Non-peptidic cyclophilin binding compounds and their use

Details Non-peptidic cyclophilin binding compounds and their use Non-peptidic cyclophilin binding compounds and their use

2000-09-07

2004-01-13

Raymond, Richard L. (Department: 1624)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Nitrogen containing other than solely as a nitrogen in an...

C514S009100, C514S011400, C514S019300, C514S604000, C514S605000, C514S616000, C514S619000, C514S621000, C514S622000, C514S626000, C514S629000, C514S688000, C514S689000, C514S718000, C514S731000, C530S317000, C564S086000

Reexamination Certificate

active

06677376

ABSTRACT:

This invention relates to the biochemistry of cyclophilin proteins, in particular, compounds which interact with or bind such proteins. Cyclophilins (CyP), which bind cyclosporin A, and FK-506 binding proteins (FKBP), which bind FK-506 and rapamycin, are both subclasses of a group of proteins termed immunophilins. Immunophilins were first identified as proteins that bind to the immunosuppressive drugs cyclosporin A, FK-506, and rapamycin. CyPs and FKBPs can also be separated based on their differing structures.
By studying the binding of test compounds to cyclophilin proteins, the inventors have identified a number of new compounds that effect the growth and health of cells in the nervous system. Building on this initial identification, the inventors developed and utilized screening procedures for rapidly identifying additional, similarly active compounds. These compounds have been specifically tested to show that they protect neuronal cells from otherwise lethal treatments, and/or that they promote the growth or regeneration of neuronal cells. In part, the invention provides compounds that interact with or bind to a cyclophilin and compounds that have activity towards neuronal cells. The compounds can be used in a variety of ways, including therapeutic and research and development applications for a number of diseases associated with neuronal degeneration.
Cyclophilin was first identified as the receptor for cyclosporin A, a potent immunosuppressive drug that is still widely used to prevent immunological rejection of transplanted tissue. The effects of the cyclosporin A:cyclophilin interaction have been well documented. Cyclosporin A binds with a dissociation constant in the range of 10
−8
mol/L, a value representing a relatively high degree of attraction (Handschumacher et al.,
Science
226:544 (1984)). While the present invention is not bound by any particular theory, it appears the complex formed between CyP and cyclosporin A exerts the effects on the organism and cells, which leads to immunosuppression. The complex interacts with the cellular enzyme calcineurin, a calmodulin-dependent phosphatase, and the interaction prevents T cell activation by blocking RNA transcription of the T cell growth factor interleukin 2 (IL-2). (Palacios,
J. Immunol.
128:337 (1982)). Without IL-2 to cause T cell proliferation, specific T cell populations cannot mount a strong immune response, resulting in immunosuppression.
A number of types of mammalian cyclophilins have been identified and cloned, cyclophilins A, B, C, D, and cyclophilin-40 (Snyder and Sabatini,
Nat. Med.
1:32-37 (1995); Friedman et al.,
Proc. Natl. Acad. Sci.,
90:6815-6819 (1993)). Cyclophilin A is a 19 kD protein, which is abundantly expressed in a wide variety of cells. Like the other cyclophilins, cyclophilin A binds the immunosuppressive agent cyclosporin A and possesses peptidyl-prolyl cis-trans isomerase (PPIase) and protein folding or “chaperone” activities. PPIase activity catalyzes the conversion of proline residues in a protein from the cis to the trans conformation (Fischer, et al.,
Biomed. Biochem. Acta
43:1101-1112 (1984)). Cyclophilin B possesses an N-terminal signal sequence that directs translocation into the endoplasmic reticulum of the cell. The 23 kD cyclophilin C is found in the cytosol of the cell. Cyclophilin D, at 18 kD, appears to target its actions in the mitochondria. And cyclophilin-40 is a component of the inactivated form of a glucocorticoid receptor.
