Reagents.

CP-456,773, NT-0796, and NDT-19795 were synthesized by NodThera; synthetic details relating to NT-0796 and NDT-19795 are provided elsewhere (Harrison et al., 2023). Z-carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-FMK), nigericin, LPS, ATP, 4-nitrophenyl butyrate (4-NPB), 4-nitrophenyl (4-NP), bis-p-nitrophenylphosphate, papain, DNase 1, and Percoll all were from Sigma Aldrich (St. Louis, MO). TAMRA-fluorophosphonate (TAMRA-FP), fluorophosphonate-biotin (FP-biotin), streptavidin-Agarose, Pierce BCA total protein assay, Alexa Fluor-680-tagged streptavidin, Roswell Park Memorial Institute (RPMI) medium containing Glutamax supplements, 1 M HEPES, penicillin/streptomycin (10,000 units/ml), and heat-inactivated fetal bovine serum (FBS) all were obtained from Thermo Fisher Scientific (Waltham, MA). Resin-bound anti-mouse albumin and IgG and mouse monoclonal anti-CES-1 IgG2B were obtained from R&D Systems (Minneapolis, MN). CES-1b and control Supersomes were obtained from Corning (Tewksbury, MA). For Western blotting, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Cell Signaling Technologies (Danvers, MA), rabbit monoclonal anti-human CES-1 from Abcam (Cambridge, UK), and DyLight 880-labeled goat anti-rabbit secondary antibody from Rockland Immunochemicals (Limerick, PA). Birb-796 was obtained from Tocris Bioscience (Minneapolis, MN). For immunohistochemical localization of human CES-1 a rabbit polyclonal antibody from Sigma Aldrich (HPA046717) was employed, and for localization of endogenous mouse ces-1 a rabbit polyclonal antibody from Thermo Fisher (PA5-79032) was employed. ELISA kits employed to assess murine IL-1β (#88-7013-88), human IL-1β (#88-7261-88), human IL-6 (#88-7066-88), and human tumor necrosis factor-α (TNFα, #88-7346-88) were from Thermo Fisher Scientific. Murine IL-6 levels were assessed with a kit from R&D Systems (#SMLB00C). ELISA assays were conducted following manufacturer’s instructions. Primer/probe sets for quantitative polymerase chain reaction (qPCR) were obtained from Invitrogen (human CES-1, Hs00275607; mouse Ces1c, Mm00468347; CD68, Mm03047340; Aif1 (Iba), Mm00479862; Tmem119, Mm00525305).

Generation of a Syntenic Mouse Line in which the Endogenous Ces-1c Locus is Replaced with Human CES-1.

Mice differ markedly from humans with respect to CES biology in that they: 1) do not express functional CES in monocytes (Needham et al., 2011) and 2) possess high plasma CES activity which stems from liver expression and subsequent release of Ces1c (Duysen et al., 2011). To mitigate both differences, a displacer vector (Supplemental Methods section) was introduced by electroporation into murine embryonic stem cells (Phnx43); this vector was designed to replace the endogenous Ces1c gene with the human CES-1 gene. To ensure wide-spread expression of human CES-1 in cells of monocyte/macrophage lineage the endogenous human CES-1 promoter was replaced with the mouse colony stimulating factor 1 receptor promoter as this promoter was successfully employed previously to drive transgene expression throughout the mouse mononuclear phagocyte system (Sasmono et al., 2003; Hawley et al., 2018). Cloned ES cells expressing a single copy of the human CES-1 locus were injected into blastocysts and the modified embryos were implanted into pseudo pregnant foster mice. Resulting offspring were crossed for six generations to C57BL/6 mice, after which heterozygous animals were intercrossed to generate cohorts of mice (referred to as hCES-1) homozygous for the humanized locus. Expression of the human CES-1 did not alter fertility, nor could mice homozygous for the locus be distinguished from C57BL/6 mice based on development size or health.

