Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2000-02-24
2003-10-07
Solola, Taofiq (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S452000, C514S460000, C546S285000, C549S228000, C549S273000
Reexamination Certificate
active
06630492
ABSTRACT:
The present invention relates to compounds which bind to all or parts of the active binding “south pole pocket” of the LFA-1 I-domain and their uses as LFA-1 antagonists.
The lymphocyte function associated antigen LFA-1 belongs to the &bgr;2-integrins and plays an important role in T-cell activation and extravasation. Interactions of LFA-1 with its counter-receptors on endothelial and antigen presenting cells such as ICAM-1 or ICAM-3 are an important process in leucocyte endothelial cellular adhesion and migration which mediates disorders or diseases, e.g. autoinmuune diseases, inflammation, ischemia/reperfusion injury and graft rejection after transplantation.
The so-called I-domain (Inserted Domain) of LFA-1 comprises a module of about 190 amino acids (Takada et al., Matrix Biology, 16, 143-151, 1997). The I-domain folds into a common structural motif comprising a central &bgr;-sheet surrounded by helices as determined by X-ray crystallography. (A. Qu & D. Leahy, Proc. NatI. Acad. Sci. USA, 92, 10277-10281, 1995).
Compactin and Mevinolin are fungal metabolites which have following formula:
They are disclosed e.g. by Y. Chapleur in “Progress in the Chemical Synthesis of Antibiotics and Related Microbial Products”, Springer Verlag, 1993, vol. 2, 829-937.
Mevinolin and most of the known analogues, e.g. pravastatin, mevastatin, simvastatin etc. have been found to be useful e.g. as 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG CoA R) inhibitors.
In accordance with the present invention, it has now surprisingly been found that mevinolin and derivatives thereof bind to LFA-1. Accordingly, the invention provides compounds for use in the treatment or prevention of autoimmune diseases, inflammation, ischemia/reperfusion injury and graft rejection which are preferably specific or substantially specific LFA-1 binding molecules, e.g. specific or substantially specific inhibitors of LFA-1/ICAM-1 or ICAM-3 interactions. Such compounds are preferably other than LFA-1 antibodies.
More particularly, it has been found that mevinolin binds to the LFA-1 I-domain between the C-terminal helix &agr;7 and one side of the &bgr;-sheet (hereinafter the “south pole pocket”). X-ray analysis of the complex of LFA-1 I-domain with mevinolin shows that mevinolin does not bind to the MIDAS-site (“metal ion dependent adhesion site”).
The complex, LFA-1 I-domain/mevinolin, is prepared by adding mevinolin (100 mM solution in DMSO) to the protein solution (12.7 mg/ml, 100 mM MgSO
4
), followed by crystallization. The structure is solved by molecular replacement (using the coordinates of apo LFA-1 I domain, A. Qu & D. Leahy, above) and has been refined to a R factor of 19.4% (R
free
=25.9%) using X-ray amplitudes in the resolution range 8 Å-2.6 Å. The final model contains 2×182 amino acids (amino acid residues 128 to 309 of the &agr;-chain of LFA-1 which corresponds to the I-domain), 2 mevinolin molecules and a total of 86 water molecules.
Data collection statistics
LFA-1 I-domain/Mevinolin
Temperature
293K
Wavelengh
1.5418Å
Resolution range
15.0Å-2.60Å
Spacegroup
P2
1
2
1
2
1
Unit cell dimensions
a = 72.7Å, b = 77.7Å, c = 91.8Å
Measurements used
87179
Unique reflections
16457
Completeness
99.9% (99.8% in shell 2.69Å-2.60Å)
Multiplicity
5.3 (5.2)
Average I/sig(I)
13.9 (2.1)
Rmerge
11.6% (48.4%)
The south pole pocket is a cavity between one side of the central &bgr;-sheet (amino acids of &bgr;
1
, &bgr;
3
, &bgr;
4
, &bgr;
5
, preferably the side chains of such amino acids) and the &agr;-helices &agr;
1
, &agr;
7
(secondary structure of LFA-1 I-domain). Preferably the south pole pocket is the cavity defined by amino acids Val 130, Leu 132, Phe 134, Phe 153, Val 157, Leu 161, Tyr 166, Thr 231, Val 233, Ile 235, Ile 255, Tyr 257, Ile 259, Lys 287, Leu 298, Glu 301, Leu 302, Lys 305, particularly Leu 132, Phe 153, Val 157, Val 233, Ile 235, Tyr 257, Ile 259, Lys 287, Leu 298, Glu 301, Leu 302, Lys 305 of LFA-1 I-domain, more particularly by the side chains of such amino acids. In this pocket, the non-hydrogen atoms of mevinolin preferably interact within a distance of <5 Å, particularly 4-4.5 Å.
