Drug screen targeting lipoprotein-membrane anchorage

Details Drug screen targeting lipoprotein-membrane anchorage Drug screen targeting lipoprotein-membrane anchorage

2000-06-07

2002-12-10

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007230, C435S007900, C435S015000, C435S021000, C436S063000, C436S503000, C436S504000

Reexamination Certificate

active

06492128

ABSTRACT:

This invention relates to screening assays. Specifically, this invention relates to screening assays to identify compounds that effect signal transducing protein phosphorylation cascades by targeting the membrane anchor of long chain fatty acid modified trigger proteins. More specifically, this invention relates to drug screens to identify drugs that effect ligand mediated signal transducing protein phosphorylation cascades (for example; Ras/Rho mediated signal transducing protein phosphorylation cascades) by effecting the long chain fatty acid dependent membrane anchorage of long chain fatty acid modified trigger proteins (for example; prenylated or palmitoylated trigger proteins).
BACKGROUND OF THE INVENTION
Multicellular organisms have a multiplicity of specialized cells functioning in cooperation. Communication in the organism to coordinate cooperative functions is mediated by a number of well known messengers, for example: hormones, neurotransmitters, cytokines, etc. The messengers communicate by binding a receptor at the cell membrane. The receptor binding activates a desired cell function. Alternatively, and/or additionally, cell functions are activated in response to signals generated and received intracellularly. The identification and characterization of the molecules involved, and the elucidation of mechanisms whereby such molecules cooperatively regulate the functions and link the signals to those functions, is one of the great scientific advances of the past few decades.
Membrane associated receptors are exquisitely specific for ligands (signal molecule) such as hormones. Any one cell exhibits a multiplicity of these receptors, the composition of which defines the types of signals to which that cell responds functionally. The receptors are often associated within the membrane superstructure with trigger proteins. When such a receptor binds the signal molecule it activates the trigger protein. These trigger proteins are in turn linked to initiate a cascade of regulatory molecules leading to the appropriate function of the cell that is activated by the hormone. A review of such a receptor→trigger→cascade→function phenomenon is provided in Denhardt, D. T.,
Signal
-
transducing Protein Phosphorylation Cascades Mediated by Ras/Rho Proteins in the Mammalian Cell: The Potential for Multiplex Signaling,
318 Biochem. J. 729-747 (1996) (“Denhardt”), the entirety of which is incorporated herein by reference.
Each trigger protein associates with specific membranes in the cell and the activity, and also often the specificity, of the protein is dependent on the trigger protein being associated with the correct membrane. To accomplish this the protein is often modified by one or more long chain fatty acids. Sometimes the protein is also carboxymethylated, glycosylated, cleaved, etc. but it appears that the most essential modification for directing many proteins to insert into the proper membrane is the long chain fatty acid modification(s).
One significant example set are cellular Ras proteins, extensively studied because mutated forms, oncogenic Ras proteins, are involved in the generation of many types of cancer. Ras is modified at its carboxy-terminus with a prenyl group and at a mid-chain Cys with a palmitoyl group. Conceptually, for Ras to be active it must be prenylated and anchored in the membrane. The prenylation of such trigger proteins as Ras, and many other related proteins, is reviewed in Omer, C A, and J. B. Gibbs,
Protein Prenylation in Eukaryotic Microorganisms: Genetics, Biology, and Biochemistry,
11 Molecular Microbiology 219-225 (1994) (“Omer & Gibbs”), the entirety of which is incorporated herein by reference.
In brief, referring to
FIG. 1
, an immature trigger protein
1
is processed in the cell by the addition of a prenyl group
2
to a C-terminal cysteine. This prenylation reaction, catalyzed by a prenylprotein-transferase
3
, is one of the processing reactions that results in the production of a mature prenylated trigger protein
4
. Prenylated trigger protein
4
attaches to a trigger protein anchorage site
6
in a cellular membrane
5
resulting in a membrane anchored prenylated trigger protein
4
′.
Membrane anchored prenylated trigger protein
4
′ is stable and fully mature, yet remains inactive until an effector
7
activates it via a signal
8
acting on membrane receptor
6
. Signal
8
can arise on either face of the membrane or can be both intracellular and extracellular. Such a signal can be activating or inactivating. If activating, as illustrated in this figure, membrane anchored prenylated trigger protein
4
′ becomes an activated prenylated trigger protein
9
. Activated prenylated trigger protein
9
has an enzymatic or binding activity that launches a regulatory cascade
10
of reactions. The reactions of regulatory cascade
10
link activated prenylated trigger protein
9
to a cellular function
11
. Cellular function
11
consists of one or a set of cellular actions that define the specialized function of the activated cell. This cascade activation system allows a single trigger event to regulate multiple cell actions that must work in concert to accomplish a particular cell function, such as cellular growth or division.
There are numerous examples of signal transducing regulatory trigger pathways of the type generalized in FIG.
1
. Denhardt defines various mammalian trigger proteins (including the gene products of Ras superfamily, and other G-proteins or GTP-binding proteins) and associated function cascades.
Briefly:
the Ras family, including H-ras, N-ras, Ka-ras, and Kb-ras, partition to specific membranes depending on the type of prenylation and, among other functions, regulate cell cycling and adhesion (mutations of ras, known as oncogenic ras, are involved in the formation of some types of tumors);
the Rap family, found in granules of Golgi and ER antagonize ras function (Rap 1A, also known as Krev-1, antagonizes the Kras oncogene);
the Ra1 family, including Ra1-a and Ra1-B appear to regulate the activity of exocytic and endocytic vesicles;
the Rho family, which includes Rho-A, Rho-B, Rho-C, Rac-1, Rac-2, CDC42, Rho-G, and TC1O, play dynamic roles in the regulation of the actin cytoskeleton and focal contacts mediating formation of filopodia and lamellipodia (Rac also controls NADPH oxidase activity in phagocytes);
the Ran proteins are involved in the transport of RNA and protein across the nuclear membrane;
the ARF/SAR proteins are important for vesicle formation and budding;
and the large and extensively studied
Rab/Ypt family are involved both in regulating intracellular vesicle trafficking between donor and acceptor membrane-enclosed compartments and in controlling the exocytosis and endocytosis of different types of vesicles.
In addition to the trigger protein—cascade systems reviewed in Denhardt, other systems have also been described and extensively studied. For example, a family of myristoylated proteins are described that have a myristoyl group (C14:0) covalently attached, via amide linkage, to the NH
2
-terminal glycine residue of certain cellular and viral proteins. The attachment is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) as a cotranslational modification. Compounds that block NMT activity have been shown to be potentially useful as anti-fungal, anti-viral, and anti-parasitic agents, and are therefore postulated to be useful for treating intracellular pathogens. A list of references and U.S. Patents detailing the myristoylation trigger protein system are found in table 1 each the entirety of which is incorporated herein by reference.
It appears that in order for most trigger proteins to become mature and capable of regulating a function cascade they must be long chain fatty acid modified and associated with the appropriate cell membrane anchor. Omer & Gibbs describe modification by prenylation of Ras superfamily encoded proteins, wherein, three enzymes are identified that mediate prenylation; farnesyl protein transferase, geranylgeranyl protein transfera

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