Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-12-15
2001-05-08
Swartz, Rodney P. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S168100, C424S199100, C424S200100, C424S248100, C435S006120, C435S007600, C435S069100, C435S091100, C435S091500, C435S252300, C435S253100, C435S863000, C435S864000, C435S865000, C435S866000, C536S022100, C536S023100, C536S023200, C536S023600
Reexamination Certificate
active
06229001
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel recombinant nucleic acids encoding the enzyme dihydrofolate reductase (DHFR) from mycobacteria, to novel recombinant DHFR peptides produced by such sequences, and to vaccines, diagnostic kits, cells and therapies utilizing these peptides and nucleic acid sequences. The invention is also directed to methods for using the sequences and peptides to develop drugs specific to
M. avium
and other species of mycobacteria, to identifying other DHFR sequences and peptides, as well as diagnostic and treatment methods incorporating the disclosed sequences and peptides.
2. Description of Background
The
Mycobacterium avium
complex represents one of the most serious opportunistic infections and is often associated with advanced stages of autoimmune deficiency syndrome or AIDS (J. J. Ellner et al., J. Infect. Dis. 163:1326-35, 1991; J. A. Havlok, Jr. et al., J. Infec. Dis. 165:577-80, 1992; C. C. Hawkins et al., Ann. Intern. Med. 105:184-88, 1986; D. S. O'Brien et al., Am. Rev. Respir. Dis. 135:1007-14, 1989; N. Rastogi et al., Res. Microbiol. 145:167-261, 1994). Unlike
Mycobacterium tuberculosis
, which can be successfully treated with two or three drug combinations (except for multidrug resistant
M. tuberculosis
; MDR-TB), the
M. avium
complex is resistant to many antimycobacterial agents (B. D. Agins et al., J. Infect. Dis. 159:784-87, 1989; C. Benson et al., Sixth International Conference on AIDS, San Francisco, 1990; J. Chiu et al., Ann. Intern. Med. 113:358-61, 1990; F. De Lalla et al., Antimicrob. Agents Chemother. 36:1567-69, 1992; L. Heifets et al., Antimicrob. Agents Chemother. 37:2364-70, 1993; D. Y. Rosenzweig, Amer. Rev. Resp. Dis. 113(Suppl.):55, 1976). Drug resistance in
M. avium
is still considered an inherent property of the wild type organism (S. L. Morris et al., Complex. Res. Microbiol. 147:68-73, 1996), resulting in large part from the refractory nature of the organism's cell envelope (H. L. David, Rev. Infect. Dis. 3:878-84, 1981; N. Rastogi et al., Res. Microbiol. 145:243-52, 1994; N. Rastogi et al., Antimicrob. Agents Chemother. 20:666-77, 1981). Although
M. avium
infections in AIDS patients are treated with 3-6 different drugs, the long term prognosis is still poor (B. D. Agins et al., J. Infect. Dis. 159:784-87, 1989; C. Benson et al., Sixth International Conference on AIDS, San Francisco, 1990; J. Chiu et al., Ann. Intern. Med. 113:358-61, 1990; F. De Lalla et al., Antimicrob. Agents Chemother. 36:1567-69, 1992; J. J. Ellner et al., J. Infect. Dis. 163:1326-35, 1991).
Tuberculosis is a disease of worldwide significance and notoriety. At any one time, about one-third of the world is infected with
M. tuberculosis
resulting in eight million new cases of tuberculosis and 2.9 million deaths annually (A. Arachi, Tubercle. 72:1-6, 1991). It is estimated that about 0.3% of U.S. residents are infected and at risk to develop active disease (CDC 1996, CDC Revises HIV Infection Estimates. HIV/AIDS Prevention. August:2). This risk becomes even greater if the person is co-infected with the human immunodeficiency virus (HIV). If so, estimates indicate that progression to tuberculosis will occur in about 30% of those cases and the risk for developing tuberculosis becomes 113 times greater (tuberculosis., N.a.p.t.c.m.-r., MMWR. 41 RR-11:1-71, 1992).
