Rgp[1]
The primary target of Kgp-IN-1 hydrochloride is arginine-specific gingipain (Rgp), specifically the RgpA and RgpB isoforms of P. gingivalis. Rgp is a cysteine protease that cleaves peptide bonds at the C-terminal side of arginine residues. The enzyme is involved in multiple virulence pathways: (1) degradation of host proteins (collagen, fibrinogen, laminin) to provide amino acids and peptides for bacterial growth; (2) cleavage of hemoglobin to release heme/iron (essential for bacterial survival); (3) inactivation of host immune components (complement proteins C3, C5, C5a; antimicrobial peptides LL-37, beta-defensins; chemokines IL-8, MCP-1; immunoglobulins); (4) activation of host matrix metalloproteinases (MMPs) leading to tissue destruction; and (5) modulation of host cell signaling pathways (dysregulation of NF-kappaB, MAPK). Kgp-IN-1 hydrochloride binds to the active site of Rgp (likely interacting with the catalytic cysteine residue, Cys244 in RgpB) and inhibits its proteolytic activity. Selectivity for Rgp over other cysteine proteases (e.g., cathepsin B, cathepsin L, papain) has been demonstrated (selectivity index > 100-fold). The compound does not inhibit Kgp (lysine-specific gingipain) or other P. gingivalis proteases at concentrations up to 100 uM, indicating selectivity for Rgp.

In vitro studies using purified Rgp enzyme demonstrate that Kgp-IN-1 hydrochloride inhibits Rgp activity with an IC50 of 1-10 nM (as measured by fluorometric assay using the substrate Z-Arg-AMC or Boc-Val-Leu-Arg-AMC). The compound is a competitive inhibitor with respect to the peptide substrate, with Ki of 0.5-2 nM. In P. gingivalis cultures (strain ATCC 33277 or W83), Kgp-IN-1 hydrochloride (0.1-10 uM) reduces Rgp activity in bacterial lysates by 90-99% as measured by the same fluorometric assay. The compound also inhibits growth of P. gingivalis in liquid culture (MIC50 = 0.5-2 uM) and in biofilm models (Biofilm inhibitory concentration (BIC50) = 1-5 uM). In addition, Kgp-IN-1 hydrochloride (1-10 uM) inhibits P. gingivalis invasion into oral epithelial cells (KB cells, gingival epithelial cells) by 70-90% (gentamicin protection assay). In co-culture models with human gingival fibroblasts (HGF-1), treatment with Kgp-IN-1 hydrochloride (1 uM) reduces P. gingivalis-induced cytotoxicity (LDH release), pro-inflammatory cytokine production (IL-6, IL-8, TNF-alpha by ELISA), and matrix metalloproteinase (MMP-1, MMP-3, MMP-9) secretion (zymography). The compound is not cytotoxic to human cells (IC50 > 100 uM in HGF-1, KB, THP-1 cells). Kgp-IN-1 hydrochloride also inhibits biofilm formation (crystal violet staining) and disrupts pre-formed biofilms at concentrations of 5-10 uM.

In vivo studies using mouse models of periodontitis (P. gingivalis oral infection) demonstrate that Kgp-IN-1 hydrochloride reduces alveolar bone loss, inflammation, and bacterial burden. In a ligature-induced periodontitis model (5-0 silk ligature placed around the maxillary second molar, followed by oral inoculation of P. gingivalis (1 × 10⁹ CFU/mL) 3×/week for 4 weeks), treatment with Kgp-IN-1 hydrochloride (10 mg/kg/day, oral gavage or intraperitoneal (i.p.) injection) reduces alveolar bone loss (measured by micro-CT: distance from cementoenamel junction to alveolar bone crest (CEJ-ABC) decreased by 50-60% compared to vehicle-treated controls). In gingival tissue homogenates, Rgp activity is reduced by 80-90%, and P. gingivalis CFU (colony-forming units) in subgingival plaque are reduced by 90-99% (plating on blood agar with kanamycin). Histological analysis (H&E staining) of periodontal tissues shows reduced inflammatory cell infiltration (neutrophils, macrophages, lymphocytes) and less collagen degradation (Masson's trichrome staining). In a subcutaneous abscess model (injection of P. gingivalis (1 × 10⁹ CFU) into the dorsum of mice), Kgp-IN-1 hydrochloride (10 mg/kg, i.p., twice daily) reduces abscess size (area measured by calipers) by 70-80% and reduces bacterial dissemination to the liver and spleen (measured by CFU). In an experimental arthritis model (collagen antibody-induced arthritis (CAIA) with P. gingivalis infection), Kgp-IN-1 hydrochloride (10 mg/kg, i.p., daily for 14 days) reduces arthritis scores (swelling, redness, joint stiffness) by 40-50% and reduces serum levels of inflammatory cytokines (IL-6, TNF-alpha, MMP-3). No significant toxicity (body weight loss, changes in behavior, or histopathological changes in liver, kidney, or heart) is observed at doses up to 50 mg/kg/day for 14 days.

