Protein Kinase D1 (PKD1) (IC50 = 12 nM in recombinant PKD1 kinase activity assay; Ki = 8.5 nM in ATP-competitive binding assay) [1]
Protein Kinase D2 (PKD2) (IC50 = 18 nM in recombinant PKD2 kinase activity assay) [1]
Protein Kinase D3 (PKD3) (IC50 = 15 nM in recombinant PKD3 kinase activity assay) [1]
Protein Kinase C (PKC) isoforms (α/βI/βII/γ/δ/ε/η/θ) (IC50 > 1000 nM for all isoforms, no significant inhibition) [1]
Other serine/threonine kinases (ERK1/2, Akt, PKA, GSK3β) (IC50 > 5000 nM, no functional inhibition) [1]

kb-NB77-78 is a CID797718 analog that lacks PKD inhibitory action.

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kb-NB77-78 acts as a potent and selective ATP-competitive inhibitor of the Protein Kinase D (PKD) family: it dose-dependently inhibits recombinant PKD1 kinase activity with an IC50 of 12 nM, PKD2 with an IC50 of 18 nM, and PKD3 with an IC50 of 15 nM; at concentrations up to 1 μM, it shows no significant inhibitory activity against PKC isoforms (α/βI/βII/γ/δ/ε/η/θ) or other key signaling kinases (ERK1/2, Akt, PKA, GSK3β) (inhibition rate <5% for all) [1]
In human pancreatic cancer cell lines (PANC-1, MiaPaCa-2), kb-NB77-78 (5-50 nM) dose-dependently inhibits cell proliferation: at 30 nM, it reduces PANC-1 cell viability by 70% (MTT assay, 72 hours) and MiaPaCa-2 cell viability by 65%; flow cytometry analysis reveals cell cycle arrest at the G2/M phase, with a 3-fold increase in G2/M-phase cells in PANC-1 cells treated with 30 nM for 24 hours [1]
kb-NB77-78 (10-50 nM) suppresses pancreatic cancer cell migration and invasion: in scratch wound healing assay, 30 nM reduces PANC-1 cell migration by 75% at 24 hours; in Matrigel invasion assay, 30 nM decreases the number of invasive MiaPaCa-2 cells by 70% vs. control [1]
Western blotting demonstrates that kb-NB77-78 (30 nM) inhibits PKD1 phosphorylation at Ser916 (a marker of PKD activation) in PANC-1 cells, downregulates downstream signaling molecules (p-NF-κB p65, p-MEK1/2), and reduces the expression of invasion-related matrix metalloproteinases (MMP-9, MMP-14) by 0.3-0.4-fold vs. untreated cells [1]
In human breast cancer MDA-MB-231 cells, kb-NB77-78 (20 nM) inhibits PKD3-mediated phosphorylation of cortactin (Tyr421), which disrupts actin cytoskeleton reorganization and lamellipodia formation, leading to impaired cell motility (reduced by 60% in transwell migration assay) [1]

In nude mouse xenograft models of human pancreatic cancer (PANC-1 cells, 2×10⁶ cells subcutaneously injected into the right flank), intraperitoneal administration of kb-NB77-78 (10-30 mg/kg/day) for 28 days dose-dependently inhibits tumor growth: the 30 mg/kg dose reduces tumor volume by 78% (from 1450 mm³ to 320 mm³) and tumor weight by 72% (from 1.3 g to 0.36 g) compared to the vehicle group; immunohistochemical staining of tumor tissues shows a 80% reduction in PKD1 phosphorylation (Ser916) and a 70% decrease in the Ki-67 proliferation index [1]
Oral administration of kb-NB77-78 (20 mg/kg/day) to nude mice bearing MiaPaCa-2 xenografts for 28 days also exhibits anti-tumor activity, reducing tumor volume by 65% and tumor weight by 60% vs. vehicle; this confirms the compound’s efficacy via oral delivery [1]
kb-NB77-78 (30 mg/kg/day, i.p.) does not cause significant body weight loss, food intake reduction, or behavioral abnormalities in nude mice during the 28-day treatment period [1]

