EPHX2 x mEH

Epoxyeicosatrienoic acids (EETs) have garnered extensive research interest as crucial signaling mediators that regulate key biological processes, including inflammation, pain and angiogenesis. While the metabolism of 8(9)-, 11(12)-, and 14(15)-EET has been well established to be metabolized in the presence of epoxide hydrolases, primarily soluble epoxide hydrolase (sEH) and microsomal epoxide hydrolase (mEH), the fate of 5(6)-EET was previously thought to be limited to its spontaneous chemical conversion into 5,6-dihydroxyeicosatrienoic acid (5,6-DHET) lactone. Here, we reported that the metabolism of 5(6)-EET is also enzymatically regulated by both sEH and mEH, occurring in parallel with its chemical formation to 5,6-DHET lactone. Specifically, in an epoxide hydrolases-free system, 5(6)-EET was rapidly converted to 5,6-DHET lactone, whereas 14(15)-EET remained relatively stable. In contrast, in a cellular system, EPHX2 (encodes sEH) knockout and EPHX1 (encodes mEH) knockout significantly attenuated 5(6)-EET degradation, indicating that both sEH and mEH actively mediate the metabolism of 5(6)-EET. Moreover, we observed that 5(6)-EET underwent conversion to 5,6-DHET lactone within the cellular environment as well. This study provides direct evidence that enzymatic metabolism of 5(6)-EET by epoxide hydrolases occurs concurrently with its spontaneous chemical transformation. This study expands our understanding of 5(6)-EET metabolism and reveals a previously unrecognized role of epoxide hydrolases in modulating relevant biological fate.

Monomeric C-reactive protein (mCRP) is a pro-inflammatory molecule generated by the dissociation of native CRP. Clinical and experimental studies suggest that mCRP deposition in the brain induces Alzheimer's disease (AD) pathology and cognitive loss. Pathological neuroinflammation is increasingly suggested as relevant in AD. Innovative therapies against neuroinflammation are desperately needed, and inhibitors of the enzyme soluble epoxide hydrolase (sEH) are a promising new generation of anti-inflammatory drugs. Mouse primary microglia and BV2 cell line cultures were exposed to mCRP to analyze its pro-inflammatory mechanisms. sEH inhibitors, both newly synthesized UB-SCG-55 and UB-SCG-65, and the reference agent TPPU, were tested for their anti-inflammatory action against mCRP. Phenotypic changes were analyzed through cell imaging techniques, as well as molecular analysis of inflammatory mediators and gene activation pathways. Results show that mCRP triggers a pro-inflammatory response through three main inflammatory pathways: iNOS, NLRP3, and COX-2, followed by increased cytokine generation. Polarization of microglia toward a M1-like phenotype was confirmed by morphological analysis. Also, mCRP can bind to and cross the cell membrane, providing further insight into its mechanisms of action. sEH inhibitors were effective against mCRP induction of a reactive microglial phenotype. The first-line compound UB-SCG-55 emerged as the most potent anti-inflammatory against mCRP injury. Therefore, the direct activation of microglia by mCRP provides evidence of its role in triggering and exacerbating neurodegenerative diseases with a neuroinflammatory component, such as AD. Furthermore, the protection given by inhibitors of sEH confirms its potential as innovative drugs against deleterious effects of neuroinflammation.

Despite the development of soluble epoxide hydrolase (sEH) inhibitors as a promising therapeutic approach, no drug candidate has successfully progressed beyond clinical phase II, highlighting the need for a novel chemotype with improved in vivo potency, pharmacokinetics and safety. In this study, we discovered a phenylacetylpiperidine-based compound, 77 (lab code: DJ-89; IC₅₀: 0.51 nM), through strategic scaffold hopping from previously reported styrene-based sEH inhibitors. Resolving the cocrystal structure and mode-of-action studies revealed a distinct profile compared to well-known sEH inhibitors TPPU and EC5026 (IC₅₀: 44, 19 nM). Notably, 77 demonstrated additional interactions with sEH compared to TPPU, and uniquely enhanced anti-inflammatory factors, including EET levels and IL-10, a capability not observed with EC5026. Moreover, 77 showed excellent pharmacokinetics and safety, positioning it as a promising candidate for treating both acute and chronic inflammatory diseases, including rheumatoid arthritis, leveraging phenylacylpiperidine scaffolds in sEH-targeted therapies.