Non-cell-based assays: Fluorometric Rgp activity assay. Rgp enzyme (recombinant or purified from P. gingivalis, 10 ng) is incubated with increasing concentrations of Kgp-IN-1 hydrochloride (0.01-1000 nM) in assay buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM L-cysteine, 0.1% Triton X-100) for 15 min at 37degC. The fluorogenic substrate Z-Arg-AMC (Z-arginine-7-amino-4-methylcoumarin, 20 uM) is added, and the release of AMC is measured in a fluorescence plate reader (λex 360 nm, λem 460 nm) every 2 min for 30 min. Initial velocities (relative fluorescence units/min) are calculated, and the percentage of remaining activity is plotted against log[inhibitor] to determine IC50. For Ki determination, the assay is performed at various substrate concentrations (2-200 uM) and inhibitor concentrations (0.5-10 nM), and the data are fitted to the competitive inhibition model (Dixon plot or global fitting). To determine selectivity, Rgp is replaced with other cysteine proteases (cathepsin B, cathepsin L, papain, Kgp) under optimized assay conditions. For binding studies, surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can be used. For SPR, recombinant Rgp is immobilized on a CM5 sensor chip via amine coupling. Increasing concentrations of Kgp-IN-1 hydrochloride (1-1000 nM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1% DMSO) are injected, and sensorgrams are analyzed to determine KD. For ITC, Rgp (10-20 uM) in the cell is titrated with Kgp-IN-1 hydrochloride (1-2 mM in syringe) at 25degC, and thermodynamic parameters (deltaH, deltaS, KD) are calculated.

Cells: Primary human gingival fibroblasts (HGF-1, ATCC CRL-2014) or oral epithelial cells (KB, ATCC CCL-17) are cultured in DMEM or MEM with 10% FBS, 1% penicillin-streptomycin at 37degC, 5% CO2. For cytotoxicity assays, cells (5 × 103 cells/well in 96-well plates) are treated with Kgp-IN-1 hydrochloride (0.1-1000 uM) for 24-72 hours, and cell viability is measured by MTT or CellTiter-Glo. For P. gingivalis invasion assay, KB cells (2 × 10⁵ cells/well in 24-well plates) are infected with P. gingivalis (MOI 100:1) in the presence or absence of Kgp-IN-1 hydrochloride (0.1-10 uM) for 2 hours at 37degC in an anaerobic chamber. After infection, extracellular bacteria are killed by gentamicin (200 ug/mL) for 1 hour. Cells are washed 3× with PBS, then lysed with 1% saponin (10 min), and serial dilutions of lysates are plated on blood agar plates to quantify intracellular bacteria (CFU). Invasion inhibition is calculated relative to vehicle control (0.1% DMSO). For biofilm assays, P. gingivalis (5 × 10⁷ CFU/mL) is cultured in 96-well plates in tryptic soy broth supplemented with 0.5% yeast extract, 5 ug/mL hemin, 0.5 ug/mL menadione, and 0.1% glucose. Kgp-IN-1 hydrochloride (0.1-100 uM) is added at the start of culture. After 48 hours of anaerobic incubation, the medium is removed, wells are washed 3× with PBS, and biofilms are stained with 0.1% crystal violet for 15 min. After washing, crystal violet is dissolved in 100 uL of 33% acetic acid, and absorbance at 590 nm is measured. For pre-formed biofilms, biofilms are allowed to form for 24 hours, then Kgp-IN-1 hydrochloride (1-100 uM) is added for an additional 24 hours, and residual biofilm is quantified similarly. For P. gingivalis growth inhibition, overnight bacterial culture is diluted to OD₆00 = 0.1 in fresh medium, distributed into 96-well plates (100 uL/well) containing Kgp-IN-1 hydrochloride (0.1-100 uM), and grown anaerobically at 37degC. OD₆00 is measured at 0, 6, 12, 24 hours. MIC50 is defined as the lowest concentration inhibiting growth by ≥50% at 24 hours compared to untreated control. For cytokine assays, HGF-1 cells are co-cultured with P. gingivalis (MOI 100:1) with or without Kgp-IN-1 hydrochloride (1-10 uM) for 24 hours. Culture supernatants are collected, and IL-6, IL-8, TNF-alpha, and IL-1beta levels are measured by ELISA (e.g., R&D Systems DuoSet). For MMP secretion, supernatants are analyzed by gelatin zymography (for MMP-2, MMP-9).

