Fitness assay and associated methods

Inactive Publication Date: 2005-07-21
THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018] The present invention is predicated on the surprising and unexpected discovery that biochemical “vitality,” as described below, can be used to determine the biological fitness of a mutant replicating biological entity relative to its predecessor under the selection pressure of an inhibitor. The present invention provides an assay for determining the biochemical fitness of a biochemical target (i.e., a biomolecule having a biochemical function), of a mutant replicating biological entity relative to its predecessor's biochemical target, in the presence of a compound that acts upon the biochemical target. The assay method of the present invention includes obtaining the predecessor, determining the biochemical vitality of the biochemical target of both the predecessor and the mutant in the presence of a compound that acts upon the biochemical target of the predecessor, and comparing the vitality of the mutant's biochemical target relative to the vitality of the predecessor's biochemical target. Where the biochemical vitality of the mutant is greater than the biochemical fitness of the predecessor, the mutant is predicted to be more biologically fit in the presence of the compound. The assay method can thus be used to predict the emergence of drug resistance for a particular replicating biological-entity (e.g., a disease-causing cell) in the presence a drug (e.g., an inhibitor). Utilization of the assay in accordance with the present invention permits the administrations of an inhibitor or combination of inhibitors to treat a disease in a way that decreases the likelihood that drug resistance will develop.
[0020] The present invention further provides a method of administering a therapeutic compound that inhibits a biochemical target of a disease-causing replicating biological entity. The therapeutic compound, when administered in accordance with the method of the present invention, minimizes the chances that the disease-causing entity will develop drug resistance. As such, the method of administering a therapeutic compound in accordance with the present invention improves the chances of long-term success in therapy.

Problems solved by technology

The development of drug resistance is one of the most perplexing challenges in the field of medicine.
One of the most common causes of drug failure in the treatment of diseases involving replicating biological entities, for example, cancer and infectious diseases, is the emergence of drug resistance.
Specifically, HIV is known to exert a profound cytopathic effect on the CD4+ helper / inducer T-cells, thereby severely compromising the immune system.
HIV infection also results in neurological deterioration and, ultimately, in the death of the infected individual.
Although very useful, the utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy.
Although these inhibitors are effective in preventing the retroviral protease from functioning, the inhibitors suffer from some distinct disadvantages.
Generally, peptidomimetics often make poor drugs, due to their potential adverse pharmacological properties, i.e., poor oral absorption, poor stability and rapid metabolism (Plattner et al, Drug Discovery Technologies, Clark et al., eds., Ellish Horwood, Chichester, England (1990)).
Typically, however, the usefulness of currently available HIV protease inhibitors in the treatment of AIDS has been limited by relatively short plasma half-life, poor oral bioavailability, and the technical difficulty of scale-up synthesis (Meek et al.
Tragically for these patients, the available options for AIDS chemotherapy and / or HIV management is severely limited or is, otherwise, completely nonexistent.
Drug resistance is unfortunately the most common reason for drug failures generally.
Once HIV resistance is obtained to first-line therapy, the chances of future success are greatly diminished because of the development of multidrug cross resistance.
Other diseases involving infectious agents (e.g., viruses, bacteria, protozoa, and prions) or other disease-causing cells (e.g., tumor cells) present similar challenges in that drug resistance is a primary cause of drug failure.

Method used

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  • Fitness assay and associated methods
  • Fitness assay and associated methods
  • Fitness assay and associated methods

Examples

Experimental program
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example 1

[0190] This example describes the synthesis of exemplary epoxide 11 (FIG. 1), which is used as an intermediate in the synthesis of a particular series of compounds within the scope of the present invention.

[0191] Anhydrous CuCN (4.86 g, 54 mmol) was added to a solution of butadiene monooxide (38 g, 540 mmol) in anhydrous tetrahydrofuran (1.2 L) and the resulting mixture was stirred at −78° C. Commercial phenyl magnesium bromide solution (Aldrich) in ether (65 mmol) was added dropwise over a period of 10 min. The resulting reaction mixture was then allowed to warm to 0° C. and it was continued to stir until the reaction mixture was homogeneous. After this period, the reaction mixture was cooled to −78° C. and 0.58 mole of phenylmagnesium bromide solution in ether was added dropwise for 30 min. The reaction mixture was allowed to warm to 23° C. for 1 h. The reaction was quenched by slow addition of saturated aqueous NH4Cl (120 mL) followed by NH4OH (70 mL), saturated NH4Cl (500 ML) a...

example 2

[0196] This example illustrates the synthesis of azidoalcohol 13 (FIG. 1), which can be used as an intermediate in the synthesis of a preferred series of the compounds of the present invention.

