (B) and human i-20S (6AVO) in (C) are superimposed on the Mtb20S. Figures were made using PYMOL (Schrödinger, New York, NY).