Metal mediated serine protease inhibitors

Details Metal mediated serine protease inhibitors Metal mediated serine protease inhibitors

1999-07-12

2002-03-12

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Enzyme inactivation by chemical treatment

C435S023000, C435S024000

Reexamination Certificate

active

06355460

ABSTRACT:

FIELD OF THE INVENTION
This application relates to serine protease inhibitors, inhibitor-enzyme complexes formed by the serine protease inhibitors, methods of using the serine protease inhibitors and the inhibitor-enzyme complexes, and methods for determining whether compounds inhibit serine proteases.
DESCRIPTION OF THE FIELD
Serine proteases are produced within the cells of many living organisms and are often secreted to act outside of the producing cell. Individual serine proteases may target specific substrates (e.g., an inactive precursor for conversion to its biologically active form) or may act non-specifically (e.g., degradation of proteins or other peptides by scission). Further, individual serine proteases may be highly selective in that they recognize only one or a few related subsequences or non-selective in that they recognize and cleave a variety of unrelated sequences.
Serine proteases have a highly conserved active site, wherein specific amino acids which catalyze the bond scission have nearly identical spatial arrangements. A complementary binding site adjacent to the active site provides for the primary specificity of any individual serine protease. A succession of indentations or “pockets” along the surface of the protease serve to bind successive amino acid side chains along the substrate polypeptide chain on either side of the peptide bond to be cleaved. Substrate side chains which contribute to the association with the protease are designated P1, P2, P3, etc., proceeding from the side chain proximate to the susceptible bond toward the amino terminal of the protein, and P1′, P2′, P3′, etc., proceeding from the side chain proximate to the susceptible bond toward the carboxyl terminal of the protein. Small molecules having suitable P binding moieties can be designed to mimic the substrate by occupying the substrate's binding site and inhibit the function of the serine protease.
Serine proteases provide a diverse array of biological functions. Important serine proteases include trypsin-like proteases, such as trypsin, tryptase, thrombin, plasma kallikrein, tissue kallikrein and factor Xa. Substrates for serine proteases are associated, for example, with blood clotting, complement mediated lysis, the immune response, glomerulonephritis, pain sensing, inflammation, pancreatitis, cancer, regulating fertilization, bacterial infection and viral maturation. Accordingly, appropriate drug therapies can comprise the inhibition of a particular serine protease implicated in the pathology and/or symptomatology of a disease. Hence, substantial interest exists in the identification of serine protease inhibitors which possess high selectivity for specific serine proteases.
The disclosure of other documents referred to throughout this application are incorporated herein by reference.
SUMMARY OF THE INVENTION
An aspect of this invention is a method for determining the serine protease inhibitory activity of a compound, which method comprises contacting the compound with a serine protease in a medium having present therein a divalent metal cation, wherein the cation has the capacity for interaction with the compound and thereby to potentiate any serine protease inhibitory activity possessed by the compound and the concentration of the divalent metal cation in the medium is modified to a level sufficient to produce any such interaction.
A second aspect of this invention is a method for determining whether the inhibitory activity of a serine protease inhibitor is potentiated by the presence of a divalent metal cation, which method comprises:
(a) assaying for the inhibition of a serine protease by the inhibitor, wherein the assay is conducted in a medium that is essentially devoid of dissociated divalent metal cations; and
(b) assaying for the inhibition of the serine protease by the compound under essentially equivalent assay conditions to those used in Step (a), with the exception that the assay performed in Step (b) is conducted in a medium that contains an effective concentration of a divalent metal cation; wherein the inhibitory activity of the compound when measured by Step (b) is significantly greater than the inhibitory activity of the compound when measured by Step (a).
A third aspect of this invention is a method for inhibiting a serine protease with a serine protease inhibitor in a medium comprising the serine protease and the inhibitor, in which the inhibitor comprises two heteroatoms in spatial relationship one to the other so as to chelate a physiologically acceptable divalent metal cation capable of chelation, the improvement which comprises having sufficient quantity of the divalent metal cation in the medium or adding a sufficient amount of the divalent metal cation to the medium to have any or all of the inhibitor which is bound to the serine protease as a divalent metal cation complex, or providing the inhibitor as a divalent metal cation binary complex, such that the divalent metal cation is bound between the inhibitor and the serine protease as a divalent metal cation ternary complex.
A fourth aspect of this invention is a divalent metal cation binary complex of a compound of Formula I:
{(B
P
)
r
—(I)}
n
(Y)
q
  I
in which:
q is 0 and n is 1 or
q is 1 and n is 2;
Y is a bond or linking group of not more than six, typically not more than three and preferably carbon, atoms in a chain;
B
P
is a binding moiety for binding to one or more P sites of a serine protease;
r is 0 or 1, with the proviso that at least one B
P
binding moiety is present; and
I is a moiety that comprises at least one heteroatom when n is 2 and comprises at least two heteroatoms when n is 1, wherein two heteroatoms are in spatial relationship one to the other so as to be able to chelate the divalent metal cation in a bidentate manner.
A fifth aspect of this invention is a divalent metal cation ternary complex comprising the compound of Formula I in association with the divalent metal cation and a serine protease.
A sixth aspect of this invention is the serine protease inhibitors identified by the methods of this invention.
A seventh aspect of this invention is a method for treating a disease in an animal in which serine protease activity contributes to the pathology and/or symptomatology of the disease, which method comprises administering to the animal a therapeutically effective amount of a serine protease inhibitor identified by the methods of this invention.
Thus, it is intended that the scope of this invention encompasses any method for determining the serine protease inhibitory activity of a compound wherein the method requires the presence of a divalent metal cation and a serine protease and one of ordinary skill in the art could demonstrate that the cation has the capacity for interaction with a compound of Formula I and thereby to potentiate the inhibitory activity of the compound or is capable of chelating simultaneously with a compound of Formula I and the active site of the serine protease and thereby potentiate the inhibitory activity of the compound.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:
“Alkyl” means a straight or branched, saturated or unsaturated hydrocarbon radical having the number of carbon atoms indicated (e.g., (C
1-8
)alkyl includes methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl, ethynyl, 1-propynyl, 2-propynyl, etc.).
“Alkylene” means a straight, saturated or unsaturated hydrocarbon divalent radical (e.g., methylene, ethylene, vinylene, ethynylene, 2-propylene, 1-propylene, tetramethylene, isopropylidene, etc.).
“Amidino” means the radical —C(NH)NH
2
.
“Amino” means the radical —NH
2
.
“Aryl” means an aromatic monocyclic or polycyclic hydrocarbon radical containing the number of carbon atoms indicated, wherein the carbon atom with the free valence is a member of an aromatic ring (e.g., (C
6-

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