Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-02
2003-09-16
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252300, C435S252340, C435S375000, C536S024500
Reexamination Certificate
active
06620585
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of enzymes which are associated with the cell (ectoenzymes) and secreted enzymes for monitoring cellular proliferation.
BACKGROUND OF THE INVENTION
Reporter enzymes are enzymes whose activities are easily assayed when present inside cells. In order to study the regulation of a gene whose expression is regulated by various environmental and/or cellular factors or influences, a gene encoding a reporter enzyme may be fused to the coding region or to the regulatory region of the regulated gene. Reporter genes may be used to determine whether a sequence contains a promoter or other cis-acting element which directs transcription, such as an enhancer. In addition, reporter genes may be used to identify regulatory sites in promoters or other cis-acting elements and to determine the effects of mutating these regulatory sites on the level of gene expression directed by the promoters or other cis-acting elements. Reporter genes may also be used to detect successful transformation, to monitor gene expression under various conditions, to assess the subcellular location of an expressed protein and to identify drugs such as antibiotics.
Since the discovery of penicillin, the use of antibiotics to treat the ravages of bacterial infections has saved millions of lives. With the advent of these “miracle drugs,” for a time it was popularly believed that humanity might, once and for all, be saved from the scourge of bacterial infections. In fact, during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated antibiotics research and development. They believed that infectious disease caused by bacteria finally had been conquered and that markets for new drugs were limited. Unfortunately, this belief was overly optimistic.
The tide is beginning to turn in favor of the bacteria as reports of drug resistant bacteria become more frequent. The United States Centers for Disease Control announced that one of the most powerful known antibiotics, vancomycin, was unable to treat an infection of the common
Staphylococcus aureus
(staph). This organism is commonly found in our environment and is responsible for many nosocomial infections. The import of this announcement becomes clear when one considers that vancomycin was used for years to treat infections caused by Staphylococcus species as well as other stubborn strains of bacteria. In short, bacteria are becoming resistant to our most powerful antibiotics. If this trend continues, it is conceivable that we will return to a time when what are presently considered minor bacterial infections are fatal diseases.
Over-prescription and improper prescription habits by some physicians have caused an indiscriminate increase in the availability of antibiotics to the public. The patients are also partly responsible, since they will often improperly use the drug, thereby generating yet another population of bacteria that is resistant, in whole or in part, to traditional antibiotics.
The bacterial pathogens that have haunted humanity remain, in spite of the development of modern scientific practices to deal with the diseases that they cause. Drug resistant bacteria are now an increasing threat to the health of humanity. A new generation of antibiotics is needed to once again deal with the pending health threat that bacteria present.
Discovery of New Antibiotics
As more and more bacterial strains become resistant to the panel of available antibiotics, new antibiotics are required to treat infections. In the past, practitioners of pharmacology would have to rely upon traditional methods of drug discovery to generate novel, safe and efficacious compounds for the treatment of disease. Traditional drug discovery methods involve blindly testing potential drug candidate-molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. The process is painstaking and laborious, with no guarantee of success. Today, the average cost to discover and develop a new drug exceeds US $500 million, and the average time from laboratory to patient is 15 years. Improving this process, even incrementally, would represent a huge advance in the generation of novel antimicrobial agents.
Newly emerging practices in drug discovery utilize a number of biochemical techniques to provide for directed approaches to creating new drugs, rather than discovering them at random. For example, gene sequences and proteins encoded thereby that are required for the proliferation of a microorganism make excellent targets since exposure of bacteria to compounds active against these targets would result in the inactivation of the microorganism. Once a target is identified, biochemical analysis of that target can be used to discover or to design molecules that interact with and alter the functions of the target. Use of physical and computational techniques to analyze structural and biochemical properties of targets in order to derive compounds that interact with such targets is called rational drug design and offers great potential. Thus, emerging drug discovery practices use molecular modeling techniques, combinatorial chemistry approaches, and other means to produce and screen and/or design large numbers of candidate compounds.
Nevertheless, while this approach to drug discovery is clearly the way of the future, problems remain. For example, the initial step of identifying molecular targets for investigation can be an extremely time consuming task. It may also be difficult to design molecules that interact with the target by using computer modeling techniques. Furthermore, in cases where the function of the target is not known or is poorly understood, it may be difficult to design assays to detect molecules that interact with and alter the functions of the target. To improve the rate of novel drug discovery and development, methods of identifying important molecular targets in pathogenic microorganisms and methods for identifying molecules that interact with and alter the functions of such molecular targets are urgently required.
To facilitate the identification of new drugs, automated assays which allow the effects of a large number of candidate compounds on microbial proliferation to be easily, rapidly and inexpensively evaluated are required. The present invention relates to the use of ectoenzymes and secreted enzymes in assays for measuring cellular proliferation.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a method for measuring cellular proliferation in a sample comprising obtaining a sample of cells which express an ectoenzyme or a secreted enzyme, determining the level of activity of the ectoenzyme or secreted enzyme in the sample and correlating the level of activity of the ectoenzyme or secreted enzyme with the extent of cellular proliferation. The step of determining the level of activity of the ectoenzyme or secreted enzyme may comprise contacting the cells with an agent which yields a detectable product when acted upon by the ectoenzyme or secreted enzyme and determining the level of the detectable product in the sample. The ectoenzyme or secreted enzyme may be selected from the group consisting of
Pseudomonas aeruginosa
metalloproteinase, Moraxella (Branhamella) catarrhalis BRO beta-lactamase,
P. aeruginosa
FpvA ferric pyoverdin receptor,
E. coli
OmpP endopeptidase, outer membrane phospholipase A,
Bacteriodes thetaiotamicron
susG starch utilization protein,
Haemophilus influenzae
phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase,
Lactococcus lactis
serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA,
S. pneumoniae
beta-N-acetylglucosaminidase,
S. pneumoniae
neuraminidase,
Streptococcus sobrinus
dextranase,
Streptococcus suis
muramidase,
Streptococcus mutans
exo-beta-D-fructosidase,
Staphylococcus aureus
murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase,
S. aureu
Elitra Pharmaceuticals Inc.
Knobbe Martens Olson & Bear LLP
LeGuyader John L.
Schultz James Douglas
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