As the projected figure for HIV infections is more than 20 million by the year 2000, it is probable that the number of tuberculosis cases worldwide will also increase. Even in 1991, the figure for people co-infected with HIV and
M. tuberculosis
was estimated to be 3.1 million (J. F. Murray, Bull. Int. Union Tuberc. Lung Dis. 66:21-15, 1991). In addition, life-threatening strains of MDR-TB are appearing. Some of these strains can result in a high mortality rate (e.g. 72-89%), with death occurring in a short period (e.g. 4-16 weeks) (CDC, Mortal. Morbid. Weekly Rep. 39:718-22, 1990; CDC, Mortal. Morbid. Weekly Rep. 40:649-652, 1991; B. R. Edlin et al., New Engl. J. Med. 326:1514-21, 1992). In summary, the impact of tuberculosis on the world today can best be appreciated by the fact that the World Health Organization declared tuberculosis a global public health emergency, a distinction never before given to any other disease (WHO., Soz Praventivmed. 38:251-52, 1993). Consequently, the development of new antimycobacterial drugs is an important research endeavor.
The dihydrofolate reductase (DHFR) enzyme is an important target for medicinal chemistry (K. Bowden et al., J. Chemother. 5:377-88, 1993) and DHFR inhibitors have been used in anticancer therapy (e.g. methotrexate (W. A. Bleyer, Cancer Treat. Rev. 41:36-51, 1978)), antibacterial therapy (e.g. trimethoprim (M. Finland et al., J. Infect. Dis. 128:S425-816, 1973)), and antimalarial therapy (e.g. pyrimethamine (A. K. Saxena, Prog. Drug Res. 30:221-80, 1986)). Dihydrofolate reductase is present in all cells and is necessary for the maintenance of intracellular folate pools in a biochemically active reduced state (M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991). Inhibition of the enzyme is effective because binding affinities for substrate analogs are so great that such analogs are not readily displaced by the natural substrates. Enzyme inhibition results in the depletion of intracellular reduced folates that are required for one carbon transfer reactions, which in turn are important for the biosynthesis of thymidylate, purine nucleotides, methionine, serine, glycine and many other compounds needed for RNA, DNA, and protein synthesis.
FIG. 1
depicts DHFR's role in the biosynthesis of tetrahydrofolate and cell metabolism. (P. G. Hartman, J. Chemother. 5:369-76, 1993). Some bacteria have an uptake system for folates, but most have to synthesize folates de novo by reduction of dihydrofolate to tetrahydrofolates.
Although DHFR is not a new drug target, enthusiasm in the development of improved derivatives to inhibit DHFR is very intense, (D. P. Baccanari et al., J. Chemother. 5:393-99, 1993; K. Bowden et al., J. Chemother. 5:377-88, 1993; M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991; J. R. Piper et al., J. Med. Chem. 39:1271-80, 1996; B. I. Schweitzer et al., FASEB. 4:2441-52, 1990; J. K. Seydel, J. Chemother. 5:422-29, 1993), and particularly with regard to mycobacteria (K. H. Czaplinski et al., Eur. J. Med. Chem. 30:779-87, 1995; M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; H. H. Locher et al., Antimicrob. Agents Chemother. 40:1376-81, 1996; S. C. C. Meyer et al., Antimicro. Agents Chemother. 39:1862-63, 1995; R. L. Then, J. Chemother. 5:361-68, 1993). A unique feature of DHFR is the selectivity possible in the design of inhibitors for this target, thus making it an ideal target for antimycobacterial agents using rational and effective drug design. Although genes for DHFR (fol A) have been identified in other bacteria, they are not equivalent to the fol A gene from
M. avium
or other mycobacteria. The enzyme product of the
M. avium
gene (i.e. DHFR) is structurally different from other known DHFRs. For these reasons and others having to do with, for example, toxicity, selective drugs can be designed for individual species. Thus, a selective drug for
M. avium
can be designed in such a way that it will not affect DHFRs in other species, such as humans. One example of the species specific nature of the enzyme is demonstrated by the fact that the diaminopyrimidines, when properly substituted, can be several thousand times more active against bacterial than mammalian DHFR (P. G. Hartman, J. Chemother. 5:369-76, 1993). In addition, there are many possible inhibitors of this enzyme that have not been synthesized or studied (K. Bowden et al., J. Chemother. 5:377-88, 1993). DHFR also represents an enzyme that has been extensively used in the development of site directed inhibitors based upon X-ray crystallographic and molecular graphic studies (K. Bowden et al., J. Chemother. 5:377-388, 1993; M. P. Bradley, J. Med. Chem. 36:3171-77, 1993; B. J. Denny et
Barrow William W.
Dooley Thomas P.
Suling William J.
Van Ginkel Sabrina Z.
Brobeck Phleger & Harrison LLP
Southern Research Institute
Swartz Rodney P.
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