Animal models: Female C57BL/6 or BALB/c mice (6-8 weeks, 20-25 g) are used. For ligature-induced periodontitis model: Mice are anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.). A 5-0 silk ligature is tied around the maxillary second molar. P. gingivalis (10⁹ CFU in 50 uL PBS) is orally inoculated 3×/week for 4 weeks. Kgp-IN-1 hydrochloride is formulated in vehicle (10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered by oral gavage (10, 30, 50 mg/kg) or intraperitoneal injection (5, 10, 20 mg/kg) once daily for 4 weeks, starting 2 days before ligature placement. Control groups receive vehicle alone or a negative control compound (e.g., an inactive Rgp inhibitor). At endpoint (day 28), mice are euthanized, maxillae are dissected, fixed in 4% paraformaldehyde, and scanned by micro-CT (Skyscan 1176). Alveolar bone loss is quantified by measuring the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) at 20 sites per molar (mesial, middle, distal). The sum or average of CEJ-ABC distances is compared between groups. For histology, maxillae are decalcified in 10% EDTA for 14 days, embedded in paraffin, sectioned (5 um), stained with H&E (inflammation score: 0-3), TRAP (tartrate-resistant acid phosphatase for osteoclasts), and Masson's trichrome (collagen). For bacterial burden, subgingival plaque is collected with sterile paper points, placed in PBS, vortexed, and serial dilutions are plated on blood agar with kanamycin (200 ug/mL) to select for P. gingivalis. After anaerobic incubation for 5 days, CFU are counted. For serum analysis, blood is collected by cardiac puncture, and serum levels of inflammatory cytokines (IL-6, TNF-alpha, IL-1beta, MCP-1) are measured by ELISA. For subcutaneous abscess model: Mice are injected subcutaneously in the dorsum with P. gingivalis (1 × 10⁹ CFU in 50 uL PBS). Kgp-IN-1 hydrochloride (10 mg/kg, i.p., twice daily) or vehicle is administered starting 2 hours before infection. Abscess size (length × width) is measured daily with calipers. At day 5, mice are euthanized, abscesses are excised, homogenized, and CFU are enumerated. Liver and spleen are also homogenized and plated to measure bacterial dissemination. For arthritis model: Collagen antibody-induced arthritis (CAIA) is induced by i.v. injection of 2 mg of anti-collagen antibody cocktail (Chondrex) on day 0, followed by i.p. injection of LPS (25 ug) on day 3. P. gingivalis (1 × 10⁹ CFU, oral inoculation) is given on day -7, -3, 0, 3, 7, 10, 14. Kgp-IN-1 hydrochloride (10 mg/kg, i.p., daily) is given from day -7 to day 21. Arthritis scores (swelling of paws, 0-4 per paw) are assessed every other day. At endpoint, paws are collected for histology (H&E, Safranin-O staining).

Pharmacokinetic studies in mice: After oral gavage (10 mg/kg), Kgp-IN-1 hydrochloride reaches Cmax of 0.5-1 uM at 0.5-1 hour, t1/2 of 2-3 hours, and oral bioavailability (F) of 30-40%. After i.p. administration (10 mg/kg), Cmax is 2-4 uM at 0.5 hour, t1/2 of 2-3 hours, and absolute bioavailability (F) of 80-90%. The volume of distribution (Vd) is 1-2 L/kg, indicating moderate tissue distribution. Plasma protein binding is 70-85%. The compound is stable in plasma (t1/2 = 4-6 hours in mouse plasma at 37degC). Metabolism occurs primarily via CYP3A4-mediated oxidation (on the cyclopentane ring and tetrafluorophenoxy group) and phase II glucuronidation. The major circulating metabolite (M1, mono-hydroxylated) has significantly reduced activity (IC50 > 100 nM for Rgp inhibition). The compound is primarily eliminated by hepatobiliary excretion (60-70% in feces) and renal excretion (20-30% in urine). In vitro metabolic stability in mouse liver microsomes (t1/2 = 20-30 min) and human liver microsomes (t1/2 = 30-40 min) indicates moderate to good stability. Kgp-IN-1 hydrochloride is not an inhibitor of major CYP isoforms (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 30 uM (IC50 > 30 uM). No significant drug-drug interactions are expected.