C57BL/6 mice lacking functional Ces1c (B6.Cg-Ces1ctm1.1Loc/J; stock #014096) were obtained from The Jackson Laboratory. Two male and six female Ces1c−/− mice were initially obtained and mated upon arrival at UNC. These mice were used to establish a breeding colony and to produce the animals for the experiments described. This ensured that the Ces1c−/− mice were born and reared in the same vivarium as the C57BL/6 wild-type and the hCES-1 mice. All mice were exposed to a similar environment with identical food, water, noise levels, light cycles, and vibrations, thereby limiting variation between groups due to different rearing environments.

Cytokine Output from Human Peripheral Blood Mononuclear Cells (PBMCs).

Heparin-stabilized blood from healthy volunteers was obtained from Bloodworks NW (Seattle); blood from both male and female donors was employed with no noticeable differences in assay performance. During the course of these studies the donor pool employed consisted of 15 individuals, of which 20% were males and 80% females. PBMCs were prepared using Ficoll-Paque Plus centrifugation. Total cell numbers were determined using a hemacytometer and adjusted to 2.6 x 106 cells/ml in RPMI Glutamax medium containing 1% FBS, 1% penicillin/streptomycin, and 20 mM HEPES, pH 7.3. To assess IL-1β output, 0.1 ml (2.6 × 105 cells) of this cell suspension was added to each well of 96-well plates. After a 2-hour incubation at 37°C in a 5% CO2 incubator to allow adherence of monocytes, media and non-adherent cells were aspirated. 0.1 ml of RPMI Glutamax medium containing 5% FBS, 1% penicillin/streptomycin, 20 mM HEPES, pH 7.3 (Base Medium) was added to all wells, and the plates were incubated overnight at 37°C in a 5% CO2 incubator. To wells designated to receive LPS, 0.04 ml of Base Medium containing 350 ng/ml LPS was introduced; wells not receiving LPS received 0.04 ml of Base Medium. Plates were incubated at 37°C for 2 hours to allow transcription/translation of proIL-1β. At this point, media were removed by aspiration and 0.143 ml of fresh RPMI GlutaMAX medium containing 1% FBS and 1% penicillin/streptomycin were added; the presence of LPS was maintained in wells previously exposed to this stimulus. Test compound at various concentrations (or 0.2% DMSO) was added to designated wells and plates were placed at 37°C in a 5% CO2 incubator for 60 minutes. To designated wells, 0.0075 ml of a 100 mM ATP solution (final concentration = 5 mM) was then introduced to promote NLRP3 activation, and the plates returned to a 37°C/5% CO2 incubator. After a 60-minute stimulation, the plates were subjected to centrifugation after which the media supernatants were harvested for assessment of IL-1β levels by ELISA.

To assess impact on IL-6 and TNFα output, 0.14 ml of a freshly isolated PBMC suspension (2.8 × 105 cells) were added to each well of 96-well plates. To wells designated to receive test compound, 0.05 ml of Base Medium containing 4X the desired final test agent concentration were added; control wells received 0.05 ml of Base Medium without compound. Plates were placed at 37°C in a 5% CO2 incubator for 30 minutes. To wells designated to receive LPS, 0.01 ml of Base Medium containing 2 µg/ml LPS were added to achieve a final LPS concentration of 100 ng/ml and the plates were returned to a 37°C/5% CO2 incubator. After 4 hours of LPS stimulation the plates were subjected to centrifugation and media supernatants were recovered and assessed for cytokine output using TNFα- and IL-6-specific ELISA kits.

NLRP3-Mediated ASC Fluorescent Speck Formation.

