In vitro and in vivo Metabolism of a Potent Inhibitor of Soluble Epoxide Hydrolase, 1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) is a potent soluble epoxide hydrolase (sEH) inhibitor that is used extensively in research for modulating inflammation and protecting against hypertension, neuropathic pain, and neurodegeneration. Despite its wide use in various animal disease models, the metabolism of TPPU has not been well-studied. A broader understanding of its metabolism is critical for determining contributions of metabolites to the overall safety and effectiveness of TPPU. Herein, we describe the identification of TPPU metabolites using LC-MS/MS strategies. Four metabolites of TPPU (M1–M4) were identified from rat urine by a sensitive and specific LC-MS/MS method with double precursor ion scans. Their structures were further supported by LC-MS/MS comparison with synthesized standards. Metabolites M1 and M2 were formed from hydroxylation on a propionyl group of TPPU; M3 was formed by amide hydrolysis of the 1-propionylpiperdinyl group on TPPU; and M4 was formed by further oxidation of the hydroxylated metabolite M2. Interestingly, the predicted α-keto amide metabolite and 4-(trifluoromethoxy)aniline (metabolite from urea cleavage) were not detected by the LC-MRM-MS method. This indicates that if formed, the two potential metabolites represent <0.01% of TPPU metabolism. Species differences in the formation of these four identified metabolites was assessed using liver S9 fractions from dog, monkey, rat, mouse, and human. M1, M2, and M3 were generated in liver S9 fractions from all species, and higher amounts of M3 were generated in monkey S9 fractions compared to other species. In addition, rat and human S9 metabolism showed the highest species similarity based on the quantities of each metabolite. The presence of all four metabolites were confirmed in vivo in rats over 72-h post single oral dose of TPPU. Urine and feces were major routes for TPPU excretion. M1, M4 and parent drug were detected as major substances, and M2 and M3 were minor substances. In blood, M1 accounted for ~9.6% of the total TPPU-related exposure, while metabolites M2, M3, and M4 accounted for <0.4%. All four metabolites were potent inhibitors of human sEH but were less potent than the parent TPPU. In conclusion, TPPU is metabolized via oxidation and amide hydrolysis without apparent breakdown of the urea. The aniline metabolites were not observed either in vitro or in vivo. Our findings increase the confidence in the ability to translate preclinical PK of TPPU in rats to humans and facilitates the potential clinical development of TPPU and other sEH inhibitors

Discovery of Potent Soluble Epoxide Hydrolase (sEH) Inhibitors by Pharmacophore-Based Virtual Screening

There is an increasing interest in the development of soluble epoxide hydrolase (sEH) inhibitors, which block the degradation of endogenous anti-inflammatory epoxyeicosatrienoic acids. Within this study, a set of pharmacophore models for sEH inhibitors was developed. The Specs database was virtually screened and a cell-free sEH activity assay was used for the biological investigation of virtual hits. In total, out of 48 tested compounds, 19 were sEH inhibitors with IC₅₀ < 10 μM, representing a prospective true positive hit rate of 40%. Six of these compounds displayed IC₅₀ values in the low nanomolar range. The most potent compound <b>21</b>, a urea derivative, inhibited sEH with an IC₅₀ = 4.2 nM. The applied approach also enabled the identification of diverse chemical scaffolds, e.g. the pyrimidinone derivative <b>29</b> (IC₅₀ = 277 nM). The generated pharmacophore model set therefore represents a valuable tool for the selection of compounds for biological testing

SCREENING 20-HETE INHIBITORS IN MICROSOMAL INCUBATES USING UPLC-MS/MS

20-hydroxyeicosatetraenoic acid (20-HETE) is a metabolite of arachidonic acid (AA) formed by cytochrome P450 (CYP) 4A11 and CYP4F2 in humans, with potent microvascular constriction activity. Inhibition of 20-HETE formation is neuroprotective in subarachnoid hemorrhage, cardiac arrest and thromboembolic stroke preclinical models. These findings suggest that inhibition of 20-HETE formation is a potential therapeutic strategy for neuroprotection after brain injury. At this point, a clinically relevant 20-HETE inhibitor is not available to be evaluated as a therapeutic intervention. Our goal is to identify a selective, metabolically stable, and potent 20-HETE inhibitor. Test compounds were obtained either via virtual screening against a CYP4F2 homology model or from scaffold hopping from structures of known inhibitors. Four different types of microsomes including human liver microsome (HLM), recombinant CYP4F2 (rCYP4F2), rat liver microsome (RLM) and rat kidney microsome (RKM) were used. AA was incubated in microsomes with/without compound for 20 min. 20-HETE formation rate was quantified using a validated UPLC-MS/MS assay and normalized by vehicle control group. Other eicosanoids including 15-, 12-HETEs, epoxyeicosatrienoic acids (EETs),and dihydroxyeicosatrienoic acids (DiHETs) were monitored simultaneously. Selected compounds were tested in HLM for metabolic stability over 60 minutes. Remaining amount of compound was quantified using UPLC-MS/MS and normalized to corresponding 0-min values. Among 26 compounds, compounds 10 and 26 both inhibited 20-HETE formation in a dosedependent manner. At 2500nM, compound 10 reduced 20-HETE formation to 19.9±1.8%, 24.0±5.5% in rCYP4F2 and HLM compared with control; compound 26 decreased 20-HETE formation to 32.4±6.5%, 34.8±5.1% in rCYP4F2 and HLM, respectively. After structure modification, compound 19 and its hydrochloride salt compound 18 were the most potent and dose-dependently inhibited 20-HETE formation. At 2500nM, compound 19 decreased 20-HETE formation to 4.4±0.4%, 8.6±1.3% in rCYP4F2 and HLM, respectively without inhibitory effects on 15-, 12-HETE, EETs or DiHETS formation. Compounds 10 and 19 were more stable with 91.4±11.0% and 100.4±1.7% remaining compound at 30min in HLM compared to 35.1±5.7% of 3-(4-n-butoxyphenyl)pyrazole. These results suggested that compounds 10, 19 and 26 are potent 20-HETE formation inhibitors, compounds 10 and 19 have improved microsomal stability, and these three compounds can serve as lead compounds for further structure modifications that may lead to novel 20-HETE formation inhibitors

