Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
2000-03-23
2004-11-30
Marschel, Ardin H. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C702S019000, C435S007100
Reexamination Certificate
active
06826488
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates the crystal structure of IMPDH isolated from bacteria. The structure is different from the structure of mammalian or fungal IMPDH, allowing design of selective inhibitors of bacterial IMPDH.
Inosine monophosphate dehydrogenase (IMPDH; Enzyme Commission (EC) 1.1.1.205) is a rate-limiting enzyme in the synthesis of guanine ribonucleotides. IMPDH has an essential role in providing critical precursors for DNA and RNA biosynthesis and in signal transduction pathways that mediate cell differentiation (Collart et at., 1990; Kiguchi et al., 1990). Because of its central role in purine metabolism, IMPDH is an attractive therapeutic target. Several recent reviews have outlined the utility of mammalian IMPDH inhibitors as anticancer (Pankiewicz, 1997) or antiviral (Andrei et al, 1993) agents or as immunosuppressive drugs (Halloran, 1996) (see Table 1).
TABLE 1
Clinically Useful Inhibitors of IMPDH
Inhibitor
Clinical Application
Ribavirin
Antiviral
Mycophenolate mofetil
Immunosuppression
Mizoribine
Imunosuppression
Tiazofurin
Anticancer
Although there are no reports of selective inhibitors of bacterial IMPDH enzymes, such compounds could have potential application as specific antimicrobial agents.
The active form of IMPDH enzymes (50-55 kDa) is a homotetramer with four active sites per tetramer. A cysteine residue in the active site forms a covalent intermediate with IMP (Wang et al., 1996). A consensus sequence of thirteen amino acid residues that includes cysteine in this active site has been proposed as a signature motif (i.e., an amino acid sequence that can be used as a fingerprint or specific identifier for this class of enzymes) for the IMPDH and guanosine monophosphate (GMP) reductase enzymes (Bairoch, 1995). This IMPDH consensus region is highly conserved in both bacteria and eukaryotes, with 90% and 85% of the respective residues being identical within each kingdom. However, only 40% of these residues remain identical when compared between the two kingdoms. This limited conservation suggests that bacterial and eukaryotic IMPDH enzymes may have distinct characteristics; a suggestion supported by their kinetic differences and differential sensitivity to inhibitors. Enzymes from mammalian sources show distinctly lower values for the K
m
for nicotinamide adenine dinucleotide (NAD) than do those enzymes from bacteria. In addition, mammalian IMPDH enzymes are 10-100 times more sensitive to inhibition by mycophenolic acid (MPA) than are bacterial IMPDH enzymes. Sequence analysis of all known IMPDH enzymes supports the distinction between bacterial and eukaryotic enzymes. A deep branching of the bacterial and eukaryotic forms of IMPDH is observed upon phylogenetic analysis of the relationships among the various IMPDH genes (Collart et al, 1996 a and b). This phylogenetic analysis indicates a general functional conservation of amino acid and suggests a unique amino acid sequence signature for these kingdoms.
The elucidation of a kingdom-specific signature for IMPDH enzymes is an important element in the development of specific inhibitors. The two partial structures of IMPDH from Chinese hamster (Sintchak et al., 1996) (85% structure complete with bound transition state analogue and mycophenolic acid, MPA) and
Tritrichomonas foetus
(Whitby et al., 1997) (68% structure complete with bound xanthosine monophosphate [XMP]) have been reported with only the coordinates of the latter available in the Protein Data Bank (PDB). These structures furnished the initial information about the structure and reaction mechanism of eukaryotic IMPDH enzymes. Inhibitors of IMPDH in bacteria are needed to treat infections, in particular, to overcome the barrier of antibiotic resistance.