A suspension of THP1-ASC-GFP cells (Invivogen) was incubated in culture medium containing 10% FBS with or without 1 µg/ml LPS for 2 hours at 37°C. Cells were collected by centrifugation, resuspended in assay medium containing 1% FBS, and dispensed into a 96-well plate (0.1 ml containing 2 × 105 cells/well). Diluted compounds or equivalent dilutions of vehicle (DMSO) were then added to the cells, and the cells were incubated for 30 minutes at 37°C; the final DMSO concentration in all wells was 0.4%. At this point the caspase inhibitor zVAD-FMK was added to all wells to yield a final concentration of 20 μM, and the cells were incubated for 5 minutes at 37°C; z-VAD-FMK was included to prevent pyroptosis and preserve speck detection. Next, 1 µl of a 500 µM nigericin solution (in 10% EtOH) was added to yield a final concentration of 5 μM. Control wells not treated with nigericin received an equivalent dilution of ethanol. After a 1-hour incubation at 37°C, cells were fixed with a final concentration of 1% paraformaldehyde and then incubated with Hoechst 33342 trihydrochloride to fluorescently label DNA. Images of the cells were acquired using an ImageXpress Micro Confocal High Content Imaging System (Molecular Devices) and analyzed using CellProfiler software.

LPS/ATP-Induced IL-1β Output in Blood.

Heparin-stabilized blood from healthy volunteers was subjected to a 2-step activation protocol as previously described (Perregaux et al., 2000; Primiano et al., 2016). Blood samples were diluted with RPMI GlutaMax medium containing 20 mM HEPES, pH 7.3 (two parts blood to one part medium) after which 0.14 ml of the diluted samples were placed in individual wells of a 96-well plate designated not to receive LPS. To the remaining diluted blood sample, an appropriate volume of 1 μg/ml LPS was added to achieve a final concentration of 100 ng/ml. 0.14 ml of the LPS-containing diluted blood sample were then placed into wells of the 96-well plate designated to receive LPS. The plates were incubated for 3 hours at 37°C in a 5% CO2 incubator to allow activation of proIL-1β transcription/translation. After the LPS activation phase, 0.05 ml of RPMI GutaMax media containing 1% FBS, 1% penicillin/streptomycin, 20 mM HEPES, pH 7.3, and test agent (at a 4-fold higher concentration than final) were added to designated wells. Wells designated not to receive test agent received 0.05 ml of RPMI GlutaMAX media containing 1% FBS, 1% penicillin/streptomycin, 20 mM HEPES, pH 7.3, and 1.6% DMSO. The plates were returned to the 37°C/5% CO2 incubator for an additional 30 minutes. At this point, 0.01 ml of 100 mM ATP and 20 mM HEPES, pH 7.3, were added to designated wells to promote NLRP3 activation. The plates were incubated for an additional 60 minutes at 37°C/5% CO2 after which they were subjected to centrifugation, and plasma supernatants were harvested for assessment of IL-1β levels by ELISA. A similar protocol was employed to assess IL-1β output from heparin-stabilized mouse blood with the following changes: blood samples from multiple mice were pooled, the LPS concentration was increased to 1 µg/ml, and cytokine levels in plasma supernatants were assessed using an ELISA specific for murine IL-1β.

IL-1β Output from Mouse Peritoneal Macrophages.

Cells within the peritoneal cavity were harvested by lavage following euthanasia. Cells were collected by centrifugation and resuspended in RPMI-1640 medium containing 5% heat-inactivated FBS, 2 mM GlutaMAX, 1% penicillin/streptomycin, and 25 mM HEPES, pH 7.3 (Culture Medium). 5 × 104 cells (in 0.1 ml of Culture Medium) were seeded per well of 96-well tissue culture plates and the plates were incubated overnight at 37°C in a 5% CO2 incubator. The following day, 1 µg/ml LPS was introduced to designated wells and the cultures incubated for an additional 2 hours. Culture supernatants then were aspirated and fresh RPMI-1640 medium containing 1% heat-inactivated FBS, 2 mM GlutaMAX, 1% penicillin/streptomycin, and 25 mM HEPES, pH 7.3 (Compound Medium) was added; where indicated, a test agent was present in the Compound Medium. The plates were returned to the 37°C incubator for 60 minutes, after which ATP was introduced to achieve a final concentration of 5 mM. The plates were incubated for an additional 60 minutes at 37°C prior to being subjected to centrifugation (5 minutes at 700 × g). Supernatants were collected and assessed for IL-1β content by ELISA.