Impact of sex differences and small molecules on proinflammatory lipid mediator biosynthesis

In the first part, the thesis examines the influence of sex on prostanoid formation. Prostaglandins (PG) are generated by cyclooxygenase (COX)-1/2, which convert arachidonic acid (AA) to PGH2 which is further metabolized to distinct PGs by terminal synthases. Recent findings suggest a sex biased regulation in animals. Hence, the regulation of the prostanoid formation in male and female human whole blood and freshly isolated cells was evaluated. Analysis of human blood cells showed higher lymphocyte and neutrophil numbers in females, whereas monocyte numbers were higher in males and long term incubations of whole blood resulted in significantly enhanced levels of PGs in males. The elevated formation in males was not influenced by pre-incubation with sex hormones, independent of COX-1/2 protein and COX-2 mRNA expression and of AA release in leukocytes. In contrast, the involvement of the 15-prostaglandin dehydrogenase and of transcellular interactions of isolated leukocytes could be shown. In the second part, potential 5-lipoxygenase (5-LO) inhibitors were evaluated. Leukotrienes (LT) are bioactive lipid mediators, which are biosynthesized involving 5-LO catalyzing the incorporation of molecular oxygen into AA. Out of three distinct series of potential 5-LO inhibitors, the two most potent compounds F-XII and F-XVI were analyzed in detail. Both compounds showed potent inhibition of 5-LO with IC50 values in the nanomolar range in cell-based and cell-free preparations (F-XII = 70 - 100 nM; F-XVI = 60 - 120 nM). 5-LO inhibition was direct, reversible and not primarily mediated due to radical scavenging and antioxidant properties. Docking simulations proposed binding to the amino acid Asp-166, which is involved in a salt bridge connecting the two domains of 5-LO. Furthermore, inhibition of 5-LO was independent of interference with cellular signals required for the 5-LO activation and were confirmed in human whole blood and in zymosan-induced peritonitis in mice

Computational Modeling of Protein Structure, Function, and Binding Hotspots

Mixed-solvent molecular dynamics (MixMD) is a cosolvent mapping technique for structure-based drug design. MixMD simulations are performed with a solvent mixture of small molecule probes and water, which directly compete for binding to the protein’s surface. MixMD has previously been shown to identify active and allosteric sites based on the time-averaged occupancy of the probe molecules over the course of the simulation. Sites with the highest maximal occupancy identified known biologically relevant sites for a wide range of targets. This is consistent with previous experimental work identifying hotspots on protein surfaces based on the occupancy of multiple organic-solvent molecules. However, previous MixMD analysis required extensive manual interpretation to identify and rank sites. MixMD Probeview was introduced to automate this analysis, thereby facilitating the application of MixMD. Implemented as a plugin for the freely available, open-source version of PyMOL, MixMD Probeview successfully identified binding sites for several test systems using three different cosolvent simulation procedures. Following identification of binding sites, the occupancy maps from the MixMD simulations can be converted into pharmacophore models for prospective screening of inhibitors. We have developed a pharmacophore generation procedure to convert MixMD occupancy maps into pharmacophore models. Validation of this procedure on ABL kinase showed good performance. Additionally, we have identified characteristic occupancy levels for non-displaceable water molecules so that these sites may be incorporated into structure-based drug design efforts. Lastly, we have explored the potential for accelerated sampling methods to be used in tandem with MixMD to simultaneously capture conformational changes while mapping favorable interactions within binding sites. These developments greatly extend the utility of MixMD while also simplifying its application. In addition, two exploratory studies were completed. First, traditional MD simulations were performed to understand the dynamics of NSD1. Crystal structures of NSD1 capture the post-SET loop in an autoinhibitory position. MD simulations allow conformational sampling of this loop, yielding insight into its dynamic behavior in solution. Second, an epidemiological study was conducted which was aimed at understanding the transmission and sequence variation of CTX-M-type β-lactamases, in fulfillment of the clinical research component of the MICHR Translational Research Education Certificate.PHDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138744/1/sarahgra_1.pd