BRIEF SUMMARY OF THE INVENTION
The invention relates for the first time a crystal structure of a bacterial IMPDH. This invention relates that bacterial and mammalian IMPDH enzymes provide the same catalytic function, but have a set of unique structural and biochemical characteristics. An embodiment is a crystal structure of IMPDH isolated from
Streptococcus pyogenes. S. pyogenes
IMP dehydrogenase represents the class of bacterial IMPDH enzymes that show distinct functional differences when compared to mammalian IMPDH enzymes. The bacterial enzymes bind NAD poorly (Zhou et al., 1997; Kerr et al., 1997) (K
M
>1 mM) and are inhibited by MPA only at very high concentrations (Ki>0.5 mM). Elucidation of the structural basis of these distinct characteristics is useful to aid in design of specific IMPDH inhibitors that will inhibit the infectious agent without harming the host's IMPDH.
The coding sequence of bacterial IMPDH specifies a protein of 493 amino acids that contain only a single cysteine residue at the active site (Ashbaugh et al., 1995). IMPDH from
S. pyogenes
is a representative bacterial enzyme because the organism is pathogenic, and therefore a good model for the investigation of enzyme inhibitors. Streptococci are the most common cause of worldwide pneumonia and a leading cause of pediatric infections. The structure of the
S. pyogenes
bacterial IMPDH provides the basis for elucidation of the structural characteristics that distinguish bacterial from eukaryotic IMPDH enzymes. Knowledge of these characteristics permits an understanding of why these enzymes exhibit functionally distinct behavior and therefore provides a foundation for the design of specific inhibitors of IMPDH that have clinical value.
In addition to inhibiting pathogens, the immunosuppressive use of IMPDH inhibitors is applicable to treat chronic inflammatory diseases such as arthritis, diabetes, or systemic lupus erythromotosis. Use of the IMPDH structure from
S. pyogenes
will facilitate identification of other pathogens that will be inhibited by drugs that inhibit
S. pyogenes.
Definitions and Abbreviations
A “binding pocket” is a space in a molecule in which an inhibitor of the molecule is bound.
The following abbreviations are used throughout the application:
A = Ala = Alanine
V = Val = Valine
L = Leu = Leucine
I = Ile = Isoleucine
P = Pro = Proline
F = Phe = Phenyalanine
W = Trp = Trytophan
M = Met = Methionine
G = Gly = Glycine
S = Ser = Serine
T = Thr = Threonine
C = Cys = Cysteine
Y = Tyr = Tyrosine
N = Asn = Asparagine
Q = Gln = Glutamine
D = Asp = Aspartic Acid
E = Glu = Glutamic Acid
K = Lys = Lysine
R = Arg = Arginine
H = His = Histidine
CBS = Cystathionine-&bgr;-synthase
GMP = Guanosine monophosphate
IMP = Inosine monophosphate
IMPDH = Inosine monophosphate dehydrogenase
MPA = Mycophenolic acid
NAD = Nicotinamide adenine dinucleotide
PDB = Protein Data Bank
XMP = Xanthosine monophosphate
REFERENCES:
patent: 6128582 (2000-10-01), Wilson et al.
Drenth, J.,Principles of Protein X-ray Crystallography[Published by Springer-Verlag New York Inc., 175 Fifth Ave., New York, NY 10010], pp. 1-19, 1994.*
Fleming et al., Biochemistry, vol. 35, pp. 6990-6997, 1996.*
Geysen et al, Cognitive features of continuous antigenic determinants. J. of Molecular Recognition, vol. 1, pp. 32-40, 1988.*
Russell et al. Structural features can be unconserved in proteins with similar folds. (Journal of Molecular Biology, vol. 244, pp. 332-350, 1994.*
Zhang et al. Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase. Biochemistry, vol. 38, pp. 4691-4700, Apr. 13, 1999.*
Allison A. and Eugui E.(1996) “Purine metabolism and immunosuppresive effects of mycophenolate mofetil (MMF),”Clin Transplantation10: 77-84.
Antonino L. et al. (1994) “Probing the active site of human IMP dehydrogenase using halogenated purine riboside 5′-monophosphates and covalent modification reagents,”Biochemistry33: 1760-1765.
Anton
Collart Frank R.
Huberman Eliezer
Joachimiak Andrezej
Westbrook Edwin M.
Zhang Rongguang
Barnes & Thornburg LLP
Marschel Ardin H.
Martin Alice O.
The University of Chicago
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