Expression Analysis by qPCR.

Total RNA was isolated from indicated cells or tissues using RNA STAT-60 (AMSBIO, Cambridge, MA) according to the instruction manual. Reverse transcription of total RNA to cDNA for quantitative real-time PCR was performed by using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. All reactions were performed with TaqMan PCR Universal Master Mix (Applied Biosystems) using the Applied Biosystems QuanStudio 6 Flex Real-Time PCR System. Reactions were carried out in duplicate in a 20 µl final volume reaction. PCR conditions for all reactions were as follows: 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Expression levels of genes of interest were normalized to 18S. Data were analyzed using the comparative CT method as described by Applied Biosystems.

Plasma carboxylesterase Detection.

EDTA-stabilized plasma was prepared from wild type (WT) and hCES-1 mice. Total plasma-associated esterase activity was assessed using a non-selective substrate 4-NPB (Williams et al., 2011). Reaction mixtures contained 200 mM 4-NPB in 50 mM HEPES, pH 7. Plasma samples were added (resulting in a 600-fold dilution) and the mixtures were incubated at 37°C. Production of 4-NP was monitored spectrophotometrically over time by assessing OD405 and converted to nmoles by comparison with a 4-NP standard curve.

WT and hCES-1 plasma samples were also treated with a serine hydrolase activity-based probe (TAMRA-FP) to visualize targeted polypeptides (Simon and Cravatt, 2010). Prior to labeling, plasma samples were depleted of serum albumin and IgG by incubation with resin-bound antibodies following the supplier’s protocol. The resulting depleted fractions were concentrated using 3 kD mol. wt. cut-off centrifugation filtration units. Protein concentrations were determined using the Pierce BCA total protein reagent. The depleted plasma samples were adjusted to 1 mg/ml of total protein and treated with 4 µM TAMRA-FP for 45 minutes at room temperature. The mixtures then were quenched by addition of an equal volume of 2X SDS sample buffer containing 50 mM dithiothreitol and heated at 70°C for 10 minutes. Forty microliters of each sample was subjected to SDS gel electrophoresis and resulting gels were washed and imaged using GE Typhoon Trio instrumentation with 532 nm and 720 nm lasers.

Western Blotting for hCES-1.

Peritoneal macrophages were isolated from WT B6 female and homozygous hCES-1 female mice. An equal number of cells (27.5 × 106) were pelleted and suspended into 0.5 ml of an extraction buffer containing 1% Triton X-100 and protease inhibitors. The cellular extracts were clarified by centrifugation and resulting supernatants were recovered and disaggregated with SDS sample buffer and subjected to SDS gel electrophoresis. Separated polypeptides were blotted onto nitrocellulose membranes and probed for human CES-1 and the housekeeper protein GAPDH.

Stability of NT-0796 in Plasma as Assessed by Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS) Analysis.

NT-0796 was added to EDTA-stabilized plasma samples from WT and hCES-1 mice which were diluted 2-fold with 50 mM HEPES, pH 7.4, 1% FBS. As a positive control, NT-0796 also was incubated with Supersomes expressing CES-1b (the major isoform of CES-1 expressed by human liver). Reaction mixtures were incubated at 37°C for 60 minutes, after which 1 mM bis-p-nitrophenyl-phosphate was introduced to stop further hydrolysis. Standard curves were generated for both NT-0796 and NDT-19795 by preparing a common serial dilution series titration from 10 to 0.1 μM in 50 mM HEPES, pH 7.4, 1% FBS. Samples were stored at −80°C until transferred to the University of Washington School of Pharmacy for LC-MS/MS analysis. Briefly, 10 µl of each sample was combined with 20 µl of an internal standard, glyburide (50 ng/ml in water), and vortexed. 0.1 ml of acetonitrile was then added to each sample, after which the samples were vortexed and clarified by centrifugation. Fifty microliters of each supernatant was collected and combined with 50 µl of water in a limited volume HPLC vial. Five microliters of these samples were injected into a Waters Xevo-TQ-XS. Recoveries of NT-0796 and NDT-19795 in the reaction mixtures were calculated based on comparison of peak areas of each analyte to standard curves generated with each compound.

