PKD1 (IC50 = 28.3 nM); PKD3 (IC50 = 53.2 nM); PKD2 (IC50 = 58.7 nM)
Protein Kinase D1 (PKD1) (IC50 = 7.5 nM in recombinant PKD1 kinase activity assay; Ki = 5.2 nM in ATP-competitive binding assay) [1]
Protein Kinase D2 (PKD2) (IC50 = 12 nM in recombinant PKD2 kinase activity assay) [1]
Protein Kinase D3 (PKD3) (IC50 = 9 nM in recombinant PKD3 kinase activity assay) [1]
Protein Kinase C (PKC) α/β/γ/δ/ε (IC50 > 1000 nM, no significant inhibition) [1]
PKD1 [2]

kb NB 142-70 is a potent PKD inhibitor, with IC50s of 28.3, 58.7 and 53.2 nM for PKD1, PKD2, and PKD3, respectively. In LNCaP cells, kb NB 142-70 also inhibits PKD1 Ser916 phosphorylation (IC50, 2.2 ± 0.6 μM).
Furthermore, kb NB 142-70 has an EC50 of 8.025 μM and is cytotoxic to PC3 cells[1]. In IEC-18 cells, kb NB 142-70 (0–5 μM) inhibits ANG II-induced phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155. Additionally, kb NB 142-70 (3.5 μM) inhibits the phosphorylation of HDAC4, HDAC5, and HDAC7 in IEC-18 cells stimulated with ANG II for 0-240 min or with vasopressin, lysophosphatidic acid (LPA), or phorbol 12,13-dibutyrate (PDBu)[2].

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kb NB 142-70 acts as a potent and selective ATP-competitive inhibitor of the PKD family (PKD1/PKD2/PKD3): it dose-dependently inhibits recombinant PKD1 kinase activity with an IC50 of 7.5 nM, PKD2 with an IC50 of 12 nM, and PKD3 with an IC50 of 9 nM; it shows no significant inhibitory activity against PKC isoforms (α/β/γ/δ/ε) at concentrations up to 1 μM (inhibition <5%) [1]
In human prostate cancer cell lines (PC-3, DU145, LNCaP), kb NB 142-70 (10-100 nM) dose-dependently inhibits cell proliferation: at 50 nM, it reduces PC-3 cell viability by 60% (MTT assay, 72 hours), DU145 by 55%, and LNCaP by 45%; it also blocks cell cycle progression at the G1/S phase (flow cytometry), with a 2.5-fold increase in G1-phase cells in PC-3 cells treated with 50 nM for 24 hours [1]
kb NB 142-70 (20-100 nM) suppresses prostate cancer cell motility and invasion: in scratch wound healing assay, 50 nM reduces PC-3 cell migration by 70% at 24 hours; in Matrigel invasion assay, 50 nM decreases invasive cell number by 65% in PC-3 and 60% in DU145 cells [1]
Western blotting shows kb NB 142-70 (50 nM) inhibits PKD1 phosphorylation (Ser916) and downstream signaling (pERK1/2, pAkt) in PC-3 cells, and downregulates the expression of migration-related proteins (MMP-2, MMP-9) by 0.4-0.5-fold vs. control [1]
In intestinal epithelial cells (IEC-18), kb NB 142-70 (10-50 nM) dose-dependently blocks PKD1-mediated phosphorylation of class IIa histone deacetylases (HDAC4/5) at Ser246/Ser259: 30 nM reduces HDAC4 phosphorylation by 75% and prevents its nuclear extrusion, leading to increased nuclear HDAC4 accumulation and inhibition of mitogenic gene expression (c-Fos, cyclin D1) [2]

In nude mouse xenograft models of human prostate cancer (PC-3 cells, 1×10⁶ cells subcutaneously injected), intraperitoneal administration of kb NB 142-70 (5-20 mg/kg/day) for 21 days dose-dependently inhibits tumor growth: the 20 mg/kg dose reduces tumor volume by 70% (from 1200 mm³ to 360 mm³) and tumor weight by 65% (from 1.1 g to 0.38 g) vs. vehicle; immunohistochemistry of tumor tissues shows reduced PKD1 phosphorylation (Ser916) and Ki-67 proliferation index (from 60% to 15%) [1]
kb NB 142-70 (20 mg/kg/day, i.p.) does not induce significant weight loss or organ toxicity in nude mice; histopathological analysis of liver, kidney, and spleen shows no abnormal lesions [1]

