Methods for the identification of antimicrobial compounds

Details Methods for the identification of antimicrobial compounds Methods for the identification of antimicrobial compounds

2001-05-25

2004-05-04

Gitomer, Ralph (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S014000, C435S069200

Reexamination Certificate

active

06730495

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for the assay of 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase activity. The present invention also relates to methods for drug screening to identify compounds having antimicrobial activity, wherein the compounds have the ability to inhibit the enzymatic activity of microbial ketol-isomerase.
BACKGROUND
Microbial infections (e.g., infections by fungal or bacterial species) account for significant morbidity and mortality throughout the world. Although significant resources have been dedicated to identifying compounds having antimicrobial properties, microbial infections continue to present a significant human health risk in both developed and undeveloped countries.
Bacterial Infections and Drug Resistance
The development of antibacterial agents, starting with the identification of penicillin in the 1920s, has played a vital role in the treatment of human infectious diseases. However, the recent emergence of pathogenic microorganisms that are resistant to known antimicrobial compounds is cause for great concern. This situation has resulted from prolonged, worldwide use of antimicrobial compounds, with the unfortunate effect of selection of resistant microorganisms. For example, penicillin resistance has become increasingly widespread in the bacterial species that were previously susceptible to the drug. Some microorganisms produce &bgr;-lactamase, an enzyme which destroys the antimicrobial agent itself, while some microorganisms have undergone genetic changes resulting in alterations in the cell receptor protein to which the penicillin binds (i.e., penicillin-binding proteins; PBPs;
Jawetz, Melnick & Adelberg's Medical Microbiology,
19th ed, Appleton & Lange, Norwalk, Conn. [1991], p. 150), such that the drugs will no longer effectively bind to the receptors. Still other microorganisms have evolved mechanisms that prevent lysis of the organism after the drug has bound to the cell. In this latter scenario, the drug inhibits the growth of the organism, but the organism is not killed, and relapse of disease occurs following discontinuation of treatment.
One well-documented example highlighting the problem of drug resistance is the development of penicillin resistance in the pathogenic bacterium
Streptococcus pneumoniae
. Initially, the introduction of penicillin to treat
S. pneumoniae
resulted in a significant decrease in the mortality due to this organism. However,
S. pneumoniae
infection remains of great concern, as it is one of the organisms most frequently associated with invasive infections; it is the most common cause of bacterial pneumonia and otitis media, as well as the second most common cause of bacterial meningitis, and the third most common isolate from blood cultures (Sessegolo et al.,
J. Clin. Microbiol.,
32:906-911 [1994]).
The first report of pneumococci with decreased susceptibilities to penicillins occurred in 1967 in Australia. Since this initial report, additional strains with decreased susceptibilities have been reported worldwide. Additionally, bacterial resistance to alternative antibacterial compounds, such as chloramphenicol, erythromycin, tetracycline, clindamycin, rifampin, and sulfamethoxazole-trimethoprim has also been reported, often in conjunction with penicillin resistance. Multiple-antimicrobial resistance in pneumococci was first reported in 1977 in South Africa. Since this initial report, multi-drug resistant strains have been reported in several other countries, including Spain, Italy, France, Belgium, Hungary, Pakistan, Czechoslovakia, Canada, the United Kingdom, and the United States (Sessegolo et al.,
J. Clin. Microbiol.,
32:906-911 [1994]).
A second example illustrating the evolution of a multiple drug-resistant organism is
Nisseria gonorrhoeae
, the causative agent of gonorrhea. Prior to the 1930s, treatment for this disease was largely ineffective. In the late 1930s, sulfonamide antibacterials were found to be effective in treating gonorrhea. A few years thereafter, sulfonamide-resistant strains of
N. gonorrhoeae
were identified. Fortunately, by this time, penicillin was available and found to be effective against these infections. However, by the 1970's, many isolates of
N. gonorrhoeae
were found to be penicillin-resistant, requiring the use of additional, alternative drugs such as spectinomycin. It can be expected that this trend will continue, with the development of strains that are resistant to sulfonamides, penicillin, spectinomycin, and other antimicrobials.
Multiple drug resistance has been reported in a large number of clinically significant bacterial species, including
Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseriae gonorrhoeae, Staphylococcus aureus, Staphylococcus hemolyticus
, and
Streptococcus pneumoniae
. Many of these organisms are isolated from hospital environments. The development of multi-drug-resistance of nosocomial (hospital-acquired) and community-acquired pathogens to antimicrobial agents is a significant public health concern with both clinical and economic consequences.
Alarmingly, in the past few years, a handful of organisms resistant to all known antimicrobial agents has emerged (Tenover et al.,
Am. J. Med. Sci.,
311:9-16 [1996]). Though such organisms are rare, the existence of conditions favoring the development and spread of these organisms forecasts the continued emergence of multi-drug resistance. This problem is further exacerbated by the scarcity of new classes of antimicrobial agents, since many pharmaceutical manufacturers have abandoned the discovery of antimicrobial drugs in favor of more profitable products.
Fungal Infections and Drug Resistance
Fungal organisms have become increasingly significant pathogens in immunocompromised patients, especially those who because of cancer, organ transplantation, chemotherapy, pregnancy, age, diabetes, complications following extensive surgery, and various immune system dysfunctions, are at risk of experiencing life-threatening diseases caused by microorganisms which do not ordinarily pose a threat to normal, immunocompetent people. Other risk factors for deeply invasive fungal infections include protracted treatment using broad spectrum antimicrobials, corticosteroids, and vascular catheters.
Indeed, immunocompromised patients provide a significant challenge to modern health care delivery. For example, fungal infections have become one of the leading factors contributing to morbidity and mortality in cancer patients, and fungi account for 4-12% of nosocomial pathogens in leukemia patients (Anaissie,
Clin. Infect. Dis.,
14[Suppl.1]:S43 [1992]). The incidence of nosocomial bloodstream infections with fungi such as Candida spp. (“candidemia”) has increased in recent years and has been reported to account for 5.6% of all primary bloodstream infections. There are an estimated 200,000 patients/year who acquire nosocomial fungal infections, with bloodstream infections having a mean mortality rate of 55% (See e.g., Beck-Sague et al.,
J. Infect. Dis.,
167:1247 [1993]; and the Centers for Disease Control website at www.cdc.gov
cidod/publications/brochures/hip.html). Fungal infections in non-humans, such as livestock and agricultural products, is also of significant health and economic concern. The most common fungal pathogens in humans are the opportunistic yeast,
Candida albicans
and the filamentous mold,
Aspergillus fumigatus
(See, Bow,
Br. J. Haematol.,
101:1 [1998]; and Warnock,
J. Antimicrob. Chemother.,
41:95 [1998]).
C. albicans
is the most common fungal pathogen in humans, with other Candida species becoming increasingly important in fungal disease in humans and other animals (See, Walsh and Dixon, “Spectrum of Mycoses,” in Baron [ed.],
Medical Microbiology,
4th ed, University of Texas Medical Branch, Galveston, Tex. [1996], pp. 919-925). Approximately 200 Candida species are known, wit

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