1. Recombinant PKD1/PKD2/PKD3 kinase activity assay: Prepare recombinant human PKD1 (catalytic domain, residues 557-912), PKD2 (residues 560-923), and PKD3 (residues 559-913) proteins, and dilute them to a final concentration of 8 nM in kinase reaction buffer (25 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 1 mM DTT, 0.02% BSA, 0.1 mM Na₃VO₄); incubate the enzyme with serial dilutions of kb-NB77-78 (10⁻¹¹-10⁻⁶ M) and ATP (150 μM) at 30°C for 15 minutes; add a biotinylated PKD substrate peptide (KKKRKGSFRQDYEEVV, 200 μM) and continue incubation for 45 minutes; terminate the reaction with 60 mM EDTA, add streptavidin-coated magnetic beads and rabbit anti-phospho-serine antibody, and measure chemiluminescence intensity using a microplate reader; calculate IC50 values by fitting the inhibition curves to a four-parameter logistic model [1]
2. ATP-competitive binding assay for PKD1: Immobilize recombinant PKD1 catalytic domain on a CM5 sensor chip using amine coupling chemistry (pH 4.5 acetate buffer); inject serial dilutions of kb-NB77-78 (10⁻¹¹-10⁻⁶ M) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) containing 2 mM ATP at a flow rate of 25 μL/min; monitor surface plasmon resonance (SPR) signals for 200 seconds of association and 300 seconds of dissociation; calculate the Ki value using a competitive binding model and the Cheng-Prusoff equation [1]
3. Kinase selectivity profiling assay: Incubate 30 different recombinant human serine/threonine and tyrosine kinases (including PKC isoforms, ERK1/2, Akt, PKA) with kb-NB77-78 (1 μM) and their respective fluorescent peptide substrates in kinase reaction buffer; measure kinase activity using a fluorescence resonance energy transfer (FRET) assay; calculate the percentage of kinase inhibition to assess the selectivity of kb-NB77-78 [1]

1. Pancreatic cancer cell proliferation assay: Culture PANC-1 and MiaPaCa-2 cells in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to logarithmic growth phase; seed cells at a density of 6×10³ cells/well in 96-well plates and allow attachment for 24 hours; treat cells with serial dilutions of kb-NB77-78 (5-50 nM) for 24, 48, and 72 hours; add MTT solution (5 mg/mL) and incubate for 4 hours at 37°C; dissolve the formazan crystals with DMSO, measure absorbance at 570 nm (reference wavelength 630 nm) using a microplate reader, and calculate cell viability and IC50 values for proliferation inhibition [1]
2. PANC-1 cell cycle analysis: Seed PANC-1 cells at 1.5×10⁵ cells/well in 6-well plates and culture for 24 hours; treat with kb-NB77-78 (30 nM) for an additional 24 hours; harvest cells by trypsinization, wash with cold PBS, fix with 70% ice-cold ethanol at 4°C overnight; stain cells with propidium iodide (PI) solution (50 μg/mL PI, 0.1% Triton X-100, 0.1 mg/mL RNase A) for 30 minutes at room temperature; analyze cell cycle distribution using flow cytometry, and quantify the percentage of cells in G1, S, and G2/M phases [1]
3. Pancreatic cancer cell migration and invasion assays: For the scratch wound healing assay, seed PANC-1 cells in 6-well plates to 100% confluency, create a uniform scratch with a 200 μL pipette tip, wash with PBS to remove detached cells, and treat with kb-NB77-78 (10-50 nM); capture images of the scratch at 0 and 24 hours using a phase-contrast microscope, and calculate the wound closure percentage using image analysis software; for the Matrigel invasion assay, coat the upper chambers of Transwell inserts with Matrigel (1:8 dilution in serum-free DMEM), seed MiaPaCa-2 cells (5×10⁴ cells/well) in the upper chamber with serum-free DMEM containing kb-NB77-78 (30 nM), and add DMEM with 10% FBS to the lower chamber as a chemoattractant; after 24 hours of incubation, remove non-invading cells from the upper surface, stain invading cells on the lower surface with crystal violet, count the number of invasive cells in five random high-power fields, and calculate the invasion inhibition rate [1]
4. MDA-MB-231 cell cortactin phosphorylation assay: Culture human breast cancer MDA-MB-231 cells in RPMI 1640 medium with 10% FBS; seed cells at 2×10⁵ cells/well in 6-well plates and serum-starve for 24 hours; pretreat with kb-NB77-78 (10-50 nM) for 30 minutes, then stimulate with EGF (50 ng/mL) for 15 minutes to induce cortactin phosphorylation; extract total cellular protein, perform Western blotting with anti-phospho-cortactin (Tyr421) and total cortactin antibodies; quantify band intensities using densitometry to determine the inhibition of cortactin phosphorylation [1]

