RAC-PK as a therapeutic agent or in diagnostics, screening method for agents and process for activating RAC-PK

a technology of rac-pk and therapeutic agents, applied in the field of rac-pk as a therapeutic agent or in diagnostics, screening methods for agents and processes for activating rac-pk, can solve the problems of unsatisfactory systems, disadvantageous use of such agents, and insulin not stimulating muscle cells and fat cells in the normal way

Inactive Publication Date: 2005-03-10
ALESSI DARIO +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many people with diabetes have normal levels of insulin in their blood, but the insulin fails to stimulate muscle cells and fat cells in the normal way (type II diabetes).
Such agents are expensive and, when it is desired to produce active kinases or to activate cells in large amounts, the use of such agents is disadvantageous.
In the case of kinases, such as those with which we are presently concerned, however, such systems are unsatisfactory because the proteins produced would be unphosphorylated and therefore inactive.

Method used

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  • RAC-PK as a therapeutic agent or in diagnostics, screening method for agents and process for activating RAC-PK
  • RAC-PK as a therapeutic agent or in diagnostics, screening method for agents and process for activating RAC-PK
  • RAC-PK as a therapeutic agent or in diagnostics, screening method for agents and process for activating RAC-PK

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0134] Specific Interaction of RAC-PK with IMPDH

[0135] a. Bacterial and Yeast Strains

[0136] All yeast strains and plasmids for two-hybrid experiments are obtained from Clontech (Palo Alto, Calif.) as components of the MATCHMAKER Two Hybrid System or from Dr. Nathans, Howard Hughes Medical Institute, Baltimore, Md. Yeast strains SFY526, e.g., MATa, Ura3-52, His3-200, Ade2-101, Lys2-801, Trp1-901, Leu2-3, 112, canr, Gal4-542, Gal80-538 and Ura3::GAL1-lacZ; HF7c, e.g., MATa, Ura3-52, His3-200, Lys2-801, Ade2-101, Trp1-901, Leu2-3, 112, Gal4-542, Gal80-538, Lys2::Gal1-His3 and Ura3::(Gal4 17-mer)3-CYC1 -LacZ; and PCY2 [see Chevray and Nathans, Proc Natl Acad Sci USA, Vol. 89, No. 13, pp. 5789-5793 (1992)], e.g., MATα, His3-200, Ade2-101, Lys2-801, Trp1-63, Leu2-3, Gal4-542, Gal80-538 and Ura3::Gal1-LacZ, are used to assay for protein-protein interactions. Yeast strain HF7c is used for library screening. SFY526 and PCY2 have the upstream activating sequence and TATA sequence of the GAL...

example 2

[0151] Inhibition of GSK3

[0152] Two major kinases known to be involved in regulating the insulin-dependent signalling pathways are MAPKAP kinase-1 and p70S6K. These kinases are respectively inhibited by PD98059 and rapamycin. Both of these agents fail to inhibit phosphorylation of GSK3, suggesting that the kinase responsible for GSK3 inactivation is not MAPKAP kinase-1 or p70S6K.

[0153] L6 myotubes are incubated with both compounds and the stimulated with insulin, as follows. Monolayers of L6 cells are grown in 6 cm petri dishes to the stage of myotubes, deprived of serum overnight and then incubated for 1 hour in 20 mM Hepes / NaOH, pH 7.4, 0.14 M NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2, 25 mM glucose (HBS buffer), in the presence or absence of 50 μM PD98059 or 100 μM LY294002. Two (2) mM 8-Br-cAMP or 0.1 μM rapamycin, when added, are included for the last 15 minutes. The cells are stimulated for 5 minutes with 0.1 μM insulin, or for time courses of up to 10 minutes. The medium is ...

example 3

[0161] To determine if RAC-PK's domain with its carboxyl-terminal extension could interact with other proteins we fused it to the Gal4 DNA binding domain and screened a HeLa cDNA library fused to the Gal4 transcriptional activation domain in the yeast reporter strain HF7c following the procedure of Example 1. Briefly, amino acids 147-480 of RACα are fused in frame to GST in the expression vector pGEX-2T (see Example 1). Appropriate BamHI-EcoRI fragment is then subcloned into the PstI-XbaI sites of yeast vector pPC62 (Dr. Nathans) using PstI-BamHI and EcoRI-XbaI linkers in order to create a Gal4 DNA binding domain-RAC-PK and C-terminal domain fusion. XhoI-XbaI fragments are then subcloned into pGBT9 (Clontech). The HeLa S3 MATCHMAKER cDNA library is used as before.

[0162] In our screen of 1.5×106 primary transformants, we identify 7 clones which show specific interaction with RAC-PK's domain plus the carboxyl-terminal extension, by activation of the reporters for His auxotrophy and La...

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Abstract

The invention concerns RAC-PK and fragments thereof, as well as activators and inhibitors of RAC-PK for use as medicaments, particularly in the treatment of diseases concerned with abnormalities in processes modulated by insulin, such as cellular proliferation, insulin deficiency and / or excess blood sugar levels. Moreover, the invention provides RAC-PK for use in screening potential mimics or modulators thereof. A method for screening for agents capable of affecting the activity of GSK3 is also disclosed. The invention further provides a screening kit comprising the RAC-PK as an active principle, and a method for screening compounds which are candidate mimics or modulators of RAC-PK activity comprising detecting specific interactions between the candidate compounds and RAC-PK. There is also provided a process for activating RAC-PK comprising treatment thereof with a phosphatase inhibitor.

Description

[0001] This application is a Continuation In Part of application (I) Ser. No.10 / 673,091, Filing Date Sep. 26, 2003, pending, which is a continuation of Ser. No. 09 / 845,667, filed Apr. 30, 2001, now abandoned, which is a continuation of Ser. No. 09 / 091,763, filed Jun. 19, 1998, now abandoned, which is a National Stage of PCT / GB96 / 03186, filed Dec. 20, 1996; (II) of application Ser. No.10 / 147,123, filed May 16, 2002, which is a continuation of Ser. No. 09 / 542,646, filed Apr. 3, 2000, now abandoned, which is a continuation of Ser. No. 09 / 091,109, filed Jun. 11, 1998, now abandoned, which is a National Stage of PCT / EP96 / 0481 1, filed November 5, 1996; and (III) of application Ser. No. 09 / 970,000, pending, which is a Continuation of Ser. No. 09 / 068,702, filed May 13, 1998, now abandoned, which is a National Stage of PCT / EP96 / 04810, filed Nov. 5, 1996. [0002] The present invention relates to the control of glycogen metabolism and protein synthesis, in particular through the use of insulin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/47C12N9/12C12Q1/42C12Q1/48
CPCA61K38/00C07K14/47C12Q1/48C12N9/1205C12Q1/42C12N9/12A61P3/00A61P35/00
Inventor ALESSI, DARIOANDJELKOVIC, MIRJANACOHEN, PHILIPCRON, PETERCROSS, DARRENHEMMINGS, BRIAN
Owner ALESSI DARIO
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