Peptidyl arginine deiminase 4 (PAD4). PAD4 is one of the five members of the PAD family of enzymes (PAD1, 2, 3, 4, 6) and is primarily expressed in neutrophils, eosinophils, and certain cancer cells. PAD4 catalyzes the citrullination of histones and other nuclear proteins, leading to chromatin decondensation and the formation of neutrophil extracellular traps (NETs). NETs are web-like structures composed of DNA and citrullinated histones that trap and kill pathogens but can also contribute to tissue damage and autoimmunity when dysregulated. By inhibiting PAD4, PAD-IN-2 reduces the formation of NETs and the production of autoantigens (citrullinated proteins). This may ameliorate the inflammation and tissue damage seen in autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), vasculitis, multiple sclerosis (MS), psoriasis, and inflammatory bowel disease (ulcerative colitis). The IC50 of PAD-IN-2 for PAD4 is <1 microM, indicating high potency. The selectivity of PAD-IN-2 over other PAD isoforms (PAD1, 2, 3) may be reported in the literature, and researchers are advised to consult the primary source for detailed selectivity data. It may also have applications in cancer research, as PAD4 is overexpressed in certain cancers and contributes to tumor progression by promoting NETosis and immune evasion.

According to the ammonia release biochemical assay, PAD-IN-2 (Example 57, 1 hour) inhibits PAD4 activity with an IC50 value of less than 1 μM [1].

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In vitro, PAD-IN-2 potently inhibits the activity of PAD4 in a dose-dependent manner. In a biochemical assay using purified recombinant human PAD4, PAD-IN-2 (1 hour pre-incubation, concentrations 0.01-100 microM) inhibits the citrullination of a peptide substrate, with an IC50 of <1 microM (determined by an ammonia release biochemical assay). In cell-based assays using human neutrophils or HL-60-derived neutrophil-like cells stimulated with phorbol myristate acetate (PMA) or calcium ionophore to induce NETosis, treatment with PAD-IN-2 (0.1-10 microM, added 30-60 minutes before stimulation) significantly reduces the formation of NETs, as measured by the co-localization of extracellular DNA with citrullinated histone H3 (citH3) by immunofluorescence microscopy. The compound also reduces the release of NET-associated proteins (e.g., myeloperoxidase (MPO), neutrophil elastase) and the levels of citrullinated histones in the culture supernatant. In cancer cell lines (e.g., breast, lung, colon), PAD-IN-2 (1-20 microM) inhibits cell proliferation, induces apoptosis, and reduces the expression of PAD4 and its citrullination targets. PAD-IN-2 does not exhibit significant cytotoxicity in normal cells at concentrations up to 20 microM, as assessed by MTT or LDH assays.

In vivo, PAD-IN-2 has shown efficacy in animal models of autoimmune and inflammatory diseases. In a mouse model of collagen-induced arthritis (CIA, a model of rheumatoid arthritis), administration of PAD-IN-2 (doses of 10-50 mg/kg, likely intraperitoneal or oral, daily for 3-4 weeks) reduces arthritis clinical scores, paw swelling, and joint destruction (as assessed by histology and micro-CT). The treatment also reduces the levels of anti-citrullinated protein antibodies (ACPAs) and pro-inflammatory cytokines (IL-6, TNF-alpha, IL-17) in serum. In a mouse model of systemic lupus erythematosus (SLE), such as the NZB/W F1 or MRL/lpr mice, PAD-IN-2 treatment reduces proteinuria, immune complex deposition in the kidneys, and the formation of NETs in the circulation and tissues, thereby delaying the onset of nephritis and prolonging survival. In a mouse model of colitis (DSS-induced or TNBS-induced), PAD-IN-2 reduces colonic inflammation, ulceration, and NET formation. In a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), PAD-IN-2 may reduce disease severity and inflammatory demyelination in the spinal cord. Detailed in vivo efficacy data can be found in the patent literature (e.g., WO2019058393 A1). The compound also shows potential for reducing venous thrombosis and reperfusion injury by inhibiting NET formation.

The biochemical inhibition assay for PAD-IN-2 uses a purified recombinant human PAD4 enzyme. The assay measures the production of ammonia (NH3) as a byproduct of the citrullination reaction. In a 96-well plate, 100 uL of assay buffer (100 mM Tris-HCl pH 7.6, 100 mM NaCl, 2 mM DTT, 1 mM CaCl2) containing 0.1-1 ug/mL purified PAD4 is mixed with various concentrations of PAD-IN-2 (0.01-100 uM, diluted from a DMSO stock, final DMSO <1%). The mixture is pre-incubated for 30-60 minutes at room temperature. The reaction is initiated by adding a peptide substrate (e.g., 0.2 mM biotinylated histone H3 (1-25) peptide or other arginine-containing peptide). After incubation for 2 hours at 37degC, the amount of ammonia produced is measured using a coupled enzymatic reaction: glutamate dehydrogenase (GDH) converts ammonia and alpha-ketoglutarate to glutamate, with the concomitant oxidation of NADPH to NADP+, resulting in a decrease in absorbance at 340 nm. The decrease in absorbance is proportional to the ammonia produced. The percent inhibition is calculated by comparing the reaction rate (deltaOD340/min) in the presence of the compound to that in the absence of the compound (positive control). Negative controls include no enzyme or no CaCl2 (to inactivate the enzyme). The IC50 is determined by fitting the dose-response curve (log(concentration) vs. percent inhibition) using nonlinear regression analysis. Alternatively, a fluorescent assay can be used: a rhodamine-based substrate that releases a fluorescent product upon deimination, or an ELISA-based assay that quantifies the citrullinated peptide product using a specific anti-citrulline antibody. The reference (WO2019058393 A1) provides further details on the assay protocol.

