Archives

  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • Currently DMPO or any of its structural analogs have

    2019-07-08

    Currently, DMPO or any of its structural analogs, have not been moved into the drug development pipeline. That may be due to DMPO synthesis being really expensive in comparison to PBN and its derivatives. PBN-analogs have been synthesized and their anti-inflammatory properties have been tested in different experimental models [4,8]. Moreover, some of them have reached phase III clinical trials, but the mechanisms behind these effects remain obscure [4]. By using DMPO as a tool we have studied the intracellular localization and identity of radicalized protein in cells, tissues and whole animals [9,10]. Interestingly, DMPO dampened LPS-driven RAW264.7-macrophage-like cell line activation, thus prevented nitric oxide and inflammatory chemokine production. These effects afforded by DMPO were linked to inhibition of nuclear factor kappa-light-chain-enhancer of activated NSC232003 (NF-ĸB) signaling pathway at early time points after the stimulus (i.e., 15 min) [5]. We have also studied the effects of DMPO on the transcriptome of macrophages and found that the spin trap was able to change the expression of 215 genes when added simultaneously with LPS on RAW 264.7 cells for 6 h. Interestingly, 75% of those genes were downregulated when compared to LPS stimulated cells. Functional analysis of these genes using Ingenuity Pathway Analysis (IPA) software was consistent with a negative regulation of the innate immune system with several toll-like receptors (TLR-4, -3 and -9) indicated as receptors being affected by DMPO [11] (Fig. 1). Currently, the exact mechanism by which spin traps exert their anti-inflammatory effects remains unknown. However, the production of mechanism-based drugs is an emerging field that will lead to safer anti-inflammatory drugs. This can be moved forward by knowing the exact changes in transcriptome, proteome, and phenome induced by DMPO in inflammatory cells, such as macrophages. In this regard, transcriptomic and functional changes induced by DMPO in macrophages, as we have recently reported [11], may be an important step towards accomplishing this goal. Toll-like receptors sense conserved pathogen- and danger-associated molecular patterns released by microbes and the host [12]. Among the most studied TLRs, TLR4 is well known because of its participation in inflammatory response triggered by LPS. Closely related, TLR2 binds peptidoglycan and it is necessary for the correct signaling triggered by LPS throughout TLR4 [13,14]. Interestingly, all TLRs have in common a cytoplasmic domain called toll-interleukin-1 receptor (TIR) that mediates interactions with adaptor molecules such as myeloid differentiation primary response 88 (MyD88) and Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) [15]. A region called BB-loop within the TIR domain has been pointed as a critical region for a successful TLR downstream signal transduction. Indeed, a site-directed mutation of one residue at the BB-loop (called after the β-sheet and α-helix motifs that compose the tridimensional structure) region (P712 in murine TLR4 and its analog on human TLR2-P681) results in signal impairment [16,17]. This evidence points the BB-loop within the TLR's TIR domain as a rational target for attenuation of TLRs signaling. To the date, TLR4-TIR domain has not yet been crystallized making impossible to perform in silico studies on this receptor's domain. On the other hand, TLR2-TIR domain has already been crystallized and has many structural and functional similarities with TLR4 [13,14] making it a suitable experimental model for docking and molecular dynamics studies on TLR-TIR signaling. These pieces of evidence led us to hypothesize that the anti-inflammatory effects of DMPO can be explained by direct binding of the spin trap to the TIR domain of TLRs. To test this hypothesis we combined in silico techniques of docking, molecular dynamics simulations and QTAIM (Quantum Theory of Atoms In Molecules) calculations to determine the site and strength of the interaction between DMPO and TLR2 TIR domain, as well as biochemical techniques to determine the functional significance of our findings.