PP 1

IC50: A potent and selective inhibitor of Src-family tyrosine kinases, with an IC50 of 5 and 6 nM respectively for p56lck and p59fynT.

PP1 inhibits Src-family tyrosine kinases effectively and selectively. The non-receptor protein tyrosine kinase Src is a crucial enzyme in the signal transduction pathways involved in cell division, motility, adhesion, and survival. Src is regarded as a promising target for cancer therapy due to its importance in tumor progression, invasion, transition, angiogenesis, and the development of metastasis. [1]

In vitro: It was reported that PP1 specifically inhibited the expression and activity of Lyn, a Src family kinase, in RBL-2H3 cells. Based on the immune-complex kinase assays in vitro, PP1 suppressed the activity of Lyn at nanomolar levels without any effect on Syk kinase activity. In contrast, phosphorylation of both Syk and Lyn kinases were both blocked in RBL cells. FcεRI- and Thy-1-mediated early and late activation events were also interrupted by PP1 in a similar mannar. Moreover, PP1 was found to inhibited RET-derived oncoproteins with IC50 of 80 nM. RET/PTC3-transformed cells received PP1 treatment with a dose of 5 μM lost proliferative autonomy and showed morphological reversion. [2, 3]

In vivo: Under in vivo conditions PP1 was suggested to suppress tyrosine phosphorylation and proliferation in T cells stimulated with anti-CD3 and mitogen. Studies using mice tumor model also showed that PP1 upregulated the expression of the IL-2 gene rather than the granulocyte macrophage colony-stimulating factor or the IL-2 receptor genes. Based on these, PP1 could be adopted as a useful agent to investigate the role of Lck and Fyn T cell activation. [2]

Clinical trial: So far, no clinical trial has been conducted.

References:
[1]Sen B and Johnson FM.  Regulation of src family kinases in human cancers. J Signal Tr. 2010 Feb; doi:10.1155/2011/865819.
[2] Amoui M, Driber P and Driberovi L.  Src family-selective tyrosine kinase inhibitor, PP1, inhibits both FcERI- and Thy-1-mediated activation of rat basophilic leukemia cells. Eur. J. Immunol. 1997. 27: 1881-1886.
[3]Carlomagno F, Vitagliano D, Guida T, Napolitano M, Vecchio G, Fusco A, Gazit A, Levitzki A and Santoro M.  The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes. Cancer Res. 2002 Feb. 62: 1077–82

The specificities of protein kinase inhibitors: an update.[Pubmed:12534346]

Biochem J. 2003 Apr 1;371(Pt 1):199-204.

We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM).

The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases.[Pubmed:12475982]

J Biol Chem. 2003 Feb 14;278(7):4847-53.

4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit. While investigating the role of Src kinases in SCF signaling, we found that PP1 completely abrogated the proliferation of M07e cells in response to SCF. PP1 inhibited SCF-induced c-Kit autophosphorylation in intact cells and blocked the activation of mitogen-activated protein kinase and Akt. In vitro kinase assays using immunoprecipitated c-Kit confirmed direct inhibition by PP1. SCF-induced c-Kit phosphorylation was also inhibited by the related inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) and by STI571 but not by the Src inhibitor SU6656. PP1 inhibited the activity of mutant constitutively active forms of c-Kit (D814V and D814Y) found in mast cell disorders, and triggered apoptosis in the rat basophilic leukemia cell line RBL-2H3 that expresses mutant c-Kit. In addition, PP1 (and PP2) inhibited the in vitro kinase activity and autophosphorylation in whole cells of p210 Bcr-Abl. PP1 reduced the constitutive activation of signal transducer and activators of transcription 5 and mitogen-activated protein kinase and triggered apoptosis in FDCP1 cells expressing Bcr-Abl. These results have implications for the use of PP1 in investigating intracellular signaling and suggest that PP1 or related compounds may be useful in the treatment of malignant diseases associated with dysregulated c-Kit or Abl tyrosine kinase activity.

The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.[Pubmed:11861385]

Cancer Res. 2002 Feb 15;62(4):1077-82.

Oncogenic activation of the RET receptor tyrosine kinase is common in different human cancers. We found that the pyrazolo-pyrimidine PP1 inhibited RET-derived oncoproteins with a half maximal inhibitor concentration of 80 nM. Furthermore, RET/PTC3-transformed cells treated with 5 microM of PP1 lost proliferative autonomy and showed morphological reversion. PP1 prevented the growth of two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements and blocked anchorage-independent growth and tumorigenicity in nude mice of NIH3T3 fibroblasts expressing the RET/PTC3 oncogene. These findings suggest targeting RET oncogenes with PP1 or related compounds as a novel treatment strategy for RET-associated neoplasms.

Structural basis for selective inhibition of Src family kinases by PP1.[Pubmed:10467133]

Chem Biol. 1999 Sep;6(9):671-8.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.

Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.[Pubmed:8557675]

J Biol Chem. 1996 Jan 12;271(2):695-701.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.