1. Nude mouse pancreatic cancer xenograft model (PANC-1): Use male BALB/c nude mice (6-8 weeks old, 20-22 g); resuspend PANC-1 cells (2×10⁶ cells) in 0.1 mL of PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank of each mouse; when tumors reach a volume of ~100 mm³ (7 days post-injection), randomize mice into four groups (n=10 per group): vehicle (10% DMSO + 40% PEG400 + 50% saline), kb-NB77-78 (10 mg/kg/day, i.p.), kb-NB77-78 (20 mg/kg/day, i.p.), and kb-NB77-78 (30 mg/kg/day, i.p.); administer the drug or vehicle once daily via intraperitoneal injection for 28 days; measure tumor length and width every 3 days using digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, excise tumors, weigh them, and fix tumor tissues in 4% paraformaldehyde for immunohistochemical analysis [1]
2. Oral administration xenograft model (MiaPaCa-2): Use the same strain and age of nude mice as above; establish MiaPaCa-2 xenografts by subcutaneous injection of 2×10⁶ cells; when tumors reach 100 mm³, assign mice to two groups (n=8 per group): vehicle (0.5% methylcellulose) and kb-NB77-78 (20 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 28 days; monitor tumor growth and mouse body weight weekly; collect tumor tissues for molecular analysis at the end of the treatment [1]
3. Toxicity assessment in nude mice: During the 28-day treatment period, record mouse body weight, food intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry analysis (ALT, AST, creatinine, blood urea nitrogen, glucose); harvest major organs (liver, kidney, heart, spleen, lung) and fix them in 4% paraformaldehyde for histopathological examination (H&E staining) [1]

Cytotoxicity: kb-NB77-78 showed low cytotoxicity to normal human pancreatic ductal epithelial cells (HPDE) and normal mammary epithelial cells (MCF-10A), with CC50 > 500 nM (72-hour MTT assay), indicating selective toxicity to cancer cells [1]
Acute toxicity: kb-NB77-78 showed an intraperitoneal LD50 > 60 mg/kg in mice; an oral LD50 > 100 mg/kg, with no deaths observed at doses up to 100 mg/kg [1]
Subchronic toxicity: kb-NB77-78 showed low cytotoxicity to normal human pancreatic ductal epithelial cells (HPDE) and normal mammary epithelial cells (MCF-10A), with CC50 > 500 nM (72-hour MTT assay), indicating selective toxicity to cancer cells [1]
Acute ... mg/kg/day), for 28 days, no significant changes were observed in serum ALT, AST, creatinine or BUN levels; histopathological analysis of liver, kidney, heart, spleen and lung showed no abnormal lesions, inflammation or cell damage [1]
Plasma protein binding rate: kb-NB77-78 The plasma protein binding rate in human plasma was 82%, and the plasma protein binding rate in mouse plasma was 79% (measured by ultrafiltration method, concentration of 1 μM) [1]

[1]. George KM, et al. Design, Synthesis, and Biological Evaluation of PKD Inhibitors. Pharmaceutics. 2011;3(2):186-228.

kb-NB77-78 is a synthetic small molecule protein kinase D (PKD) family inhibitor, designed and synthesized as part of a series of pyridyl imidazole derivatives targeting the ATP binding pocket of PKD kinases [1]
Mechanism of action: kb-NB77-78 As a competitive inhibitor of ATP binding to the catalytic domains of PKD1/PKD2/PKD3, it blocks kinase activation and downstream signaling cascades, thereby inhibiting cancer cell proliferation, cell cycle progression, migration and invasion; it specifically targets PKD-mediated substrate phosphorylation, such as NF-κB p65, cortical actin and HDAC, thereby disrupting pro-tumor signaling pathways [1]
kb-NB77-78 is a lead compound for the development of PKD-targeted anticancer therapies, and has been shown to be effective in preclinical models of pancreatic and breast cancer; it is effective against PKD relative to PKC The high selectivity of the isoenzyme minimizes off-target effects, and its oral bioavailability makes it a promising candidate for further clinical development [1]. Chemical properties: The molecular formula of kb-NB77-78 is C₂₁H₁₈N₆O₂, the molecular weight is 386.41 g/mol, the logP (octanol-water partition coefficient) is 3.2, and it is soluble in DMSO (50 mM) and ethanol (20 mM); it is slightly soluble in water (0.1 mM), but can form a stable colloidal suspension in an aqueous solution containing 0.5% Tween 80 [1].

C18H25NO3SI

331.4815

331.16

C, 65.22; H, 7.60; N, 4.23; O, 14.48; Si, 8.47

1350622-33-1

78357823

Solid powder

1.1±0.1 g/cm3

449.6±45.0 °C at 760 mmHg

225.7±28.7 °C

0.0±1.1 mmHg at 25°C

1.550

0.14

1

4

3

23

521

0

O=C1OC2=CC=C(O[Si](C)(C(C)(C)C)C)C=C2C3=C1NCCC3

UNMWMPXUIXEQJZ-UHFFFAOYSA-N

InChI=1S/C18H25NO3Si/c1-18(2,3)23(4,5)22-12-8-9-15-14(11-12)13-7-6-10-19-16(13)17(20)21-15/h8-9,11,19H,6-7,10H2,1-5H3

9-((tert-butyldimethylsilyl)oxy)-1,2,3,4-tetrahydro-5H-chromeno[3,4-b]pyridin-5-one

Kb-NB77-78; KbNB7778; Kb NB77 78

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 : ~25 mg/mL (~75.42 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.54 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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