Bortezomib Warhead-Switch Confers Dual Activity against Mycobacterial Caseinolytic Protease and Proteasome and Selectivity against Human Proteasome

Abstract

Mycobacteria harbor two main degradative proteolytic machineries, the caseinolytic protease ClpP1P2 and a proteasome. We recently showed that Bortezomib inhibits ClpP1P2 and exhibits whole cell activity against Mycobacterium tuberculosis. Bortezomib, a dipeptide with a boronic acid warhead, is a human proteasome inhibitor approved for cancer therapy. The boronic acid warhead of the compound has been shown to drive potency against both the human proteasome and ClpP1P2 protease. Selectivity for the bacterial ClpP1P2 protease over the human proteasome is lacking but needs to be achieved to move this new anti-tuberculosis lead forward. In this study we explored whether an alternative warhead could influence Bortezomib's selectivity. We synthesized an analog containing a chloromethyl ketone instead of the boronic acid warhead and determined potencies against the bacterial and human enzymes. Surprisingly, the analog retained activity against mycobacterial ClpP1P2 and was active against the mycobacterial proteasome, but was devoid of activity against the human proteasome. Interrogation of a set of chloromethyl ketone peptides identified three additional compounds similarly inhibiting both ClpP1P2 and the proteasome in the bacteria while leaving the human proteasome untouched. Finally, we showed that these compounds display bactericidal activity against M. tuberculosis with cytotoxicity ranging from acceptable to undetectable. These results suggest that selectivity over the human proteasome is achievable. Selectivity, together with dual-targeting of mycobacterial ClpP1P2 and proteasome makes this new scaffold an attractive starting point for optimization.

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

“…Spectra from these 8 two preparations are very similar. We have used proton-detected three-and four-dimensional 9 MAS NMR approaches (26) to obtain the sequence-specific resonance assignment for ( 1 H N , 15 N, 10 13 C⍺, 13 CO) nuclei of 97 residues spread throughout the molecule, including the active site and 11 the central helix ⍺E which undergoes most changes in the compressed-to-extended transition. 12…”

“…4C, insert 1). The geometry and distances between the in this helix has been reported also in EcClpP by methyl-directed solution-state NMR (15). Due 17 to the lack of reliable parametrization of the boronic acid group of bortezomib, we focused on 18 simulating tripeptides bound to the active site.…”

“…TtClpP and ∆TtClpX were 25 purified using native NiNTA affinity chromatography followed by size exclusion chromatography 26 in a 16/600 S200 200pg Superdex column. U-[ 2 H, 15 N],Ile-δ1-[ 13 CH 3 ]TtClpP was expressed as 27 described(46). When expressed in D 2 O TtClpP was insoluble and the protein was recovered 28 from E. coli inclusion bodies using denaturing conditions.…”

“…The angle of the proton excitation pulse was set to 3 30°, and the recycling delay was optimized to 0.6 s to achieve the highest sensitivity (49). 250 4 μM of U-[ 2 H,15 N], Ile-δ1-[ 13 CH 3 ]TtClpP and U-[ 2 H, 15 N],Ile-δ1-[ 13 CH 3 ]S97ATtClpP were mixed with 5 2 mM bortezomib (or DMSO equivalent) in Tris buffer (pH 8.5 mM 100 mM, NaCl 100 mM and Computational assays. The three-dimensional structure utilized in the simulations was obtained 10 from apo-TtClpP, which corresponds to an extended conformation of the protein, bereft of 11 substrate.…”

Pyrazinamide/Pyrazinoic Acid Resistance in Mycobacterium Tuberculosis: Recent Findings and Implications for Improving the Treatment of Tuberculosis

“…Unfortunately, as this compound is used as a proteasome inhibitor approved by the U.S. FDA for the treatment of human multiple myeloma [1] it lacks (myco)bacterial specificity. However, recent work on derivatives of bortezomib, by the group who identified the potential of this compound, demonstrates scope for improving its specificity [43].…”