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US 8,710,188 B2Granted
Factor IXa crystals, related complexes and methods
AssigneeMerck Sharp & Dohme Corp.
InventorsBrian M. Beyer, Alan W. Hruza, Richard N. Ingram, Vincent S. Madison, Andrew J. Prongay, Paul Reichert
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Abstract
The present invention relates to factor IXa complexes and crystals thereof as well as methods for identifying inhibitors of factor IXa.
Claims5
A method for determining the structure of a complex comprising a compound and a peptide bound to factor IXa protein, wherein said factor IXa is selected from the group consisting of SEQ ID NO:
- 14 and 18, said peptide comprises SEQ ID NO:
- 13 and said compound is selected from the group consisting of:
- said method comprising:
- (a) mixing the factor IXa protein with the compound;
- (b) crystallizing said factor IXa protein-compound complex;
- and (c) determining the three-dimensional structure of the complex which results in atomic coordinate 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 wherein the resultant crystal of part (b) forms in one of in the following crystalline space groups and unit cell parameters:
- (i) space group P212121 having unit cell dimensions a=48.1 Å, b=69.8 Å, c=92.1 Å and α=β=γ=90° or space group P43212 having unit cell dimensions a=100.4 Å, b=100.4 Å, c=97.3 Å and α=β=γ=90°;
- (ii) space group P43212 having unit cell dimensions a=100.6 Å, b=100.6 Å, c=98.1 Å and α=β=γ=90°;
- (iii) space group P43212 having unit cell dimensions a=99.2 Å, b=99.2 Å, c=97.3 Å and α=β=γ=90°;
- and
- (iv) space group P43212 having unit cell dimensions a=100.5 Å, b=100.5 Å, c=98.4 Å and α=β=γ=90°.
8 more elements
A method for determining the structure of a complex comprising a first compound and a peptide bound to factor IXa protein and a second compound, wherein said factor IXa is selected from the group consisting of SEQ ID NO:
- 14 and 18, said peptide comprises SEQ ID NO:
- 13 and said first compound is selected from the group consisting of:
- and wherein said second compound bound to said factor IXa protein is complexed with said first compound, said method comprising;
- (a) providing a crystal of factor IXa protein complexed with the first compound and the peptide, wherein said crystal forms in one of in the following crystalline space groups and unit cell parameters:
- (i) space group P212121 having unit cell dimensions a=48.1 Å, b=69.8 Å, c=92.1 Å and α=β=γ=90° or space group P43212 having unit cell dimensions a=100.4 Å, b=100.4 Å, c=97.3 Å and α=β=γ=90°;
- (ii) space group P43212 having unit cell dimensions a=100.6 Å, b=100.6 Å, c=98.1 Å and α=β=γ=90°;
- (iii) space group P43212 having unit cell dimensions a=99.2 Å, b=99.2 Å, c=97.3 Å and α=β=γ=90°;
- and
- (iv) space group P43212 having unit cell dimensions a=100.5 Å, b=100.5 Å, c=98.4 Å and α=β=γ=90°;
- (b) soaking the crystal 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 to determine the three-dimensional structure which results in atomic coordinate which varies 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.
9 more elements
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 compound and a peptide, wherein said factor IXa is selected from the group consisting of SEQ ID NO:
- 14 and 18, said peptide comprises SEQ ID NO:
- 13 and said first compound is selected from the group consisting of:
- and said crystal forms in one of in the following crystalline space groups and unit cell parameters:
- (i) space group P212121 having unit cell dimensions a=48.1 Å, b=69.8 Å, c=92.1 Å and α=β=γ=90° or space group P43212 having unit cell dimensions a=100.4 Å, b=100.4 Å, c=97.3 Å and α=β=γ=90°;
- (ii) space group P43212 having unit cell dimensions a=100.6 Å, b=100.6 Å, c=98.1 Å and α=β=γ=90°;
- (iii) space group P43212 having unit cell dimensions a=99.2 Å, b=99.2 Å, c=97.3 Å and α=β=γ=90°;
- and
- (iv) space group P43212 having unit cell dimensions a=100.5 Å, b=100.5 Å, c=98.4 Å and α=β=γ=90°;
- (b) soaking the crystal comprising the first compound 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 to determine the three-dimensional structure which results in atomic coordinate which varies 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,
- wherein 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.
10 more elements
A method for identifying a substance which binds to factor IXa or inhibits blood clotting comprising:
- (a) employing on a computer the structural coordinates of a complex comprising:
- a compound selected from the group consisting of I-IV, a peptide (SEQ ID NO:
- 13) and factor IXa (SEQ ID NO:
- 14 or 18) as set forth in any of Tables 2-6 and having a root-mean square deviation of less than 1.5 Å from the backbone atoms or alpha carbon atoms thereof, to generate three-dimensional models thereof in silico;
- (b) utilizing the model(s) as generated in step (a) to analyze the interaction(s) of a candidate substance with the serine protease domain of factor IXa by computational docking means to determine the potential binding of said substance to factor IXa;
- and
- (c) optionally, synthesizing said candidate substance and/or determining if said candidate substance binds to factor IXa or inhibits factor IXa proteolytic activity in vitro;
- And wherein compounds I-IV are:
5 more elements
Dependencies are inferred from the claim text.