CNS Expression of Human CES-1.

Right hemispheres of brains isolated from WT and hCES-1 mice were homogenized in a lysis buffer composed of 10 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, and 1% NP-40 and the lysates clarified by centrifugation. Recovered supernatant fractions (1 mg of total protein) were incubated with 0.06 ml of streptavidin-agarose, after which the beads were removed by centrifugation. To the precleared supernatants, FP-biotin was added (2 μM) and the mixtures were incubated at 25°C for 45 minutes. Reaction mixtures were quenched by addition of an equal volume of 10 mM urea in lysis buffer. The mixtures were then re-treated with streptavidin-agarose to capture biotinylated polypeptides. Beads were collected by centrifugation and washed 3X by repeated centrifugation with 5 M urea in lysis buffer. Final bead pellets were resuspended in 0.04 ml of SDS sample buffer containing 50 mM dithiothreitol and boiled for 5 minutes. Aliquots of the soluble extracts were subjected to SDS gel electrophoresis, and the resulting gels blotted onto nitrocellulose membranes. These blots were probed with Alexa Fluor-680 streptavidin (diluted 1 to 4000) and re-probed with rabbit anti-human CES-1 (diluted 1 to 5000) followed by a DyLight 880-labeled goat anti-rabbit secondary antibody (diluted 1 to 10,000).

Enriched preparations of microglial cells from WT and hCES-1 mice were isolated as described previously (Lee and Tansey, 2013). Briefly, each perfused brain was diced into small pieces and transferred to a 15-ml tube containing 3 ml of dissociation medium (Dulbecco’s Modified Eagles Medium/F12, 1 mg/mL papain, 1.2 U/ml dispase II and 20 U/mL DNase1) for 20 minutes at 37°C with gentle inversion every 5 minutes. At the end of the incubation period, 5 ml of neutralization media (Dulbecco’s Modified Eagles Medium/F12, 10% FBS and 4.5 mg/ml glucose) was added to the tube to neutralize the enzymes, followed by 5 minutes of centrifugation at 250 × g. Pelleted tissues were resuspended in 6 ml of Dulbecco’s Modified Eagles Medium/F12 media and dissociated by pipetting up and down using a Pasteur pipette. The cell suspension then was passed through a 40 µm cell strainer and the filtrate was centrifuged at 250 × g for 4 minutes. The cell pellet was resuspended in 4 ml of 37% isotonic Percoll and 4 ml of 70% isotonic Percoll was gently underlaid. Then 4 ml of 30% isotonic Percoll was overlaid on top of the 37% layer, followed by 2 ml of phosphate-buffered saline. The gradient was centrifuged at 300 × g for 40 minutes at 18°C with no brake. Microglia were collected at the 37%–70% interface; based on enrichment of Tmem119 expression, the isolated cells were estimated to be 30% of microglial origin.

Immunohistochemical Assessments.

