Identification of novel antimicrobial agents using membrane...

Details Identification of novel antimicrobial agents using membrane... Identification of novel antimicrobial agents using membrane...

2000-07-27

2002-09-24

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S032000, C435S004000, C435S911000, C435S968000, C549S227000, C430S585000

Reexamination Certificate

active

06455271

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to screening methods involving use of membrane potential indicator dyes for identifying antimicrobial agents, including antifungal and antibacterial compounds.
BACKGROUND OF THE INVENTION
Fungi are not only important human and animal pathogens, but they are also among the most common causes of plant disease. Fungal infections (mycoses) are becoming a major concern for a number of reasons, including the limited number of antifungal agents available, the increasing incidence of species resistant to known antifungal agents, and the growing population of immunocompromised patients at risk for opportunistic fungal infections, such as organ transplant patients, cancer patients undergoing chemotherapy, burn patients, AIDS patients, or patients with diabetic ketoacidosis. The incidence of systemic fungal infections increased 600% in teaching hospitals and 220% in non-teaching hospitals during the 1980's. The most common clinical isolate is
Candida albicans
(comprising about 19% of all isolates). In one study, nearly 40%of all deaths from hospital-acquired infections were due to fungi. [Sternberg,
Science,
266:1632-1634 (1994).]
Known antifungal agents include polyene derivatives, such as amphotericin B (including lipid or liposomal formulations thereof) and the structurally related compounds nystatin and pimaricin; flucytosine (5-fluorocytosine); azole derivatives (including ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole, itraconazole, voriconazole [Pfizer] and SCH56592 [Schering-Plough]); allylamines-thiocarbamates (including toinaftate, naftifine and terbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins (including MK-0991 [Merck]); and nikkomycins. Recently discovered as antifungal agents are a class of products related to bactericidal/permeability-increasing protein (BPI), described in U.S. Pat. Nos. 5,627,153, 5,858,974, 5,652,332, 5,763,567 and 5,733,872, the disclosures of all of which are incorporated herein by reference.
Resistance of bacteria and other pathogenic organisms to antimicrobial agents is an increasingly troublesome problem. The accelerating development of antibiotic-resistant bacteria, intensified by the widespread use of antibiotics in farm animals and overprescription of antibiotics by physicians, has been accompanied by declining research into new antibiotics with different modes of action. [
Science,
264: 360-374 (1994).]
Gram-positive bacteria have a typical lipid bilayer cytoplasmic membrane surrounded by a rigid cell wall that gives the organisms their characteristic shape, differentiates them from eukaryotic cells, and allows them to survive in osmotically unfavorable environments. This cell wall is composed mainly of peptidoglycan, a polymer of N-acetylglucosamine and N-acetylmuramic acid. In addition, the cell walls of gram-positive bacteria contain teichoic acids which are anchored to the cytoplasmic membrane through lipid tails, giving rise to lipoteichoic acids. The various substituents on teichoic acids are often responsible for the biologic and immunologic properties associated with disease due to pathogenic gram-positive bacteria. Most pathogenic gram-positive bacteria have additional extracellular structures, including surface polysaccharides, capsular polysaccharides, surface proteins and polypeptide capsules.
Gram-negative bacteria also have a cytoplasmic membrane and a peptidoglycan layer similar to but reduced from that found in gram-positive organisms. However, gram-negative bacteria have an additional outer membrane that is covalently linked to the tetrapeptides of the peptidoglycan layer by a lipoprotein; this protein also contains a special lipid substituent on the terminal cysteine that embeds the lipoprotein in the outer membrane. The outer layer of the outer membrane contains the lipopolysaccharide (LPS) constituent.
Antibacterial agents are generally directed against targets not present in mammalian cells. One major difference between bacterial and mammalian cells is the presence in bacteria of a rigid wall external to the cell membrane. Thus, chemotherapeutic agents directed at any stage of the synthesis, export, assembly, or cross-linking of peptidoglycan lead to inhibition of bacterial cell growth and, in most cases, to cell death. These agents include bacitricin, the glycopeptides (vancomycin and teichoplanin), &bgr;-lactam antibiotics (penicilins, cephalosporins, carbapenems, and monobactams). Virtually all the antibiotics that inhibit bacterial cell wall synthesis are bactericidal. However, much of the loss of cell wall integrity following treatment with cell wall-active agents is due to the bacteria's own cell wall-remodeling enzymes (autolysins) that cleave peptidoglycan bonds in the normal course of cell growth. In the presence of antibacterial agents that inhibit cell wall growth, autolysis proceeds without normal cell wall repair; weakness and eventually cellular lysis occur. There are also antibacterial agents that do not affect cell wall synthesis but instead are believed to alter cell membrane permeability, such as the polymyxins (polymyxin B and colistin, or polymyxin E) and gramicidin A.
Another group of antibacterial agents are those that inhibit protein synthesis; most of these interact with the bacterial ribosome. The difference between the composition of bacterial and mammalian ribosomes gives these compounds their selectivity. These agents include the aminoglycosides (e.g., gentamicin, kanamycin, tobramycin, streptomycin, netilmicin, neomycin, and amikacin), the macrolides (e.g., erythromycin, clarithromycin, and azithromycin), the lincosamides (e.g., clindamycin and lincomycin), chloramphenicol, the tetracyclines (e.g., tetracycline, doxycycline, and minocycline) and mupirocin (pseudomonic acid).
Another group of antibacterial agents are antimetabolites that interfere with bacterial synthesis of folic acid. Inhibition of folate synthesis leads to cessation of cell growth and, in some cases, to bacterial cell death. The principal antibacterial antimetabolites are sulfonamides (e.g., sulfisoxazole, sulfadiazine, and sulfamethoxazole) and trimethoprim.
Yet a further group of antibacterial compounds affects nucleic acid synthesis or activity. These agents include the quinolones (e.g., nalidixic acid and its fluorinated derivatives norfloxacin, ciprofloxicin, ofloxacin, and lomofloxacin), which inhibit the activity of the A subunit of DNA gyrase, rifampin, nitrofurantoin, and metronidazole (which not only has activity against the electron transport system but also is believed to cause DNA damage).
BPI protein products are also described to have antibacterial activities in U.S. Pat. Nos. 5,198,541 and 5,523,288 and International Publication No. WO 95/08344 (PCT/US94/11255), all of which are incorporated by reference herein, disclosing activity against gram-negative bacteria, and U.S. Pat. Nos. 5,578,572 and 5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656), all of which are incorporated by reference herein, disclosing activity against gram-positive bacteria and mycoplasma, and co-owned, co-pending U.S. application Ser. No. 08/626,646, which is in turn a continuation of U.S. application Ser. No. 08/285,803, which is in turn a continuation-in-part of U.S. application Ser. No. 08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463), all of which are incorporated by reference herein, disclosing activity against mycobacteria.
BPI protein products have been shown to have additional antimicrobial activities. For example, U.S. Pat. No. 5,646,114 and International Publication No. WO 96/01647 (PCT/US95/08624), all of which are incorporated by reference herein, disclose activity of BPI protein products against protozoa.
Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutro

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