A66

Biological Activity

A66 is a novel small molecule inhibitor of PI3K p110α with IC50 values of 32, 30 and 43 nM for p110α, p110α/E545K and p110α/H1047R. A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. A66 is more than 100 fold less active against the other class-I PI 3-kinase isoforms and had not inhibitory activity against 200 protein kinases when tested at 10 micromolar. This makes it the most selective and specific p110α inhibitor available for research purposes.

Product Citations

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  Cell Commun Signal. 2022 Jul 7;20(1):102.

  Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells
  A66 purchased from AbMole

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  Front Cell Dev Biol. 2021 Aug 6;9:649656.

  PI3K Plays an Essential Role in Planarian Regeneration and Tissue Maintenance
  A66 purchased from AbMole

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  OncoImmunology 2018 Jan 1.

  A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice
  A66 purchased from AbMole

Customer Product Validations & Biological Datas

	Source	OncoImmunology (2017). Figure 4. A66 (Abmole Bioscience, Houston, TX, USA)
	Method	oral gavage
	Cell Lines	BALB/C mice
	Concentrations	100 mg/kg
	Incubation Time	3 d
	Results	Under CD8+ T cell depletion, the anti-tumor efficacy of antineu antibody alone or in combination with GDC-0941 was slightly reduced, whereas that of antibody plus A66 was completely abolished

	Source	OncoImmunology (2017). Figure 3. A66 (Abmole Bioscience, Houston, TX, USA)
	Method	sulforhodamine B assay
	Cell Lines	TUBO-P2J cells
	Concentrations	1 μM
	Incubation Time	48 h
	Results	Furthermore, A66 in combination with the antibody prolonged survival

	Source	OncoImmunology (2017). Figure 1. A66 (Abmole Bioscience, Houston, TX, USA)
	Method	oral gavage
	Cell Lines	BALB/C mice
	Concentrations	100 mg/kg
	Incubation Time	3 d
	Results	In contrast to the pan-PI3K inhibitor, a PI3K p110α isoform-selective inhibitor (A66) alone had less effect on cell viability; however, anti-neu antibody combined with A66 acted synergistically to inhibit tumor cell survival

Protocol (for reference only)

Cell Experiment
Cell lines	CD4+ T lymphocytes
Preparation method	T lymphocyte isolation and activation To obtain naive CD4+ T lymphocytes (CD4+CD62L+ T cells), spleens were passed through a 70 lm mesh. After centrifugation, red blood cells were lysed and the cells washed in culture medium (Click’s medium supplemented with 10% heat inactivated FCS). Then, naive CD4+ T cells were isolated using Miltenyi CD4+CD62L+ T cell isolation kit II for mouse cells (Ref. 130-093-227, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturers’ instructions. The isolated cells were routinely >97% CD4+, >95% CD62L+. Cells (106) were cultured in 1 ml culture medium in 24- well culture plates (Costar) pre-coated with anti-CD3 antibody (YCD3-1, 5 lg ml 1). Where indicated, anti-CD28 (H57, 2.5 lg ml 1, eBioscience, San Diego, California), DMSO or inhibitors dissolved in DMSO (1 ll per culture) were added. At 24 h, the cultures were resuspended, centrifuged and the supernatants assayed for cytokine content.
Concentrations	0~10 μ M
Incubation time	24 h

Animal Experiment
Animal models	mice bearing SK-OV-3, HCT-116 and U87MG xenograft models
Formulation	20% 2-hydroxypropyl-β-cyclodextrin (Sigma–Aldrich) in water
Dosages	10 ml/kg once daily (QD) or twice daily (BID) daily for 21 days or twice daily for 16 days (SK-OV-3), daily for 14 days (U87MG) and daily for 7 days (HCT-116)
Administration	intraperitoneal injection

Chemical Information

Molecular Weight	393.53
Formula	C17H23N5O2S2
CAS Number	1166227-08-2
Solubility (25°C)	DMSO 50 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

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.

References

[1] Aragoneses-Fenoll L, et al. Biochem Pharmacol. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

[2] Smith GC, et al. Biochem J. Effects of acutely inhibiting PI3K isoforms and mTOR on regulation of glucose metabolism in vivo.

[3] Jamieson S, et al. Biochem J. A drug targeting only p110α can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types.