Synthesis of kavain analogs

A useful approach to optimize potential lead compounds is commonly referred to as a structure–activity-relationship (SAR) study. Specifically, efforts are directed toward identifying the correlations between substructures of compounds and their biological properties. A series of chemical modifications on the specific sites of the lead compounds are made to improve the potency and pharmaceutical properties. In our previous SAR study16, we found that one ring-opened analogue bearing an α,β-unsaturated ester displayed ≥50% suppression of TNF-α secretion. In addition, medicinal chemistry studies have revealed the effect of a methyl group in promoting and enhancing compound potency19,20. In light of these findings, we generated a focused library of cyclohexenones (5,5-dimethyl substituted cyclohexenones) substituted with a benzoate derivative at the 3-position of the cyclohexanone, which included the analog Kava-205Me (Fig. 1A). Kava-205Me was tested in vitro at the concentration ranging from 10, 50, 200 μg/ml.

Figure 1 (A) The synthesis of Kava-205Me is based on the O-acylation of the highly enolizable cyclic 1,3-diketones. Accordingly, treatment of 1,2-dichloroethane solution of commercially available 1,3-cyclohexanedione with 4-methylbenzoyl chloride in the presence of pyridine efficiently provided the O-acylated enol derivative Kava-205Me. (B) Kava-205 chlorinated and methylated forms reduce TNF-α in BMM infected with P. gingivalis. Kavain derived compounds identified through SAR were tested (40 μg/ml). Compounds were plotted according to z-score of TNF-α inhibition. Chemical formula and structure of others tested analogs are presented in Supplementary Fig. 1. Full size image

Porphyromonas gingivalis culture

The Porphyromonas gingivalis 381 strain was cultured and maintained in brain-heart infusion media supplemented with hemin (5 μg/mL, Sigma-Aldrich, St. Louis, MO), and menadione (1 μg/mL, Sigma-Aldrich, St. Louis, MO) in an anaerobic environment (AnaeroPack-Anaero, Mitsubishi Gas Chemical Co.; New York, NY) as previously described10.

Mouse bone marrow macrophage (BMM) isolation and infection

BMM were isolated from mouse bone marrow as previously described10. Briefly, after euthanasia, femurs and tibias were harvested and the bone marrow was flushed from the medullar cavity with collection media (DMEM, 10% FBS, and 1% penicillin-streptomycin). Cells were cultured in 30% L-929 conditioned RPMI media at a density of 105 cells/mL. L-929 (ATCC no. CCL-1) is a murine fibroblast cell line that is used as a source of macrophage colony-stimulating factor (M-CSF)21. After one week, cells had differentiated into BMM. The day of infection, cells were seeded in a 24-wells plate and after PBS wash, P. gingivalis 381 was added for 4 h to the BMM cultures at a MOI = 20:122.

THP-1 cell culture

THP-1 (ATCC® TIB-202™) cells were grown in RPMI medium containing 1% penicillin/streptomycin, 10% fetal bovine serum and β-mercaptoethanol (0.05 mM) in 5% CO 2 at 37°. Infection was performed as described for BMM.

TNF-α ELISA

The supernatants from infected cells and mouse serum were evaluated by ELISA for the detection of TNF-α concentration with an Invitrogen kit (KMC3011, ThermoFisher, Dublin, OH, USA). ELISA immunoreactivity was quantified using a microplate reader (Bio-Rad, Hercules, CA, USA).

Bioplex pro Mouse cytokine 23-plex assay

A cytokine 23-plex kit (BioRad, CA, USA, Cat #M60009RDPD) was used according to manufacturer’s instructions to measure the concentrations of eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, KC MCP-1 (MCAF), MIP-1α, MIP-1β, RANTES, and TNF-α in supernatants from BMM. The fluorescent signal intensity was measured using Bioplex 200 system. Cytokine concentrations were determined using standard curves generated using Bioplex manager software (V4.1).

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA from BMM was isolated and purified with a Rneasy Mini kit according to the manufacturer’s instructions (Qiagen, Hilden, DE, USA). cDNA from total RNA was synthesized (50–100 ng RNA/20 μl) using a QuantiTect Reverse transcription kit according to the manufacturer’s instructions (Qiagen). qRT-PCR was performed using the Taqman Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA) and was run for the following gene using the probe provided by Thermofisher: acid phosphatase 5 (ACP5) (Mn00475698_m1). qRT-PCR assays were performed in duplicates on an Applied Biosystems QuantStudio 5 Real-Time PCR system. The data were analyzed using QuantStudio 5 software V1.4. The gene expression levels were normalized to β-actin for BMM samples respectively and expressed relative to unstimulated controls following the 2-ΔΔCT method.

