Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-25
2002-11-05
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S069100, C435S069700, C435S320100, C435S252300, C424S093100, C424S093200, C424S093210, C424S093400, C424S093420
Reexamination Certificate
active
06475726
ABSTRACT:
BACKGROUND OF THE INVENTION
In the discovery and development of new drugs, it is a common strategy to first try to identify molecules or complexes of molecules, naturally occurring within cells, that are involved in producing symptoms of a disease. These naturally occurring molecules can be thought of as “targets.” A second major part of the strategy is then to find molecules that bind to the targets. These molecules are candidates for drug development, on the theory that a molecule that binds to a target can modulate (inhibit or enhance) the function of the target, thereby causing a change in the biological status of the cell containing the target. The change caused in the cell (e.g., a change in phenotype towards wild type, or a change in growth rate) may be therapeutically beneficial to the animal or human host of the cell.
The genomics revolution, by determining the DNA sequences of great numbers of genes from many different organisms, has considerably broadened the possibilities for drug discovery by identifying large numbers of molecules that are potential targets of drug action. These technical advances in genomics however, have posed an entirely new set of challenges. Specifically, how can one prove that a chosen target molecule is essential to maintaining the disease or disorder to be treated? That is, how does one validate a target? (See “target validation” in Definitions.)
Although methods currently available to validate targets do provide some guidelines in selection of drug targets, they are usually not conducted under the conditions in which a drug actually interacts with its target, and therefore provide a limited set of information. In addition, they do not directly address, among other things: 1) if a wild type (normal) target is essential for cell growth and viability during the disease state; 2) if the wild type gene products themselves are suitable targets for drug discovery; 3) if specific sites on a target are suitable for drug interaction (for example, in a pathogenic organism, there can be one gene coding for a single protein target with two activities—one activity essential for growth and infectivity, the second activity non-essential); 4) if a compensatory mechanism in the cell, either in vitro or in vivo, can overcome or compensate for target modulation or, 5) if a disease state can be cured by modulation of function of the candidate target. These methods also do not provide a direct route for testing wild type target proteins in high throughput screening assays.
An analysis of the discovery of novel antimicrobial agents illustrates the problems researchers in all fields of drug development face today. The increasing prevalence of drug-resistant pathogens (bacteria, fungi, parasites, etc.) has led to significantly higher mortality rates from infectious diseases and currently presents a serious crisis worldwide. Despite the introduction of second and third generation antimicrobial drugs, certain pathogens, such as vancomycin resistant strains of Enterococcusfacieum, have developed resistance to all currently available drugs. New antimicrobial drugs must be discovered to treat such infections by such organisms, and new methods are urgently needed to facilitate making such discoveries.
Neither whole cell screening, chemistry nor target based drug discovery approaches as currently applied, have met the challenge of controlling infectious diseases, particularly those caused by drug resistant microorganisms. Whole cell screening assays have been limited by the fact that they are unable to identify compounds that can effectively modulate a target function inside the cell but cannot permeate the cell membrane to get to the target. Therefore entire classes of potent, intracellular target modulators, which could be subsequently modified by medicinal chemistry to increase cell membrane permeability, go undetected. Chemistry based approaches have focused on chemically modifying the molecular structure of existing antimicrobial drugs or combining existing antimicrobials with another agent to circumvent established resistance mechanisms. Technical advances in molecular biology, automated methods for high throughput screening and chemical syntheses have led to an increase in the number of target based screens utilized for antimicrobial drug discovery and in the number of compounds being analyzed. However, despite these advances, only a limited number of antimicrobial drugs acting by a novel mechanism have been identified during recent years.
How does one efficiently establish screening assays for drugs that can be used with a variety of different targets having different properties, enzymatic activities, or even unknown functions? A number of potentially novel, valuable targets are incompatible with current methods to screen for drug candidates because either the target's exact function and molecular mechanism of action are unknown, or there are technical obstacles preventing the development of effective high throughput screening methods. It can take anywhere from six months to several years to develop a screening assay, which is impractical when the goal is to rapidly screen multiple targets in a cost-effective manner.
The path in the progression from target identification through assay development, high throughput screening, medicinal chemistry, lead optimization, preclinical and clinical drug development is expensive, time consuming and full of technical challenges. Many different targets must be screened against multiple chemical compounds to identify new lead compounds for drug development. New, efficient technologies are needed that can be broadly applied to a variety of different targets to validate targets in the direct context of the desired outcome of drug therapy and to rapidly develop screening assays using these targets for drug discovery. Such developments will allow the wealth of genomics information to be leveraged for drug discovery and will lower the risk and costs while expediting the timelines of the drug discovery process.
SUMMARY OF THE INVENTION
The invention relates to methods that couple the validation of a target (see Definitions) for drug discovery with the development of an assay to identify compounds that cause a phenotypic effect on the target cell. These procedures can be applied to identifying compounds that bind to and modulate the function of target components of a cell whose function is known or unknown, and cell components that are not amenable to other screening methods.
The invention relates to procedures for identifying a compound that binds to and modulates (inhibits or enhances) the function of a component of a cell, thereby producing a phenotypic effect in the cell. Within these procedures are methods for identifying a biomolecule (See Definitions section) that 1) binds to, in vitro, a component of a cell that has been isolated from other constituents of the cell and that 2) causes, in vivo, as seen in an assay upon intracellular expression of the biomolecule, a phenotypic effect (See Definitions section) in the cell which is the usual producer and host of the target cell component. In an assay demonstrating characteristic 2) above, intracellular production of the biomolecule can be in cells grown in culture or in cells introduced into an animal. Further methods within these procedures are those methods comprising an assay for a phenotypic effect in the cell upon intracellular production of the biomolecule, either in cells in culture or in cells that have been introduced into one or more animals, and an assay to identify one or more compounds that behave as competitors of the biomolecule in an assay of binding to the target cell component.
One procedure envisioned in the invention is a process for identifying one or more compounds that produce a phenotypic effect on a cell. The process is at the same time a method for target validation (See Definitions section). The process is characterized by identifying a biomolecule which binds an isolated target cell component, constructing cells comprising the t
Connelly Gene
Gallant Paul L.
Shen Xiaoyu
Tally Francis P.
Tao Jianshi
Cubist Pharmaceuticals, Inc.
Guzo David
Leffers, Jr. Gerald G.
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