[0197] To a stirred solution of above azidoepoxide 11 (700 mg, 3.7 mmol) in ispropanol (70 mL) was added isobutyl amine (Aldrich, 0.74 mL 7.4 mmol) and the resulting mixture was heated at 80° C. for 12 h. After this period, the reaction mixture was concentrated under reduced pressure and the residue was chromatographed over silica gel to provide azidoalcohol 13 (800 mg) as an oil.

example 3

[0198] This example illustrates the synthesis of azidosulfonamide 14, the structure of which is shown in FIG. 1.

[0199] To a stirred solution of 13 (600 mg, 2.28 mmol) in CH2Cl2 (20 mL) was added 4-methoxybenzenesulfonyl chloride (Aldrich, 530 mg, 2.52 mmol) and saturated aqueous NaHCO3 (6 mL). The resulting heterogeneous mixture was stirred at 23° C. for 12 h. The reaction was diluted with CH2Cl2 and the layers were separated. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated to dryness. The residue was chromatographed over silica gel (25% ethyl acetate / hexane) to provide 900 mg of azidosulfonamide 14.

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Abstract

The present invention provides an assay for determining the biochemical fitness of a biochemical species in a mutant replicating biological entity relative to its predecessor. The present invention further provides a continuous fluorogenic assay for measuring the anti-HIV protease activity of a protease inhibitor. The present invention also provides a method of administering a therapeutic compound that reduces the chances of the emergence of drug resistance in therapy. The present invention also provides a compound of the formula: or a pharmaceutically acceptable salt, a prodrug, a composition, or an ester thereof, wherein A is a group of the formula: R1, R2, R3, R5, or R6 is H, or an optionally substituted and / or heteroatom-bearing alkyl, alkenyl, alkynyl, or cyclic group; Y and / or Z are CH2, O, S, SO, SO2, amino, amides, carbamates, ureas, or thiocarbonyl derivatives thereof, optionally substituted with an alkyl, alkenyl, or alkynyl group; n is from 1 to 5; X is a bond, an optionally substituted methylene or ethylene, an amino, 0 or S; Q is C(O), C(S), or SO2; m is from 0 to 6; R4 is OH, ═O (keto), NH2, or alkylamino, including esters, amides, and salts thereof; and W is C(O), C(S), S(O), or SO2. Optionally, R5 and R6, together with the N—W bond of formula (I), comprise a macrocyclic ring.

Description

TECHNICAL FIELD OF THE INVENTION [0001] The present invention relates to a biochemical fitness assay and related methods. BACKGROUND OF THE INVENTION [0002] The development of drug resistance is one of the most perplexing challenges in the field of medicine. One of the most common causes of drug failure in the treatment of diseases involving replicating biological entities, for example, cancer and infectious diseases, is the emergence of drug resistance. One of the most dramatic and tragic examples of drug resistance can be found in connection with the antiviral therapy of acquired immune deficiency syndrome (AIDS). [0003] AIDS is a fatal disease, reported cases of which have increased dramatically within the past several years. Estimates of reported cases in the very near future also continue to rise dramatically. [0004] The AIDS virus was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphocyte virus (HTLV-III), and it has the c...

Claims

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Application Information

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IPC IPC(8): A61K31/34A61K31/4525G01N33/15A61P31/18A61P37/04A61P43/00C07D493/04C12Q1/02C12Q1/37
CPCA61K31/34A61K31/496A61K31/426A61K31/4523A61K31/4525A61K31/47C12Q1/025C12Q1/37G01N2333/16G01N2333/8142A61K31/341A61K31/343A61K31/427A61K31/4725A61K31/35A61P31/00A61P31/04A61P31/12A61P31/14A61P31/18A61P33/06A61P35/00A61P37/04A61P43/00Y02A50/30
Inventor ERICKSON, JOHN W.GULNIK, SERGEI V.MITSUYA, HIROAKIGHOSH, ARUN K.
Owner THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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