Mouse calvarial bone resorption model

The 8- to 12-week-old wild-type C57BL/6 J mice used in this study were purchased from Taconic Laboratories (Germantown, NY). All mice were housed 3/cage at the New York Medical College Animal Facility, on a 12 h light dark cycle and ad libitum access to food and water. All animals were cared by veterinary staff for house boundary, biological analysis and maintenance. All procedures were approved by the NYMC IACUC committee and follow ARRIVE guidelines. The mice were randomly allocated into the following three groups (5 mice/group): (i) PBS (ii) P. gingivalis (iii) P. gingivalis + Kava-205Me concurrently. Mice were anesthetized by intraperitoneal injection of ketamine-xylazine. The heads of mice were shaved, and then live bacteria resuspended in 100 μl of PBS were injected subcutaneously with a 30.5-gauge needle at a point on the midline of the skull between the ears and eyes, as we have described previously10. The dose of P. gingivalis (5 × 108) was injected. In the treatment group, 1 mg of Kava-205Me was concurrently injected. Mice were euthanized 4 days post injection. The size of the lesion (area in square millimeters) resulting from the injection in each animal was analyzed using ImageJ software.

Induction of arthritis and scoring

Six-weeks-old, pathogen-free DBA1/BO male mice were obtained from Taconic Laboratories. Arthritis was induced by two consecutive intraperitoneal injections of ArthritoMab (AB) antibody cocktail (Chondrex, WA, USA). 3.5 mg were injected at baseline and a second injection of 1 mg was done at day 4. A sample of 5 mice/group was considered based on our previous data18 and all animals were randomly allocated in each group. For P. gingivalis injected groups, 3 intraperitoneal injections of 5 × 108 bacteria/100 μl were administered. Mice were euthanized after 35 days by CO 2 inhalation. All four paws were evaluated by a reviewer blinded to the treatment group to score arthritis using a visual qualitative assessment scoring as follows: (0) no paw swelling, (1) mild swelling, (2) moderate swelling, (3) severe swelling.

Injection of P. gingivalis and Kava-205Me compound

In Kava-205Me treated groups, 8 intraperitoneal injections (40 mg/kg) were administered. The first injection was performed 3 days after the first AB injection (day 0) and at days 5, 7, 10, 13, 14, 18. As a control, same volume of the DMSO used to dissolve Kava-205 powder was used.

Tissue preparation

Phalangeal joints and intact surrounding tissues were fixed with 4% freshly prepared paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in PBS (pH 7.2) for 24 h at 4 °C. Following fixation, specimens were consecutively washed with 5%, 10%, and 15% glycerol (American Bioanalytical, Natick, MA) in PBS, each for 15 min at 4 °C and decalcified in an EDTA solution (Sigma-Aldrich, St. Louis, MO) for 14 days at 4 °C. Samples were then immersed in 30% sucrose (Sigma-Aldrich, St. Louis, MO) in PBS until embedding. Tissue blocks were embedded with a HISTOPREP® compound (Fisher Scientific, Hanover Park, IL) for cryostat sectioning. Serial mesiodistal sections (5 μm) parallel to the long axis of the phalangeal joint were made and stained with hematoxylin (Fisher Scientific, Pit, IL) – eosin (ACROS Organics, Morris Plains, NJ).

TRAP staining

Osteoclasts were detected by TRAP staining. 5 µm thick histological slides were exposed to the TRAP solution containing N,N-dimethylformamide (EM Science), 3.7 mM of fast red violet LB di-azonium salt (Sigma), 6.4 mM of tartaric acid (Sigma), and 0.4% MgCl 2 in 0.2 M sodium acetate buffer (pH 5.0) for 10 minutes at 37 °C. The slides were then washed for 30 minutes before being counter-stained with hematoxylin for 5 seconds. Osteoclasts were identified as being positively stained for TRAP and possessing a ruffled border with an underlying lacuna.

Histological scoring

Samples were scored for inflammation, bone destruction, bone formation/repair and for cartilage destruction. To score inflammation, a 0–4 scale was used with 0 corresponding to no signs of inflammation, 1 to mild infiltration of inflammatory cells, 2 to mild inflammation with small hyperplasia in the synovial lining layer, 3 to synovial edema, hyperplasia and more pronounced inflammation, and 4 to severe synovial hyperplasia and cellular infiltration. Bone destruction was scored using a 0 to 4 scale with 0 corresponding to no signs of bone destruction, 1 to osteoclast activation, 2 to presence of some osteoclast lacunae, 3 to presence of many osteoclast lacunae and signs of bone resorption, and 4 to severe bone resorption and erosion.

Bone histomorphometry

For each animal, two slides, each containing three tissue sections with the largest number of bone marrow cells (six specimens total), were analyzed. For each tissue section, the microscopic fields with the most resorption were studied. The osteoclast index, which represents the number of osteoclasts per millimeter of trabecular bone surface, was measured.

The percentage of bone surface covered by osteoclasts was also quantified. This was calculated as the sum of the lengths of the osteoclasts containing lacunae (active eroded area) divided by the total trabecular bone perimeter.

Statistical analysis

All experiments have been performed at least in triplicate. Data were analyzed for statistical significance with XLStat (Addinsoft, New York, NY, USA). P values were calculated with the Mann-Whitney U-test or ANOVA one-way t-test for multiple comparisons. Results were considered significant at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Data are presented as mean ± standard error of the mean (SEM).