Psoralen Derivatives as Inhibitors of Mycobacterium tuberculosis Proteasome

Abstract

Protein degradation is a fundamental process in all living organisms. An important part of this system is a multisubunit, barrel-shaped protease complex called the proteasome. This enzyme is directly responsible for the proteolysis of ubiquitin- or pup-tagged proteins to smaller peptides. In this study, we present a series of 92 psoralen derivatives, of which 15 displayed inhibitory potency against the Mycobacterium tuberculosis proteasome in low micromolar concentrations. The best inhibitors, i.e., 8, 11, 13 and 15, exhibited a mixed type of inhibition and overall good inhibitory potency in biochemical assays. N-(cyanomethyl)acetamide 8 (K_i = 5.6 µM) and carboxaldehyde-based derivative 15 (K_i = 14.9 µM) were shown to be reversible inhibitors of the enzyme. On the other hand, pyrrolidine-2,5-dione esters 11 and 13 irreversibly inhibited the enzyme with K_i values of 4.2 µM and 1.1 µM, respectively. In addition, we showed that an established immunoproteasome inhibitor, PR-957, is a noncompetitive irreversible inhibitor of the mycobacterial proteasome (K_i = 5.2 ± 1.9 µM, k_inact/K_i = 96 ± 41 M^-1·s^-1). These compounds represent interesting hit compounds for further optimization in the development of new drugs for the treatment of tuberculosis.

Keywords: Mycobacterium tuberculosis; nonpeptidic proteasome inhibitors; proteasome; protein degradation; psoralens.

Figure 1

The ubiquitin – proteasome pathway in protein degradation [7,8,9]. The ubiquitination cascade is triggered by the ATP-mediated conjugation of a target protein with a single ubiquitin molecule by the Ub-activating enzyme E1. This tagged protein is then transferred to the Ub-carrier proteins E2 that subsequently forms a complex with Ub-ligases E3. The ligases recognize the protein and perform a sequence of ubiquitin additions until the final polyubiquitinated protein is formed. Finally, in human a rapid degradation of ubiquitinated protein by the proteasome is executed at six proteolytic sites of the enzyme. The degradation process results in 3 to 25 amino acids-long peptides, which are quickly digested into amino acids or can serve as antigen presentation molecules.

Figure 2

The pupylation pathway of protein degradation in Mtb.

Figure 3

Superimposition of the β5i subunit of human IP (blue, PDB code 5M2B) and Mtb proteasome β subunit (grey, PDB code 6ODE) showing high similarity between the two enzymes and their active sites. The respective ligands Ro19 (purple) and B6 (green) are also presented. Catalytic threonine residues (Thr1) are red.

Figure 4

The summary of the SAR study of psoralen derivatives targeting the Mtb proteasome.

Figure 5

Dilution assay: Enzyme recovery after preincubation with the inhibitor for 0, 25, 30 or 60 min was observed and compared to DMSO control. Over longer preincubation time bortezomib caused significant loss of enzyme activity (up to approximately 45%) compared to the DMSO control at concentrations equal or higher than the inhibitor’s IC₅₀ value. The recovery of proteasome seems to be slowly increasing with longer preincubation time. On the other hand, PR-957 completely inhibited the enzyme at higher concentrations regardless of the preincubation time. However, a time-dependent inhibition was observed at lower concentrations (top right graph).