# US 2013/0157281 A1 · Factor IXa Crystals, Related Complexes and Methods

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- Number: US 2013/0157281 A1
- Document ID: US20130157281A1
- Status: Published application
- Kind: A1
- Application number: 13/149,011
- Filed: 2011-05-31
- Published: 2013-06-20
- Earliest claimed: 2008-05-07
- Assignees: Schering Corporation
- Inventors: BRIAN M. BEYER; ALAN W. HRUZA; RICHARD N. INGRAM; VINCENT S. MADISON; ANDREW J. PRONGAY; PAUL REICHERT
- Web page: https://patentgrep.com/patent/US20130157281A1

## CPC

- C12Q 1/56: involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
- C12N 9/644: {Coagulation factor IXa (3.4.21.22)}
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2299/00: Coordinates from 3D structures of peptides, e.g. proteins or enzymes
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to factor IXa complexes and crystals thereof as well as methods for identifying inhibitors of factor IXa.

## Claims

### Claim at position 1

1-16. (canceled)

### Claim 17

A method for determining the structure of a complex comprising a compound bound to factor IXa protein, said method comprising: (a) mixing the factor IXa protein with the compound; (b) crystallizing a factor IXa protein-compound complex; and (c) determining the structure of the complex by employing the data of any one of Tables 2-6 optionally varied by a root mean square deviation of conserved residue backbone atoms or of alpha carbon atoms of less than about 1.5 Å when superimposed on backbone atoms described in Table 2, 3, 4, 5 or 6.

### Claim 18

A method for identifying a substance which inhibits blood clotting or binds to factor IXa comprising
(a) mixing factor IXa protein with the substance;
(b) crystallizing a factor IXa protein-substance complex; and
(c) determining the structure of the complex by employing the data of any one of Tables 2-6 optionally varied by a root mean square deviation of conserved residue backbone atoms or of alpha carbon atoms of less than about 1.5 Å when superimposed on backbone atoms described in Table 2, 3, 4, 5 or 6; and, if said substance is observed to complex with factor IXa; optionally determining if the substance selectively inhibits factor IXa proteolytic activity but not factor Xa proteolytic activity; wherein the substance is identified as a blot clotting inhibitor or factor IXa binding substance if said complex and/or selective inhibition is observed.

### Claim 19 (depends on 18)

The method of claim 18 wherein said proteolytic activity is determined by measuring proteolysis of Methylsulfonyl-D-cyclohexylglycyl-glycyl-arginine-paranitroanilide.

### Claim 20

A method for determining the structure of a complex comprising a second compound bound to factor IXa protein which is complexed with a first compound, said method comprising; (a) providing a crystal of factor IXa protein complexed with the first compound; (b) soaking the crystal comprising the first compound with a second compound to form a complex between said factor IXa protein and said second compound; and (c) determining the structure of the complex comprising the second compound by employing the structural data of any one of Tables 2-6 optionally varied by a root mean square deviation of conserved residue backbone atoms or of alpha carbon atoms of less than about 1.5 Å when superimposed on backbone atoms described in Table 2, 3, 4, 5 or 6.

### Claim 21

A method for identifying a substance which inhibits blood clotting or binds to factor IXa comprising
(a) providing a crystal of factor IXa protein complexed with a first substance;
(b) soaking the crystal comprising the first substance with a second substance to form a complex between said factor IXa protein and said second substance; and (c) determining the structure of the complex comprising the second substance by employing the data of any one of Tables 2-6 optionally varied by a root mean square deviation of conserved residue backbone atoms or of alpha carbon atoms of less than about 1.5 Å when superimposed on backbone atoms described in Table 2, 3, 4, 5 or 6; and, if second said substance is observed to complex with factor IXa, optionally determining if the second substance selectively inhibits factor IXa proteolytic activity but not factor Xa proteolytic activity; wherein the second substance is identified as a blot clotting inhibitor or factor IXa binder if said complex and/or selective inhibition is observed.

### Claim 22 (depends on 21)

The method of claim 21 wherein said proteolytic activity is determined by measuring proteolysis of Methylsulfonyl-D-cyclohexylglycyl-glycyl-arginine-paranitroanilide.

### Claim at position 8

23-27. (canceled)

### Claim 28

A method for identifying a substance which binds to factor IXa or inhibits blood clotting comprising: determining the structure of a complex comprising a compound represented by structural formula A, B, C or D by employing the data of any one of Tables 2-6 optionally varied by a root mean square deviation of conserved residue backbone atoms or of alpha carbon atoms of less than about 1.5 Å when superimposed on backbone atoms or alpha carbon atoms described in Table 2, 3, 4, 5 or 6; and, employing a computational means to dock a candidate substance with the factor IXa serine protease domain to determine if said candidate substance binds to said serine protease domain; and, optionally, synthesizing said candidate substance; and, optionally, determining if said candidate substance binds to factor IXa or inhibits factor IXa proteolytic activity.