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Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System*.

ABSTRACT: Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.

SUBMITTER: Janssen GV

PROVIDER: S-EPMC8596589 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The pup-proteasome system of Mycobacterium tuberculosis.

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The <i>Mycobacterium tuberculosis</i> Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway.

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Project description:The Mycobacterium tuberculosis dihydrodipicolinate synthase (Mtb-dapA) is an essential gene. Mtb-DapA catalyzes the aldol condensation between pyruvate and L-aspartate-beta-semialdehyde (ASA) to yield dihydrodipicolinate. In this work we tested the inhibitory effects of structural analogues of pyruvate on recombinant Mtb-DapA (Mtb-rDapA) using a coupled assay with recombinant dihydrodipicolinate reductase (Mtb-rDapB). Alpha-ketopimelic acid (α-KPA) showed maximum inhibition of 88% and IC50 of 21 μM in the presence of pyruvate (500 μM) and ASA (400 μM). Competition experiments with pyruvate and ASA revealed competition of α-KPA with pyruvate. Liquid chromatography-mass spectrometry (LC-MS) data with multiple reaction monitoring (MRM) showed that the relative abundance peak of final product, 2,3,4,5-tetrahydrodipicolinate, was decreased by 50%. Thermal shift assays showed 1 °C Tm shift of Mtb-rDapA upon binding α-KPA. The 2.4 Å crystal structure of Mtb-rDapA-α-KPA complex showed the interaction of critical residues at the active site with α-KPA. Molecular dynamics simulations over 500 ns of pyruvate docked to Mtb-DapA and of α-KPA-bound Mtb-rDapA revealed formation of hydrogen bonds with pyruvate throughout in contrast to α-KPA. Molecular descriptors analysis showed that ligands with polar surface area of 91.7 Å(2) are likely inhibitors. In summary, α-hydroxypimelic acid and other analogues could be explored further as inhibitors of Mtb-DapA.

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Project description:The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.

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Project description: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 (Ki = 5.6 µM) and carboxaldehyde-based derivative 15 (Ki = 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 Ki 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 (Ki = 5.2 ± 1.9 µM, kinact/Ki = 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.

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A conserved loop sequence of the proteasome system depupylase Dop regulates substrate selectivity in Mycobacterium tuberculosis.

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