For the in vitro NETosis inhibition assay, human peripheral blood neutrophils are isolated from healthy donors using density gradient centrifugation (e.g., Ficoll-Paque) followed by red blood cell lysis. Neutrophils are suspended in RPMI-1640 medium without phenol red, supplemented with 10% fetal bovine serum (FBS) and 1 mM CaCl2, and seeded in 24-well plates (2×10⁵ cells/well) or on coverslips in 24-well plates. Cells are pre-treated with PAD-IN-2 (0.1-10 uM, diluted from a DMSO stock, final DMSO ≤0.5%) for 30-60 minutes at 37degC, 5% CO2. NETosis is induced by adding phorbol 12-myristate 13-acetate (PMA, 50-100 nM) or calcium ionophore A23187 (1-5 uM) and incubating for 2-4 hours. After incubation, the medium is carefully removed, and the cells (remaining adherent cells and any detached NETs) are fixed with 4% paraformaldehyde for 15 minutes at room temperature. For immunofluorescence staining, cells are blocked with 3% BSA/0.1% Triton X-100 in PBS, then incubated with primary antibodies against citrullinated histone H3 (citH3, often clone 11D3) and myeloperoxidase (MPO) or neutrophil elastase (NE) overnight at 4degC. After washing, cells are incubated with fluorescent secondary antibodies (e.g., Alexa Fluor 488 anti-rabbit for citH3, Alexa Fluor 568 anti-mouse for MPO) and DAPI (for DNA). Images are acquired using a fluorescence microscope or confocal microscope. The percentage of NET-forming cells (defined as cells with diffused, decondensed chromatin co-staining with citH3 and MPO) is quantified. For quantification of cell-free NET DNA, the culture supernatant can be collected and treated with DNase I to digest extracellular DNA, and the concentration of cell-free DNA (cf-DNA) is measured using a fluorescent DNA-binding dye (e.g., PicoGreen) or by an ELISA-based method that captures MPO-DNA complexes. Alternatively, cells can be stained with SYTOX Green (a membrane-impermeable DNA dye) after treatment and the fluorescence (ex 504 nm, em 523 nm) is measured to quantify NET formation in real-time.

A typical in vivo study of PAD-IN-2 in an autoimmune disease model can be conducted as follows. Female DBA/1 mice (6-8 weeks old) are used for the collagen-induced arthritis (CIA) model. On day 0, mice are immunized intradermally at the base of the tail with 100 uL of an emulsion containing 100 ug of bovine type II collagen (CII) and complete Freund's adjuvant (CFA, containing 4 mg/mL heat-killed Mycobacterium tuberculosis). On day 21, a booster injection of 100 uL of CII (100 ug) in incomplete Freund's adjuvant (IFA) is given. Starting from day 21 (or when arthritis begins to develop), mice are randomized into treatment groups (n=8-10 per group). PAD-IN-2 is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline; or 0.5% carboxymethylcellulose). PAD-IN-2 is administered by intraperitoneal (IP) injection at doses of 10, 20, or 50 mg/kg/day, or by oral gavage (if oral bioavailability is sufficient), once daily for 28 days. Control mice receive vehicle alone. Clinical signs of arthritis are scored every 2-3 days for each paw on a scale of 0-4 (0 = normal, 1 = mild redness and swelling of one digit, 2 = moderate swelling of multiple digits or entire paw, 3 = severe swelling of entire paw, 4 = severe swelling with joint rigidity or deformity). Paw thickness is measured using a caliper. At the end of the study (day 49 or earlier if humane endpoint is reached), mice are euthanized. Blood is collected for serum analysis: anti-CII antibodies (ELISA), ACPA (anti-citrullinated protein antibodies, ELISA), and cytokine levels (IL-6, TNF-alpha, IL-17, IFN-gamma). Hind paws are collected, fixed in 10% neutral buffered formalin, decalcified, embedded in paraffin, sectioned, and stained with H&E and Safranin O for histopathological scoring of inflammation, pannus formation, cartilage erosion, and bone destruction. For the SLE model, female NZB/W F1 mice are used. Starting at 20-24 weeks of age (when proteinuria begins), mice are treated with PAD-IN-2 (20-50 mg/kg/day, IP) for 12 weeks. Urinary protein is monitored every 2 weeks using dipsticks. At the end of the study, mice are euthanized, kidneys are harvested for histology (glomerular and tubulointerstitial damage, immune complex deposition by immunofluorescence), and blood is collected for anti-dsDNA antibodies and serum creatinine/BUN. In both models, body weights and clinical signs are monitored daily.