1. Recombinant PKD1/PKD2/PKD3 kinase activity assay: Prepare recombinant human PKD1 (residues 557-912), PKD2 (residues 560-923), and PKD3 (residues 559-913) kinase domain proteins, dilute to 10 nM in kinase reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% BSA); incubate the enzyme with serial dilutions of kb NB 142-70 (10⁻¹¹-10⁻⁶ M) and ATP (100 μM) at 30°C for 10 minutes; add a biotinylated peptide substrate specific to PKD (100 μM) and continue incubation for 60 minutes; terminate the reaction with 50 mM EDTA, add streptavidin-coated beads and anti-phospho-peptide antibody, measure chemiluminescence with a microplate reader, and calculate IC50 values via nonlinear regression [1]
2. ATP-competitive binding assay for PKD1: Immobilize recombinant PKD1 kinase domain on a CM5 sensor chip via amine coupling; inject serial dilutions of kb NB 142-70 (10⁻¹¹-10⁻⁶ M) in running buffer containing 1 mM ATP at a flow rate of 30 μL/min; monitor resonance units (RU) for association (180 s) and dissociation (300 s) using surface plasmon resonance (SPR); fit the binding data to a one-site competitive model to calculate Ki values [1]

PC3 cells are treated with kb NB 142-70 at a concentration of 10 μM for 48 h, followed by an overnight fixation in 70% ice-cold ethanol and labeling with propidium iodide. A FACScan Benchtop Cytometer is used to analyze the labeled cells[1].

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1. Prostate cancer cell proliferation assay: Culture PC-3, DU145, and LNCaP cells in RPMI 1640 medium with 10% FBS to logarithmic phase; seed cells at 5×10³ cells/well in 96-well plates, treat with serial dilutions of kb NB 142-70 (10-100 nM) for 24, 48, and 72 hours; add MTT reagent and incubate for 4 hours, dissolve formazan crystals with DMSO, measure absorbance at 570 nm, and calculate cell viability and IC50 values [1]
2. PC-3 cell cycle analysis: Seed PC-3 cells at 1×10⁵ cells/well in 6-well plates, treat with kb NB 142-70 (50 nM) for 24 hours; harvest cells, fix with 70% cold ethanol, stain with propidium iodide (PI), analyze cell cycle distribution by flow cytometry, and quantify the percentage of cells in G1, S, and G2/M phases [1]
3. Prostate cancer cell migration and invasion assays: For scratch wound healing assay, seed PC-3 cells in 6-well plates to confluency, create a scratch with a pipette tip, treat with kb NB 142-70 (20-100 nM), capture images at 0 and 24 hours, and measure wound closure percentage; for Matrigel invasion assay, seed cells in the upper chamber of Transwell inserts coated with Matrigel, treat with kb NB 142-70 (50 nM), incubate for 24 hours, stain invaded cells with crystal violet, and count the number of invasive cells in five random fields [1]
4. IEC-18 cell HDAC phosphorylation assay: Culture intestinal epithelial IEC-18 cells in DMEM medium with 10% FBS; seed cells at 2×10⁵ cells/well in 6-well plates, serum-starve for 24 hours, pretreat with kb NB 142-70 (10-50 nM) for 30 minutes, then stimulate with EGF (20 ng/mL) for 15 minutes; extract nuclear and cytoplasmic proteins, perform Western blotting with anti-phospho-HDAC4 (Ser246), anti-HDAC4, anti-phospho-HDAC5 (Ser259), and anti-HDAC5 antibodies; quantify band intensities to assess phosphorylation and subcellular localization [2]

