Towards Selective Mycobacterial ClpP1P2 Inhibitors with Reduced Activity against the Human Proteasome

Abstract

Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti-M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti-M. tuberculosis activity.

Keywords: antimycobacterial; caseinolytic protease; dipeptidyl boronic acid.

FIG 1

Structure of bortezomib (compound 1).

FIG 2

Genetic engineering of M. smegmatis prcAB null mutant strain by recombineering. (A) Schematic representation of the recombineering strategy used to delete prcAB genes in M. smegmatis. (B) PCR verification of the M. smegmatis prcAB null mutant. The primers used to generate the AES and to verify the null mutant are described in Table S1 in the supplemental material. (C) Compound 1 growth inhibition of the M. smegmatis wild-type and prcAB null mutant strains. WT, wild type.

FIG 3

ClpP1P2 and proteasome inhibition assays. (A) ClpP1P2 inhibition assay principle. Without any interference, ClpP1P2 recognizes and degrades SsrA-tagged (YALAA) RFP protein, resulting in a low fluorescence level. In the presence of a ClpP1P2 inhibitor (compound 1, bortezomib), RFP is not degraded. Its accumulation results in an increase in fluorescence. (B) Proteasome inhibition assay principle. Without any interference, the proteasome recognizes the Z-LLVY tag and cleaves it. The aminoluciferin is used as a substrate by the luciferase enzyme to generate luminescence. In the presence of a proteasome inhibitor (compound 1), the cleavage of Z-LRR is prevented. The lack of the luciferase substrate results in a reduced luminescence emission. RFU, relative fluorescence units; RLU, relative luminescence units.

FIG 4

Docking of compound 1 into M. tuberculosis ClpP1P2 (top) (PDB accession number 4U0G) and the human proteasome (bottom) (PDB accession number 4R67). Compound 1 is shown as a thick tube with a plum carbon. (Left) Molecular surface of M. tuberculosis ClpP1P2 and the human proteasome in gray, blue, and red, indicating neutral, positive, and negative electrostatics, respectively, with substrate sites 1 to 3 (S1 to S3) labeled in yellow. There is more available space in the S1 and S3 sites of ClpP1P2 than in the human proteasome. (Right) Selected residues of M. tuberculosis ClpP1P2 and the human proteasome are shown as a thin tube, with gray carbon and hydrogen bonds as dashed magenta lines. Residues are from the same protein subunit unless marked by a suffix indicating the PDB chain. The hydrogen bond network between compound 1 and the human proteasome is retained when compound 1 binds to M. tuberculosis ClpP1P2, and consequently, we did not try to modify the backbone of compound 1. The orientation of the P2 side chain differs, but there is little interaction between this and either protein, and modeling indicates that it is free to move around in the binding site.

FIG 5

Synthesis of pyrazine acid 6. Reagents and conditions: (a) BSA, DCM, rt, 16 h; (b) CDI, DCM, rt, 16 h; (c) −40°C to rt, 16 h.

FIG 6

General synthesis of carboxylic acid derivatives 10a to -o. Reagents and conditions: (a) 8a-d, TBTU, DIPEA, CH₂Cl₂, 0°C to rt; (b) LiOH, THF:MeOH:H₂O, 0°C to rt; ¹lithium salt of 5-phenyloxazole-2-carboxylic acid; ²piperidine-1-carbonyl chloride.

FIG 7

General synthesis of amino boronate salts 16a to -g. Reagents and conditions: (a) CuSO₄·5H₂O, 4 Å mol. sieves, CH₂Cl₂, rt; (b) CuSO₄, BnNH₂, PCy₃·HBF₄, 5:1 toluene/H₂O, rt; (c) (ICy)CuOt-Bu, dry toluene, rt; (d) 4.0 M HCl in dioxane, dry MeOH, dry dioxane, rt.

FIG 8

General synthesis of boronic acids 25 to 58. Reagents and conditions: (a) 6/10a-o,TBTU, iPr₂EtN, CH₂Cl₂, 0°C to rt; (b) 20, TBTU, iPr2EtN, dry CH₂Cl₂, 0°C to rt; (c) 4.0 M HCl in dioxane, CH₂Cl₂, rt; (d) 6/7b/23/24, TBTU, iPr₂EtN, dry CH₂Cl₂, 0°C to rt; (e) 17, TBTU, iPr₂EtN, CH₂Cl₂, 0°C to rt; (f) iBuB(OH)₂, 1 N HCl, CH₃OH, pentane, rt.

FIG 9

Docking of compound 58 into M. tuberculosis ClpP1 (PDB accession number 4U0G). Compound 58 is shown as a thick tube with a plum carbon. (Left) Electrostatic surface of M. tuberculosis ClpP1P2, with neutral charges in gray, positive partial charges in blue, and negative partial charges in red. (Right) M. tuberculosis ClpP1P2 is shown with selected residues shown as a thin tube with a gray carbon. Residues are from the same protein subunit unless marked by a suffix indicating the PDB chain. The P1 side chain is surrounded by hydrophobic S1 residues I71, M75, M99, F102, P125, L126, and M150.

FIG 10

Intravenous/oral pharmacokinetic profile for compound 58 in mice (3 mice per time point). (A) i.v./p.o. concentrations of compound 58 plotted against time for up to 24 h, and pharmacokinetic parameters at a dose of 10 mg/kg; (B) tissue distribution plot of concentrations against time and pharmacokinetic parameters for orally administered compound 58 in plasma, lung, and brain at the higher dose of 100 mg/kg; (C) dose linearity plot of C_max versus dose; (D) dose linearity plot of AUC_0–inf versus dose. CL, clearance; F, variance ratio.