Medical Research Council

Novartis Ag

University of Dundee

Dario Alessi

R Jeremy Nichols

Philip Cohen

Brian Arthur Hemmings

Mirjana Andjelkovic

David Komander

Darren Cross

Nicolas Dzamko

Ricardo Biondi

Daan Van Aalten

Marin Deak

Matilde Caivano

Peter David Cron

A method for assessing the effect of a test compound on LRRK2 in a cell-based system, the method comprising the steps of a) assessing the effect of exposing the cell-based system comprising LRRK2 to the test compound on the phosphorylation state of Ser910 and/or Ser935 of the LRRK2; and/or b) assessing the effect of exposing the cell-based system comprising LRRK2 to the test compound on the binding of the LRRK2 to a 14-3-3 polypeptide. The method may comprise or further comprise the step of assessing the effect of exposing the cell-based system comprising LRRK2 to the test compound on the subcellular location of LRRK2. The method is considered to be useful in assessing the effect of putative LRRK2 inhibitors in cell based systems, including in vivo systems.

Methods for identifying modulators of LRRK2

A method for identifying a compound expected to be useful in modulating, for example inhibiting, LRRK2 protein kinase activity, the method comprising the steps of (1) determining whether a test compound modulates, for example inhibits, the protein kinase activity of a LRRK2 polypeptide on a substrate polypeptide and (2) selecting a compound which modulates, for example inhibits, the said LRRK2 polypeptide protein kinase activity, wherein the substrate polypeptide comprises the sequence (W/F/R/K)(W/F/R/K)(R/K)(F/W/H/R)(Y/W/R)()(L/V/I) (R/K)(R/K)(A/Y) or (W/R)(X)(X)(F/Y/H/T)(Y/W/R)()(X)(R/T)(R)(X), where X represents any amino acid. Such a compound may be useful in treating Parkinson's Disease or Parkinsonism. The substrate polypeptide may consist or comprise the sequence RLGWWRFYLRRARQGNTKQ.

Methods of identifying LRRK2 inhibitors

A method for identifying a compound expected to be useful in modulating a LRRK2 protein kinase activity, the method comprising the steps of (1) determining whether a test compound modulates the protein kinase activity of a LRRK2 polypeptide on a substrate Ezrin/Radixin/moesin (ERM) family polypeptide and (2) selecting a compound which modulates the LRRK2 polypeptide protein kinase activity. Such a compound may be useful in treating Parkinson's Disease or Parkinsonism. A catalytically active fragment of LRRK2 is identified, requiring the GTPase, COR and kinase domains as well as the WD_40-like motif and C-terminal tail.

Methods

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.

Activation of mutated RAC-PK

The present invention provides for isolated polynucleotides encoding a human 3-phosphoinositide-dependent protein kinase, and vectors and host cells comprising the isolated polynucleotides.

Polynucleotides encoding a human 3-phosphoinositide- dependent protein kinase

A method for selecting a compound for modulating the activity of phosphoinositide dependent protein kinase 1 (PDKI) is provided. The method may comprise modelling a three dimensional structure of a plurality of molecules in a computer, comparing with the three dimensional structure of the compounds with that of a reference structure such as at least part of a protein kinase catalytic domain of PDK1, and selecting the compound based on a predicted interacting ability of the molecules to the protein kinase catalytic domain. Also a method for selecting a compound for modulating the activity of hydrophobic pocket containing protein kinase is provided. In this method, the reference structures may be one or more of a phosphate binding pocket of PDK1, a hydrophobic pocket of PDK1, and &#x3b1;C helix or region interacting therewith of PDK1.

Method for designing a compound based on the three dimensional structure of phosphoinositide dependent protein kinase 1 (PDK1)

The present invention provides for a substantially pure human or rabbit 3-phosphoinositide-dependent protein kinase.

3-phosphoinositide-dependent protein kinase

A substantially pure 3-phosphoinositide dependent protein kinase that phosphorylates and activates protein kinase B&#x3b1;. A recombinant polynucleotide encoding a protein kinase as defined. Uses of the protein kinase and polynucleotide, especially in screening for new drugs.

Enzyme

Substantially pure two kinase domain protein kinases comprising the amino acid sequences provided in the description, variants, fusions, fragments, or derivatives thereof useful in screening assays for drugs. Applications thereof in the modulation of CREB, COX2 and IL2 activities.

Polypeptides, polynucleotides and uses thereof

A substantially pure 3-phosphoinositide dependent protein kinase that phosphorylates and activates protein kinase B&agr;. A recombinant polynucleotide encoding a protein kinase as defined. Uses of the protein kinase and polynucleotide, especially in screening for new drugs.

Growing community of inventors

Dundee, United Kingdom

Dario Alessi