The complex south pole pocket/mevinolin is energetically favored by hydrophobic, van der Waals and/or electrostatic interactions and possibly also by indirect hydrogen bonding.
As it will be appreciated, there are 2 complexes LFA-1 I-domain/mevinolin per asymemetric unit which are related by a non-crystallographic two fold axis.
As a result of its shape the south pole pocket as defined above favorably associates not only with mevinolin but with other chemical entities or ligands. Such entities or compounds are LFA-1 inhibitors or LFA-1/ICAM-1 or ICAM-3 interaction inhibitors.
The present invention provides any chemical entity or ligand, which binds in whole or in part to the south pole pocket of LFA-1 I-domain as defined above. Preferably the chemical entity or ligand interacts within a distance <5 Å, particularly 4-4.5 Å. Suitable examples of such chemical entities include e.g. mevinolin derivatives. The elucidation of the mevinolin binding interactions on the LFA-1 I-domain south pole pocket provides the necessary information for designing new chemical entities and compounds that may interact in whole or part with the south pole pocket. Thus, the present invention permits the use of molecular design techniques e.g. computer modeling techniques, as a means of identifying, selecting and designing chemical entities or compounds capable of binding to the south pole pocket.
The design of compounds that bind to the south pole pocket according to the invention generally involves consideration of two factors. First, the entity must be capable of physically and structurally associating with parts or all of the south pole pocket. Non-covalent molecular interactions important in this association include hydrophobic, van der Waals interactions, hydrophobic interactions and/or electrostatic interactions and possibly also hydrogen bonding.
Second, the entity must be able to assume a conformation that allows it to associate with the south pole pocket directly. Although certain portions of the entity will not directly participate in these associations, those portions of the entity may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the south pole pocket, or the spacing between functional groups of an entity comprising several chemical entities that directly interact with the south pole pocket.
The chemical entities which interact in whole or in part with the south pole pocket, preferably in a way similar to that of mevinolin may further be tested for their ability to inhibit LFA-1/ICAM-1 or ICAM-3 interactions, using the Jurkat or Hut 78 cell assay described below under A). Representative compounds which bind to the south pole pocket according to the invention are those which inhibit the adhesion of Jurkat or Hut 78 cells to ICAM-1 with an IC
50
≦30 &mgr;M. These compounds are indicated as LFA-1 antagonists or LFA-1/ICAM-1 or ICAM-3 interaction inhibitors.
Preferred compounds of the invention for use in accordance with the invention are mevinolins (hereinafter referred to as “mevinolins of the invention”), preferably those having no or only limited HMG CoA R inhibitory activity.
Accordingly, the invention provides:
1. A compound for use in the treatment and/or prevention of autoimmune diseases, acute or chronic inflammatory diseases, ischemia/reperfusion injury, acute or chronic rejection of organ or tissue allo- or xenografts or infection diseases by virtue of its LFA-1 inhibitory activity.
1.1 A compound for use in the treatment and/or prevention of autoimmune diseases, acute or chronic inflammatory diseases, ischemia/reperfusion injury, acute or chronic rejection of organ or tis
Bauer Wilfried
Cottens Sylvain
Geyl Dieter
Hommel Ulrich
Kallen Jörg
Novartis AG
Savitsky Thomas R.
Solola Taofiq
LandOfFree
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