Inhibitors of human phosphatidyl-inositol 3-kinase delta

An alkylene, unsubstituted technology applied in the field of phosphatidylinositol 3-kinase, which can solve the problem of low activity

Inactive Publication Date: 2011-01-19
ICOS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention provides novel compounds that are PI3K delta inhibitors that are highly selective for delta isoforms and less active against other PI3K isoforms

Method used

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  • Inhibitors of human phosphatidyl-inositol 3-kinase delta
  • Inhibitors of human phosphatidyl-inositol 3-kinase delta
  • Inhibitors of human phosphatidyl-inositol 3-kinase delta

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0229] Preparation and purification of recombinant PI3Kα, PI3Kβ and PI3Kδ

[0230] Use BAC-to-BAC The HT baculovirus expression system (GIBCO / BRL) overexpresses a recombinant PI3K heterodimer complex consisting of the p110 catalytic subunit and the p85 regulatory subunit, which is then purified for biochemical analysis. The four class I PI3-kinases were cloned into the following baculovirus vectors:

[0231] p110δ: Using standard recombinant DNA techniques, the FLAG -tagged human p110δ (SEQ ID NO: 1) (see Chantry et al., J Biol Chem, 272: 19236-41 (1997)) was subcloned into BamH1 of the insect cell expression vector pFastbac HTb (Life Technologies, Gaithersburg, MD) -Xba1 site, so that the clone is in frame with the His tag of the vector. The FLAG is described in US Patent Nos. 4,703,004, 4,782,137, 4,851,341 and 5,011,912 system, and reagents were obtained from Eastman Kodak Co.

[0232] p110α: Similar to the method used above for p110δ, the FLAG -tagged human p110α...

Embodiment 2

[0249] High Throughput Screening (HTS) and Selectivity Analysis of PI3Kδ

[0250] A high-throughput screen of a proprietary chemical library was performed to identify candidate inhibitors of PI3Kδ activity. PI3Kδ catalyzes phosphorylation from γ-[ 32 P]ATP is transferred to the PIP 2 PIP on the D3' site of the aliphatic ring of inositol 2 / PS liposomes. The reaction depends on MgCl 2 and ended in high molar potassium phosphate buffer, pH 8.0, containing 30 mM EDTA. In said screening, the reaction is performed in the presence or absence of library compounds. Reaction products (and all unlabeled products) were transferred to 96-well pre-wet PVDF (polyvinylidene (di)fluoride) filter plates, filtered, and washed in high molarity potassium phosphate. Scintillant was added to the dried wells and the overall radioactivity quantified.

[0251] Most analytical operations use BIOMEK A 1000 robotic workstation (Beckman) was performed and all plates were read using the Wallac liq...

Embodiment 3-7

[0260] Because PI3Kδ is expressed at significant levels in leukocytes, it is important to study the effects of selective inhibitors of PI3Kδ on leukocyte function. Accordingly, the effect of PI3Kδ inhibitors in several types of leukocytes was examined. Neutrophils were examined to determine the possible role of selective inhibition of PI3K[delta] (Example 3 below). It was surprisingly found that selective inhibition of PI3Kδ activity was significantly associated with inhibition of some, but not all, functions of activated neutrophils. In addition, the effects of PI3K[delta] inhibitors on B cell and T cell function were analyzed (Examples 4-5 below). Furthermore, since PI3Kδ is also expressed in osteoclasts, the effect of PI3Kδ inhibition on these specific cellular functions was also investigated (Example 6 below). The effect of PI3K[delta] on basophil function was also investigated (Example 7 below).

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Abstract

Compounds that inhibit PI3Kd activity, including compounds that selectively inhibit PI3Kd activity, are disclosed. Methods of inhibiting phosphatidylinositol 3-kinase delta isoform (PI3Kd) activity, and methods of treating diseases, such as disorders of immunity and inflammation in which PI3Kd plays a role in leukocyte function, using the compounds also are disclosed.

Description

technical field [0001] The present invention relates to phosphatidylinositol 3-kinase (PI3K), and more particularly to selective inhibitors of PI3Kδ activity, and to methods of using such inhibitors. Background technique [0002] 3'-phosphorylated phosphoinositides are involved in cellular signaling in a variety of cellular processes, such as malignant transformation, growth factor signaling, inflammation and immunity (see Rameh et al., J. Biol Chem, 274: 8347-8350 (1999)). The enzyme responsible for the production of these phosphorylated signal products is phosphatidylinositol 3-kinase (PI3-kinase; PI3K). Initially, PI3K was identified to have activities associated with viral oncogene proteins and with growth factor receptor tyrosine kinases capable of mobilizing phospholipids at the 3'-hydroxyl end of the inositol ring Inositol (PI) and its phosphorylated derivatives are phosphorylated (Panayotou et al., Trends Cell Biol 2:358-60 (1992)). [0003] Treatment of cells wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D487/04C07D487/02C07D403/08C07D403/12
CPCC07D519/00A61P1/04A61P1/16A61P11/00A61P11/02A61P11/06A61P11/08A61P13/12A61P17/02A61P17/04A61P17/06A61P19/00A61P19/02A61P19/06A61P19/08A61P21/00A61P25/00A61P27/02A61P27/14A61P27/16A61P29/00A61P31/04A61P35/00A61P35/02A61P37/00A61P37/02A61P37/06A61P37/08A61P43/00A61P5/14A61P7/04A61P9/00A61P9/08A61P9/10A61P9/12A61P9/14A61P3/10
Inventor S·L·怀特F·阮E·A·凯西奇E·托尔塞特F·法鲁兹
Owner ICOS CORP
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