Immunophilins were discovered because of their interaction with known therapeutic drugs. Thus, knowledge about the interaction between drug and protein spawned a number of drug discovery efforts. Initially, the focus was on identifying new immunosuppressive drugs. A number of facts have influenced the search for improved immunosuppressive drugs. One factor was the importance of proline. The native substrate for the PPIase activity in cells is the amino acid proline in a protein. Cyclophilins A-D all contain a conserved proline binding site. The conversion between the cis and trans forms of proline, which PPIase performs, allows a protein to change shape and fold properly.
However, the first identified ligand for cyclophilins, cyclosporin A, which is a cyclic peptide, does not contain a proline. Both FK-506 and rapamycin, which bind FKBP, are also cyclic non-peptidic macrolide antibiotics. The FKBP proteins also possess PPIase activity, although the FKBPs share no significant sequence homology to CyPs. Since FK-506 is a more potent immunosuppressive compound than cyclosporin A, a number of analogs of FK-506 have been developed. So, the cyclic structure also became an important factor in designing potential new drugs.
Later, therapeutic applications in the nervous system were identified (Lyons et al.,
PNAS
91:3191-3195 (1994)). A number of animal models have proven the effectiveness of FKBP ligands in promoting nerve regeneration and nerve growth. (See, for example, Steiner et al.,
PNAS
94:2019-2024 (1997); Hamilton et al.,
Bioorg. Med. Chem. Lett.
7:1785-1790 (1997); Gold et al.,
Experiment. Neurol.
147:269-278 (1997); and Wang et al.,
J. Pharm. Exp. Therap.
282: 1084-1093 (1997).) However, whether or not ligands specific for CyP possess similar activity in the nervous system has been controversial (Hamilton and Steiner,
J. Med. Chem.
41:5119-5143 (1998); Gold,
Mol. Neurobiol.
15:285-306 (1997); and Carreau et al.,
Neuropharmacol.
36:1755-62 (1997)). Earlier published work by some of the inventors showed how compounds with an affinity for the cyclophilin immunophilins can be useful in effecting neuronal activity (PCT published applications WO 97/18828 and WO 98/25950). The work of the present invention further demonstrates that ligands specific for CyP are active in the nervous system and expands on the earlier work by providing additional structural and functional aspects.
Researchers have also noted a functional association of cyclophilin A with the Gag protein of the HIV virus (Thali et al.,
Nature
372:363-365(1994)). This has taken drug development approaches in a new direction (See, for example, U.S. Pat. No. 5,767,069). Many researchers now seek to develop drugs that target the interaction between cyclophilin A and Gag in order to disrupt the HIV life cycle (Sternberg,
BioWorld Today
7:1 (1996)).
SUMMARY OF THE INVENTION
The invention provides a number of compounds that bind to CyP proteins as well as compounds that are structurally or functionally related to those specifically described and shown. The compounds of this invention preferably do not suppress the immune system and preferably do not possess a biological activity involving binding to a FKBP, i.e., the compounds have an IC
50
greater than 10 &mgr;M towards FKBP. A number of methods for determining the binding to CyPs are presented and so are a number of ways for exploiting the binding through in vitro and in vivo methods and uses. Preferred compounds function to promote or affect neuronal cell growth or growth of nervous system cells, regenerate damaged or diseased neurons, or protect neurons or neuronal cells from damage. Furthermore, aspects of this disclosure can be used in methods to identify and isolate additional CyP binding compounds or additional uses of the compounds.
The invention also provides a number of uses for these compounds, including uses that comprise the step of allowing the compound to contact an immunophilin protein. A variety of permutations of this method can be devised. In particular, the compounds can be used to affect neuronal cells, either in culture or in an animal. Thus, the compounds can be administered to cells or animals to affect a number of conditions associated with the decline, damage, or degeneration of nervous system cells or function.
In one aspect, this invention provides compounds of Formula I and Formula II, shown and described below.
where n in C
n
is 0 or 1;
the dashed bond symbol represents an optional bond;
X and Y may independently be N, NH, O, S, or a direct bond;
R
1
is the same or different fr

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