JFD, a Novel Natural Inhibitor of Keap1 Alkylation, Suppresses Intracellular Mycobacterium Tuberculosis Growth through Keap1/Nrf2/SOD2-Mediated ROS Accumulation

Abstract

It is an effective strategy to treat tuberculosis by enhancing reactive oxygen species- (ROS-) mediated killing of Mycobacterium tuberculosis in macrophages, but there are no current therapeutic agents targeting this pathway. Honeysuckle has been used as the traditional medicine for tuberculosis treatment for 1500 years. Japoflavone D (JFD) is a novel biflavonoid isolated from Honeysuckle promoting ROS accumulation by Nrf2 pathway in hepatocarcinoma cells. However, its activity to kill M. tuberculosis in macrophages and molecular mechanism has not been reported. Our results showed that JFD enhances the M. tuberculosis elimination by boosting ROS levels in THP-1 cells. Moreover, the massive ROS accumulation activates p38 to induce apoptosis. Notably, the mechanism revealed that JFD suppresses the nuclear transport of Nrf2, thereby inhibiting SOD2 transcription, leading to a large ROS accumulation. Further studies showed that JFD disrupts the Keap1 alkylation at specific residues Cys14, Cys257, and Cys319, which is crucial for Nrf2 activation, thereby interrupts the nuclear transport of Nrf2. In pharmacokinetic study, JFD can stay as the prototype for 24 h in mice and can be excreted in feces without any toxicity. Our data reveal for the first time that a novel biflavonoid JFD as a potent inhibitor of Keap1 alkylation can suppress the nuclear transport of Nrf2. And it is the first research of the inhibitor of Keap1 alkylation. Furthermore, JFD robustly promotes M. tuberculosis elimination from macrophages by inhibiting Keap1/Nrf2/SOD2 pathway, resulting in the ROS accumulation. This work identified Keap1 alkylation as a new drug target for tuberculosis and provides a preliminary basis for the development of antituberculosis lead compounds based on JFD.

Druggable redox pathways against Mycobacterium abscessus in cystic fibrosis patient-derived airway organoids

“…Only few studies investigated the role of NRF2 in mycobacteria infection [ 78 ]. In Mtb-infected macrophages, it has been shown that activation of NRF2 is associated with enhanced bacterial load and cell survival [ 79 ], while inhibition of NRF2 or NQO1 ensured a better control of Mtb or BCG infection and host cell survival [ 80 , 81 ], suggesting that NRF2-NQO1 axis, by detoxifying ROS, might play a detrimental role during mycobacteria infection. By contrast, Zhou et al showed that the activation of NRF2-NQO1 axis induced better control of Mtb [ 82 ].…”

Post-translational modifications of Keap1: the state of the art

“…These investigations establish a solid theoretical foundation for the therapeutic and interventional approaches targeting Keap1 PTMs in oxidative stress-related ailments. Notably, numerous naturally occurring compounds derived from plants, such as genistein ( Tocmo and Parkin, 2019 ), Japoflavone D ( Wan et al, 2023 ), and luteolin ( Nakamura et al, 2018 ), have been experimentally validated to exhibit favorable effects on diseases through the activation of the Nrf2 pathway in both cellular and animal models. However, it should be noted that these substances do not directly alter or engage with Nrf2.…”

“…Instead, they indirectly impact Nrf2 by regulating the PTMs of Keap1. Consequently, they hold potential for the treatment of oxidative stress-related ailments like diabetic heart (I/R) injury ( Nakamura et al, 2018 ), tuberculosis ( Wan et al, 2023 ), and cerebral ischemia cognitive decline ( Tocmo and Parkin, 2019 ) ( Figure 2 ). This suggests that Keap1 could be a promising therapeutic target.…”

“…Other electrophiles, such as CDDO (a representative Michael receptor) and DMF (dimethylfumarate), have been reported to modify Keap1 with alkylation ( Dunlap et al, 2012 ; Ahuja et al, 2016 ). However, the alkylation of Cys14, Cys257, and Cys319 residues on Keap1 induced by CDDO-EA can be inhibited by JFD (a novel biflavonoid isolated from Honeysuckle), leading to ROS accumulation and thereby enhancing mycobacterium tuberculosis elimination ( Wan et al, 2023 ).…”

Selection and validation of genes related to oxidative stress production and clearance in macrophages infected with Mycobacterium tuberculosis

“…The genes GPX3, SOD2 , and CAT of the above three enzymes were verified by RT-qPCR, and it was found that the expression of GPX3 and SOD2 increased after infection, whereas the expression of CAT decreased. Many studies ( Ren et al., 2021 ; Wan et al., 2023 ) have shown that SOD2 is upregulated and regulates ROS in macrophages infected with MTB, but there are few reports of GPX3 and CAT . Among them, the high expression of GPX3 and SOD2 may be an adaptive change of macrophages in response to OS caused by MTB, to reduce ROS levels, maintain the normal function of macrophages, and prevent death.…”