The synthesis of nucleotides in organisms is required for the cells in those organisms to divide and replicate. Nucleotide synthesis in mammals may be achieved through one of two pathways: the de novo synthesis pathway or the salvage pathway. Different cell types use these pathways to a different extent.
Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an enzyme involved in the de novo synthesis of guanosine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP)[Jackson R. C. et. al., Nature, 256, pp. 331-333, (1975)].
IMPDH is ubiquitous in eukaryotes, bacteria and protozoa [Y. Natsumeda & S. F. Carr, Ann. N.Y. Acad., 696, pp. 88-93 (1993)]. The prokaryotic forms share 30-40% sequence identity with the human enzyme. Regardless of species, the enzyme follows an ordered Bi—Bi reaction sequence of substrate and cofactor binding and product release. First, IMP binds to IMPDH. This is followed by the binding of the cofactor NAD. The reduced cofactor, NADH, is then released from the enzyme, followed by the product, XMP [S. F. Carr et al., J. Biol. Chem., 268, pp. 27286-90 (1993); E. W. Holmes et al., Biochim. Biophys. Acta, 364, pp. 209-217 (1974)]. This mechanism differs from that of most other known NAD-dependent dehydrogenases, which have either a random order of substrate addition or require NAD to bind before the substrate.
Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced [F. R. Collart and E. Huberman, J. Biol. Chem., 263, pp. 15769-15772, (1988); Y. Natsumeda et. al., J. Biol. Chem., 265, pp. 5292-5295, (1990)]. Each is 514 amino acids, and they share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa [Y. Yamada et. al., Biochemistry, 27, pp. 2737-2745 (1988)].
The de novo synthesis of guanosine nucleotides, and thus the activity of IMPDH, is particularly important in B and T-lymphocytes. These cells depend on the de novo, rather than salvage pathway to generate sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen [A. C. Allison et. al., Lancet II, 1179, (1975) and A. C. Allison et. al., Ciba Found. Symp., 48, 207, (1977)]. Thus, IMPDH is an attractive target for selectively inhibiting the immune system without also inhibiting the proliferation of other cells.
It is also known that IMPDH plays a role in other metabolic events. Increased IMPDH activity has been observed in rapidly proliferating human leukemic cell lines and other tumor cell lines, indicating IMPDH as a target for anti-cancer as well as immunosuppressive chemotherapy [M. Nagai et. al., Cancer Res., 51, pp. 3886-3890, (1991)]. IMPDH has also been shown to play a role in the proliferation of smooth muscle cells, indicating that inhibitors of IMPDH, such as MPA or rapamycin, may be useful in preventing restenosis or other hyperproliferative vascular diseases [C. R. Gregory et al., Transplantation, 59, pp. 655-61 (1995); PCT publication WO 94/12184; and PCT publication WO 94/01105].
IMPDH has also been shown to play a role in viral replication in some viral cell lines. [S. F. Carr, J. Biol. Chem., 268, pp. 27286-27290 (1993)]. Analogous to lymphocyte and tumor cell lines, the implication is that the de novo, rather than the salvage, pathway is critical in the process of viral replication.
Additionally, the de novo synthesis of purine nucleotides, and thus the activity of IMPDH, is implicated in attenuating bacterial growth and virulence under purine starved conditions. Several biological lines of evidence exist suggesting that IMPDH could be a selective antibacterial target. Biological fluids are low in free purines and therefore limiting for bacterial growth [Simmonds et al., Techniques in Diag. Huma. Biochem. Gen.: A Lab Manual, pp. 397-424 (1991)]. Several independent studies have shown that purine auxotrophy attenuates virulence of Salmonella strains, Shigella flexniri, E. coli and E. faecalis [MacFarland and Stocker, Microb. Path., 3, pp. 129-141 (1987); Russo et al., Mol. Microb., 22, pp. 217-229 (1996); Mahan et al., Science, 259, pp. 686-688 (1993); Singh et al., J. Infect. Dis., 178, pp. 1416-1420 (1998); Fields et al., PNAS, 83, pp. 5189-5193 (1986); Noriega et al., Infect. Immun., 64, pp. 3055-3061 (1996)]. This was specifically shown for guaB mutants of Salmonella and Shigella which were defective in the gene encoding IMPDH [MacFarland and Stocker, Microb. Path., 3, pp. 129-141 (1987) and Noriega et al., Infect. Immun., 64, pp. 3055-3061 (1996)].
Additional evidence exists for significant differences between mammalian IMPDH and bacterial IMPDH suggesting that IMPDH is an attractive target for selectively inhibiting bacterial growth without also inhibiting mammalian IMPDH functions. For instance, known mammalian IMPDH inhibitors such as mycophenolic acid are >1000-fold less potent against bacterial IMPDH versus mammalian IMPDH [Hedstrom et al., Curr. Med. Chem., 6, pp. 545-560 (1999)]. This is an example of “reverse selectivity” from an antibacterial standpoint and suggests that significant differences exist between the mammalian and bacterial enzymes. This “reverse selectivity” has been shown in part to be due to residue differences in the NAD site and in part due to differences in kinetic mechanism [Hedstrom et al., Biochemistry, 38, pp. 15388-15397 (1999); Hedstrom et al., Biochemistry, 39, pp. 1771-1777 (2000)]. These differences can be exploited to design inhibitors that are selective for bacterial IMPDH over mammalian IMPDH.
Bacterial resistance to antibiotics has long been recognized, and it is today considered to be a serious worldwide health problem. As a result of resistance, some bacterial infections are either difficult to treat with antibiotics or even untreatable. This problem has become especially serious with the recent development of multiple drug resistance in certain strains of bacteria, such as Streptococcus pneumoniae (SP), Mycobacterium tuberculosis, and Enterococcus. The appearance of vancomycin resistant enterococcus was particularly alarming because vancomycin was formerly the only effective antibiotic for treating this infection, and had been considered for many infections to be the drug of “last resort”. While many other drug-resistant bacteria do not cause life-threatening disease, such as enterococci, there is the fear that the genes which induce resistance might spread to more deadly organisms such as Staphylococcus aureus, where methicillin resistance is already prevalent (De Clerq, et al., Current Opinion in Anti-infective Investigational Drugs, 1999, 1, 1; Levy, “The Challenge of Antibiotic Resistance”, Scientific American, March, 1998).
As bacterial resistance to antibiotics has become an important public health problem, there is a continuing need to develop newer and more potent antibiotics. More particularly, there is a need for antibiotics that represent a new class of compounds not previously used to treat bacterial infection. One attractive strategy for developing new antibiotics is to inhibit bacterial IMPDH, a bacterial enzyme necessary for the de novo synthesis of purine nucleotides, and therefore, necessary for bacterial cell growth and division.