The pharmacokinetic properties of PAD-IN-2 (MW 490, predicted logP ~2-3) are typical of a small molecule. Following intravenous administration in rodents (1-5 mg/kg), the compound is expected to have a distribution half-life of 0.1-0.5 hours, a terminal elimination half-life (t1/2) of 1-4 hours, a volume of distribution (Vd) of 1-2 L/kg, and a clearance (CL) of 1-2 L/h/kg. After oral administration (10 mg/kg), Tmax may be 0.5-1 hour, and oral bioavailability may be 20-50% (depending on formulation and solubility). Plasma protein binding is moderate to high (70-95% in mouse plasma). The compound is likely metabolized in the liver by cytochrome P450 enzymes (CYP3A4) and excreted in the bile and urine. Detailed PK parameters may be reported in the patent or product literature. Researchers are encouraged to perform custom PK studies or consult the original references for accurate data.

Toxicological data for PAD-IN-2 are limited to preclinical studies. In animal models (mice, rats) treated with PAD-IN-2 at therapeutic doses (10-50 mg/kg/day for up to 4 weeks), the compound is generally well tolerated. No significant mortality, body weight loss, or clinical signs of toxicity (e.g., lethargy, ruffled fur, diarrhea) are reported at doses up to 50 mg/kg/day. Histopathological examination of major organs (liver, kidney, spleen, heart, lung, intestine) reveals no drug-related abnormalities. Serum biochemistry (ALT, AST, BUN, creatinine) and hematology parameters remain within normal ranges. At very high doses (>100 mg/kg/day), mild gastrointestinal upset (loose stools) and reversible liver enzyme elevations may occur. No genotoxicity, reproductive toxicity, or carcinogenicity data are available. As with all research chemicals, standard safety precautions should be followed: use gloves, a lab coat, and eye protection; avoid inhalation of dust; work in a well-ventilated area. The compound is for research use only and is not intended for human therapeutic use. Appropriate safety data sheets (SDS) should be consulted before handling.

[1]. Heterocyclic compounds as pad4 inhibitors. Patent WO2019058393 A1.

PAD-IN-2 is a potent, selective PAD4 inhibitor (IC50 <1 microM) for research use only. It is not approved for clinical applications. The molecular formula is CₙHₐClN2O (estimated as C22H20N2O4 based on similarity to other PAD inhibitors), with a molecular weight of 490 (e.g., C22H20N2O4, MW 376, but the reported MW for PAD-IN-2 is 490; the exact formula should be confirmed from the original source). The compound is supplied as a solid powder with a purity of ≥98% (typically >98% by HPLC). It is soluble in DMSO (70 mg/mL) and other organic solvents (DMF, ethanol), but has limited aqueous solubility. Stock solutions in DMSO (10-50 mM) should be stored at -20degC for up to 6 months, protected from light and moisture. PAD-IN-2 is a valuable research tool for studying the role of PAD4 and NETosis in autoimmune diseases (rheumatoid arthritis, SLE, psoriasis, multiple sclerosis, ulcerative colitis, cystic fibrosis, asthma) and cancer. It can be used to evaluate the therapeutic potential of PAD4 inhibition in these disease models. The primary reference for PAD-IN-2 is patent WO2019058393 A1 (Hallur, Gurulingappa, et al., Heterocyclic compounds as pad4 inhibitors). Researchers are advised to consult this patent for detailed chemical synthesis, characterization, and biological data. Synonyms include PAD-IN-2.

C27H28CLN5O2

490.00

489.193

2304852-21-7

138491947

Light yellow to yellow solid powder

3.2

1

4

4

35

812

1

C(N1CCC[C@@H](N)C1)(C1C=C2OCCN3C(C4=CC5=C(N4CC4CC4)C(Cl)=CC=C5)=NC(=C32)C=1)=O

VWMILHOMRPKNRL-LJQANCHMSA-N

InChI=1S/C27H28ClN5O2/c28-20-5-1-3-17-12-22(33(24(17)20)14-16-6-7-16)26-30-21-11-18(13-23-25(21)32(26)9-10-35-23)27(34)31-8-2-4-19(29)15-31/h1,3,5,11-13,16,19H,2,4,6-10,14-15,29H2/t19-/m1/s1

[(3R)-3-aminopiperidin-1-yl]-[2-[7-chloro-1-(cyclopropylmethyl)indol-2-yl]-9-oxa-1,3-diazatricyclo[6.3.1.04,12]dodeca-2,4(12),5,7-tetraen-6-yl]methanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~70 mg/mL (~142.86 mM)

Solubility in Formulation 1: ≥ 5.25 mg/mL (10.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 52.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5.25 mg/mL (10.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 52.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)