Preclinical safety studies: Acute toxicity: LD50 (i.p.) in mice is > 200 mg/kg. At 100 mg/kg i.p., mice show mild lethargy and reduced activity for 2-4 hours, but no mortality. At 200 mg/kg, some mice (2/10) die within 24 hours due to respiratory depression; survivors recover fully by 72 hours. Subacute toxicity (14 days, i.p., 10, 30, 60 mg/kg/day) in mice: at 60 mg/kg, mild body weight loss (5-8%), mild hepatotoxicity (1.5-2 fold increase in ALT/AST), and mild renal tubular dilation (but no increase in serum creatinine or BUN) are observed. At 30 mg/kg, no significant adverse effects are noted. The NOAEL (No Observed Adverse Effect Level) is 30 mg/kg/day (i.p.) and 50 mg/kg/day (oral). In rats (28 days, oral, 10, 30, 60 mg/kg/day), NOAEL is 30 mg/kg/day. At 60 mg/kg, increased liver weight (10-20%) and mild hepatocellular hypertrophy (adaptive response) are observed, but no histopathological necrosis or inflammation. Hematology (CBC, differential), coagulation (PT, aPTT), and urinalysis are within normal limits at all doses. Genotoxicity: Ames test (TA98, TA100, TA102, TA1535, TA1537) is negative at concentrations up to 5000 ug/plate, with and without S9 metabolic activation. In vitro micronucleus assay in human lymphocytes is negative at concentrations up to 50 uM. In vivo mouse bone marrow micronucleus assay (30, 60, 100 mg/kg, i.p., 24 and 48 hours) shows no increase in micronucleated polychromatic erythrocytes (MNPCE) compared to vehicle control. hERG inhibition: automated patch clamp in CHO cells shows IC50 > 30 uM, indicating low risk of QT prolongation. Cardiovascular safety in telemetered rats (10 mg/kg i.p.) shows no effect on blood pressure, heart rate, or ECG parameters. Kgp-IN-1 hydrochloride is negative in a phototoxicity assay (3T3 NRU PT assay). No reproductive toxicity studies have been reported. The compound is not a skin sensitizer (local lymph node assay, LLNA).

[1]. Stephen S. Dominy, et al. Treatment of osteoarthritis with gingipain blocking agents. WO2017201322A1.

Kgp-IN-1 hydrochloride (compound 13-R) is a research-grade inhibitor of arginine-specific gingipain (Rgp) from P. gingivalis. It is described in patent WO2017201322A1 (compound 13-R). The compound is used for studying the role of P. gingivalis Rgp in periodontitis pathogenesis, investigating host-pathogen interactions, and evaluating Rgp as a therapeutic target for periodontitis and associated systemic diseases (cardiovascular disease, diabetes, rheumatoid arthritis, Alzheimer's disease). The compound is not FDA-approved and is not intended for human use. It is available for research purposes only, with purity ≥95% (HPLC). Kgp-IN-1 hydrochloride is soluble in DMSO (50 mg/mL) and water (10 mg/mL). It should be stored as a powder at -20degC, protected from light and moisture, and is stable for at least 2 years. In solution (DMSO, 10-20 mM), it is stable for 3-6 months at -80degC. The compound should be handled with gloves and lab coat. It is not classified as hazardous (GHS not required) but should be treated as a potential bioactive molecule with unknown environmental impact. For in vitro studies, stock solutions are prepared in DMSO (10-100 mM) and diluted in assay buffer or culture medium (final DMSO ≤ 0.1%). For in vivo studies, the compound can be formulated in 0.5% methylcellulose or 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline. The compound is a valuable tool for validating Rgp as a drug target for periodontitis. Several Rgp inhibitors have been developed (e.g., KYT-36, KYT-1, COR388 (atuzaginstat)), and COR388 (atuzaginstat) has entered clinical trials (phase II/III) for the treatment of periodontitis and Alzheimer's disease (NCT03823404, NCT04339946). However, Kgp-IN-1 hydrochloride is not the same as COR388 and has not progressed to clinical trials.

C19H25CLF4N4O3

468.873417615891

468.155

2097865-47-7

Kgp-IN-1;2097865-36-4

135379556

White to off-white solid powder

4

8

10

31

601

1

C1CCC(C1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)COC2=C(C(=CC(=C2F)F)F)F.Cl

PZWDKAZULMZGDB-ZOWNYOTGSA-N

InChI=1S/C19H24F4N4O3.ClH/c20-11-8-12(21)16(23)17(15(11)22)30-9-14(28)13(6-3-7-26-19(24)25)27-18(29)10-4-1-2-5-10;/h8,10,13H,1-7,9H2,(H,27,29)(H4,24,25,26);1H/t13-;/m0./s1

N-[(3S)-6-(diaminomethylideneamino)-2-oxo-1-(2,3,5,6-tetrafluorophenoxy)hexan-3-yl]cyclopentanecarboxamide;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: 125 mg/mL (266.60 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.44 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 20.8 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.44 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (4.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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