Liver, lungs, and brains from WT and hCES-1 mice were harvested and subjected to formalin fixation and paraffin embedding. All staining procedures were conducted using a Leica Bond Automated Immunostainer. Briefly, sections were deparaffinized then processed for immunolocalization of human CES-1 using a primary rabbit polyclonal antibody against CES-1. Liver sections also were probed with a polyclonal antibody against endogenous mouse Ces-1. After staining with an appropriate goat anti-rabbit secondary antibody, slides were treated with an anti-goat antibody conjugated to horseradish peroxidase and antibody complexes then were visualized using 3,3′-diaminobenzidine as substrate. Tissues were counterstained with hematoxylin. Slides were scanned in brightfield with a 20X objective using a NoanZoomer Digital Pathology System (Hamamatsu City, Japan). The digital images were then imported into Visiopharm software (version 2023.01.1.13563; Hoersholm, Denmark) for analysis. Images were quantified as a percentage of positive stain to tissue area by a blinded analyst using the same Visiopharm software.

In Vivo Studies.

To compare pharmacokinetics of NT-0796 in WT (C57BL/6J) and hCES-1 mice, animals were intravenously administered 3 mg/kg NT-0796; the dosing formulation was composed of 1 mg/ml NT-0796 in vehicle consisting of 40% polyethylene glycol 400/10% Cremophor RH-40/50% water. Three male mice of each genotype were dosed with this formulation after which blood samples were harvested at various times by saphenous vein collection. Twenty-microliter aliquots of the harvested blood samples were collected into prechilled polypropylene tubes containing 2 µl of 0.5 M potassium EDTA (anti-coagulant) and 3 µl of a quench solution containing 950 mM sodium fluoride, 722 mM potassium oxalate, and 5 mM phenylmethylsulfonyl fluoride (to neutralize esterase activity). Two hundred microliters of 75% methanol/25% acetonitrile (spiked with internal calibration standards) were added to each stabilized blood sample, the samples vortexed, and subsequently subjected to a 10-minute centrifugation at 12,000 × g at 4°C. Resulting supernatants were analyzed by LC-MS/MS analysis. Plasma T1/2 estimates were generated using Phoenix Winnonlin 6.3 software. PK studies were conducted under contract by WuXi AppTech in Shanghai, China.

Pharmacodynamic impact of NDT-19795 and NT-0796 was evaluated in an acute LPS/ATP peritonitis model (Primiano et al., 2016). Mice for these studies were bred and maintained in specific pathogen-free housing in ventilated cages. Studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals; these studies were also approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina, Chapel Hill. Additional details relating to animal care are provided in the Supplemental Methods section. Cohorts of male hCES-1 mice were dosed by oral gavage with NDT-19795 or NT-0796 suspended in 0.5% methylcellulose, 2% Tween 80; doses of 10, 30, and 100 mg/kg were employed. NT-0796 doses were selected based on IC50 values observed in the context of the hCES-1 mouse blood assay and knowledge of compound bioavailability. One-hour post-dosing, the animals received an intraperitoneal injection of 1 µg of LPS followed 2 hours later by intraperitoneal injection of 0.5 ml of 30 mM ATP. Thirty minutes later, the animals were euthanized (inhaled isoflurane anesthesia followed by physical euthanasia) and 3 ml of phosphate-buffered saline containing 10% FBS, 25 units/ml heparin and protease inhibitors was injected to facilitate peritoneal lavage. Levels of IL-1β and IL-6 recovered in the isolated lavage fluids subsequently were determined by ELISA. Similarly, age- and sex-matched (female) cohorts of hCES-1 (backcrossed to a C57BL/6 background), Ces1c−/−, and WT C57BL/6 mice were dosed orally with NT-0796 and then subjected to the LPS/ATP challenge model.

Statistical Analysis.

Data presented were graphed and analyzed using GraphPad Prism software (version 10 sourced from Graphpad Software, Boston, MA). Individual points corresponding to compound titration curves represent the mean and standard deviation of triplicate determinations. Concentration titration curves were fit and IC50 values determined using a four-parameter logistic, variable slope model. Statistical analyses were conducted using ordinary one-way ANOVA with Dunnett’s multiple comparisons test or Welch’s one-way ANOVA and Dunnett’s T3 multiple comparisons test.