LY333531 hydrochloride

Biological Activity

LY333531 hydrochloride competitively and reversibly inhibits PKCβI and PKCβII (IC50 values are 4.7 and 5.9 nM respectively). Selective for PKCβ over other PKC isozymes (IC50 values are 0.052, 0.25, 0.30, 0.36, 0.60 and >100 μM for PKCη, -δ, -γ, -α, -ε and -ζ respectively). LY333531 exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase).

Product Citations

• 

  J Mol Cell Cardiol. 2015 Oct;138-47.

  Irisin improves endothelial function in type 2 diabetes through reducing oxidative/nitrative stresses.
  LY333531 hydrochloride purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Journal of Molecular and Cellular Cardiology (2015). Figure 7.LY333531 (Abmole Company, Shanghai, China)
	Method	Western blot
	Cell Lines	HUVECs
	Concentrations	5 µM
	Incubation Time	24 h
	Results	We found that PKC-β inhibitor LY333531 significantly suppressed augmented gp91phox expression.

Protocol

Cell Experiment
Cell lines	HUVECs
Preparation method	Western blot Aortas and HUVECs were lysed in lysis buffer or membrane and cytosol protein extraction kit and total proteins in the supernatant were extracted to perform Western blot analysis. Equal amounts of proteins were separated through electrophoresis on SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, MA, USA). After blocked with 5% skim milk, the membranes were incubated with the appropriate primary antibodies respectively against PKC-β (Abcam, CA, USA), and β-actin (Zhong Shan Golden Bridge Biotechnology, Beijing, China) with recommended dilution at 4 °C overnight. The membranes were washed and further incubated with corresponding secondary antibodies [Anti-Rabbit IgG; Anti-mouse IgG (Zhong Shan Golden Bridge Biotechnology, Beijing, China)] with horseradish peroxidase (HRP) conjugation at 37 °C for 60 min. The films were developed using an ECL Plus chemiluminescence reagent kit (Millipore, MA, USA) and visualized using ChemiDocXRS (Bio-Rad Laboratory, CA, USA). Densitometries were analyzed using Quantity One software.
Concentrations	5µM
Incubation time	24h

Animal Experiment
Animal models	Male Sprague-Dawley rats diabetes models
Formulation	dissolved in citrate buffer (0.1 mol/L, pH 4.5)
Dosages	1 mg/kg/day
Administration	oral gavage for 4 weeks

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta.
Jirousek MR, et al. J Med Chem. 1996 Jul 5;39(14):2664-71. PMID: 8709095.