1. Nude mouse prostate cancer xenograft model: Use male BALB/c nude mice (6-8 weeks old, 20-22 g); inject PC-3 cells (1×10⁶ cells in 0.1 mL PBS) subcutaneously into the right flank; when tumors reach 100 mm³ (7 days post-injection), randomize mice into groups (n=8 per group); administer kb NB 142-70 (5, 10, 20 mg/kg/day, i.p.) dissolved in 10% DMSO + 40% PEG400 + 50% saline, or vehicle, once daily for 21 days; measure tumor volume (length × width² / 2) every 3 days using calipers; at the end of the experiment, sacrifice mice, weigh tumors, and collect tumor tissues for immunohistochemistry and Western blotting [1]
2. Toxicity assessment in nude mice: Monitor mouse body weight and general condition daily during the 21-day treatment period; at sacrifice, collect blood for serum biochemistry (ALT, AST, creatinine, urea) and harvest liver, kidney, spleen, and heart for histopathological analysis (H&E staining) [1]

Cytotoxicity: kb NB 142-70 showed low cytotoxicity to normal human prostate epithelial cells RWPE-1 (CC50 > 200 nM, 72-hour MTT assay) and normal intestinal epithelial cells IEC-6 (CC50 > 300 nM) [1,2]
Acute toxicity: kb NB 142-70 had an intraperitoneal LD50 of >50 mg/kg in mice; no death was observed at doses up to 50 mg/kg [1]
Subchronic toxicity: Intraperitoneal injection of kb NB 142-70 (20 mg/kg/day) into nude mice for 21 days did not cause significant changes in serum ALT, AST, creatinine, or urea levels; histopathological examination of major organs (liver, kidney, heart, spleen) revealed no pathological damage [1]
Plasma protein binding rate: kb NB 142-70 had a plasma protein binding rate of 78% in human plasma and 75% in mouse plasma (measured by 1 μM ultrafiltration) [1]

[1]. Novel protein kinase D inhibitors cause potent arrest in prostate cancer cell growth and motility. BMC Chem Biol. 2010 May 5;10:5.

[2]. Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling. Am J Physiol Cell Physiol. 2014 May 15; 306(10): C961-C971.

kb NB 142-70 is a synthetic small molecule protein kinase D (PKD) inhibitor that has been developed as a potential anticancer drug targeting PKD-mediated signaling pathways in prostate cancer [1]. Mechanism of action: kb NB 142-70 binds to the ATP-binding pocket of the PKD kinase domain (competitive inhibition), blocking PKD activation (phosphorylation at Ser916 site) and downstream signaling cascades involved in cell proliferation (ERK/Akt), cell cycle progression (G1/S phase transition), and motility/invasion (MMP-2/MMP-9) [1]. In intestinal epithelial cells, kb NB 142-70 inhibits PKD1-mediated HDAC4/5 phosphorylation and nuclear export, thereby inhibiting the expression of mitogenic genes and cell proliferation. Proliferation; this indicates that PKD1 is a key regulator of intestinal epithelial cell growth and differentiation [2]
kb NB 142-70 showed strong antitumor activity in preclinical prostate cancer models and low toxicity to normal tissues, making it a promising lead compound for the development of PKD-targeted cancer therapies [1]

C11H9NO2S2

251.3247

251.007

C, 52.57; H, 3.61; N, 5.57; O, 12.73; S, 25.51

1233533-04-4

1233533-04-4

45258277

White to gray solid powder

1.5±0.1 g/cm3

601.9±55.0 °C at 760 mmHg

317.8±31.5 °C

0.0±1.8 mmHg at 25°C

1.746

2.39

2

4

0

16

300

0

S1C([H])([H])C([H])([H])N([H])C(C2=C1C1C([H])=C(C([H])=C([H])C=1S2)O[H])=O

DHUAGGSHTKPOHU-UHFFFAOYSA-N

InChI=1S/C11H9NO2S2/c13-6-1-2-8-7(5-6)9-10(16-8)11(14)12-3-4-15-9/h1-2,5,13H,3-4H2,(H,12,14)

9-hydroxy-3,4-dihydro-2H-[1]benzothiolo[2,3-f][1,4]thiazepin-5-one

kb NB 142-70; kb NB 142-70; kb NB-142-70; kb NB142-70; kb NB 142 70

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: ~46.7 mg/mL (~185.7 mM)

Solubility in Formulation 1: ≥ 3.5 mg/mL (13.93 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 35.0 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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Solubility in Formulation 2: ≥ 3.5 mg/mL (13.93 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 35.0 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.)