Identification of Novel Mycobacterial Inhibitors Against Mycobacterial Protein Kinase G

Abstract

Protein kinase G (PknG) is a eukaryotic-like serine/threonine kinase that is expressed by Mycobacterium tuberculosis and promotes survival of mycobacteria in host macrophages by suppressing phagosome-lysosome fusion. Thus, compounds showing inhibitory activity against PknG are promising anti-mycobacterial agents. We therefore aimed to develop anti-mycobacterial agents by identifying new PknG inhibitors. A luciferase-based PknG kinase assay was used to screen potential inhibitors of PknG. We found that four compounds, namely AZD7762, R406, R406-free base, and CYC116, inhibited PknG activities. AZD7762, R406, and R406-free base promoted transfer of mycobacteria to lysosomes. These compounds also inhibited survival of M. bovis Bacillus Calmette–Guérin (BCG) inside human macrophages. Furthermore, R406 and R406-free base showed bactericidal activity against BCG in infected human macrophages without cytotoxicity. The PknG inhibitors identified in this study by the luciferase-based PknG kinase assay may be promising leads for the development of anti-mycobacterial agents.

NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G

“…These observations are in concordance with the current notion that apoptosis of the infected phagocytes prevents mycobacteria from establishing a suitable niche for long-term survival in the host (45). In agreement, other studies have also identified PknG-specific inhibitors, such as AX20017, aminopyrimidine derivatives, AZD7762, R406, R406f, CYC116, and Sclerotiorin, as promising antimycobacterial agents (31,(48)(49)(50)(51)56). Taken together, we show for the first time that small-molecule targeting of PknD and PknG has no side effects and inhibits bacterial growth in lungs and spleens of infected mice.…”

“…Previously, it was reported that PknG is secretory in nature and promotes survival of M. tuberculosis in macrophages by inhibiting phagosomelysosome fusion (42)(43)(44). Previous studies have shown that compounds modulating host apoptosis and targeting PknG are promising antimycobacterial agents (31,(44)(45)(46)(47)(48)(49)(50)(51). Since NU-6027 possessed both of these activities, we sought to assess its antimycobacterial activity in THP-1 macrophages infected with either M. bovis BCG or M. tuberculosis.…”

High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis

“…Some host-targeting compounds have been demonstrated to possess activity against bacterial kinases, such as the cyclin-dependent kinase 1 inhibitor NU-6027 ( Kidwai et al, 2019 ) and the CHK1 inhibitor AZD7762 ( Kanehiro et al, 2018 ), which inhibit the M.tb kinase, PknG. To examine if DDUG can inhibit bacterial growth in broth, disk diffusion and resazurin assays were conducted, examining a range of pathogenic bacteria and M.tb ( Table 3 ).…”

“…The activity of DDUG against M.tb in broth at concentrations similar to those achieved intracellularly suggests an alternative, CHK2-independent mode of activity. Similarly, the activity of DDUG against some bacteria further suggest DDUG might have promiscuous activity on bacterial kinases, similar to NU-6027 (Kidwai et al, 2019) and AZD7762 (Kanehiro et al, 2018), or otherwise be toxic due to physical properties such as crystallization if concentrated by the macrophage. Conversely, DDUG is not active against M.tb's close family members, M. abscessus and M. bovis-BCG, or Salmonella, which it did inhibit in macrophages, and is active against a small array of seemingly unrelated bacteria ( Table 3).…”

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…Another line of currently investigated mycobacterial inhibitors are targeted toward the cytosolic PknG kinase [86,241,242]. In contrast to PknA and PknB, PknG is not essential but is instead important for host intracellular survival by blocking macrophage phago-lysosomal fusion [86].…”