Optical molecular sensors for cytochrome P450 activity

Abstract

The invention provides a compound, useful as an optical probe or sensor of the activity of at least one cytochrome P450 enzyme, and methods of using the compound to screen candidate drugs, and candidate drugs identified by these methods. The optical probe of the invention is a compound having the generic structure Y-L-Q, wherein Y is selected from the group consisting of Q as herein defined, saturated C1-C20 alkyl, unsaturated C1-C20 alkenyl, unsaturated C1-C20 alkynyl, substituted saturated C1-C20 alkyl, substituted unsaturated C1-C20 alkenyl, substituted unsaturated C1-C20 alkynyl, C1-C20 cycloalkyl, C1-C20 cycloalkenyl, substituted saturated C1-C20 cycloalkyl, substituted unsaturated C1-C20 cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; L is selected from the group of (—OCR2H)p—, wherein for each p, all R2 are separately selected from the group consisting of a hydrogen atom, saturated C1-C20 alkyl, unsaturated C1-C20 alkenyl, unsaturated C1-C20 alkynyl, substituted saturated C1-C20 alkyl, substituted unsaturated C1-C20 alkenyl, substituted unsaturated C1-C20 alkynyl, C1-C20 cycloalkyl, C1-C20 cycloalkenyl, substituted saturated C1-C20 cycloalkyl, substituted unsaturated C1-C20 cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and p is a positive integer no greater than twelve; and Q is a chemical moiety that gives rise to optical properties in its hydroxy or hydroxylate, phenol or phenoxide form that are different from the optical properties that arise from its ether form. Most preferably, p is one, R2 is hydrogen, and Q is the ether form of a phenoxide fluorophore.

Description

[0001]This application claims priority to all of the following applications and, specifically, is a continuation of pending application Ser. No. 09 / 458,927, filed Dec. 10, 1999, which application is a continuation-in-part of U.S. patent application Ser. No. 09 / 301,525, OPTICAL MOLECULAR SENSORS FOR CYTOCHROME P450 ACTIVITY, filed Apr. 28, 1999, by Makings et al., now U.S. Pat. No. 6,420,130, which applications claim priority from U.S. Provisional Application Ser. No. 60 / 112,252, OPTICAL MOLECULAR SENSORS FOR CYTOCHROME P450 ACTIVITY, filed Dec. 14, 1998, by Makings et al. The entire disclosures of all of the applications to which priority is claimed are considered as being part of the disclosure of the accompanying application and are each hereby incorporated by reference therein.STATEMENT OF GOVERNMENT RIGHTS[0002]This invention was developed in part, under grant number 1R43GM60114-01 from the National Institutes of General Medicine Sciences. The government may have certain rights ...

AI Technical Summary

Benefits of technology

[0012]The invention provides a compound, useful as an optical probe, modulator or sensor of the activity of at least one cytochrome P450 enzyme. The optical probe of the invention is a compound having the generic structure Y-L-Q, wherein Y is selected from the group consisting of Q as herein defined (such that the probe has the general structure Q-L′-Q), and saturated C1-C20 alkyl, unsaturated C1-C20 alkenyl, unsaturated C1-C20 alkynyl, substituted saturated C1-C20 alkyl, substituted unsaturated C1-C20 alkenyl, substituted unsaturated C1-C20 alkynyl, C1-C20 cycloalkyl, C1-C20 cycloalkenyl, substituted saturated C1-C20 cycloalkyl, substituted unsaturated C1-C20 cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; L is selected from the group of (—OCR2H)p—, (—O(substituted ortho-phenyl)CR2H)p—, (—O(substituted meta-phenyl)CR2H)p—, and (—O(substituted para-phenyl)CR2H)p—, and L′ is selected from the group of —(CR4H)(—OCR2H)p—, —(CR4H)(—O(substituted ortho-phenyl)CR2H)p—, (CR4H)(—O(substituted meta-phenyl)CR2H)p—, and —(CR4H)(—O(substituted para-phenyl)CR2H)p—, wherein for each p, each R2 is separately selected from the group consisting of a hydrogen atom, saturated C1-C20 alkyl, unsaturated C1-C20 alkenyl, unsaturated C1-C20 alkynyl, substituted saturated C1-C20 alkyl, substituted unsaturated C1-C20 alkenyl, substituted unsaturated C1-C20 alkynyl, C1-C20 cycloalkyl, C1-C20 cycloalkenyl, substituted saturated C1-C20 cycloalkyl, substituted unsaturated C1-C20 cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl and p is a positive integer no greater than twelve; and Q is a chemical moiety that gives rise to optical properties in its hydroxy or hydroxylate, phenol or phenoxide form that are different from the optical properties that arise from its ether form. Most preferably, p is one, R2 is hydrogen, and Q is the ether form of a phenoxide fluorophore. Most preferably, the ether form of the phenoxide fluorophore are ether derivatives of the well-known phenoxide type fluorophores 7-hydroxycoumarin, fluorescein, and resorufin.

[0013]The invention also provides methods for using the optical sensor compounds of the invention to determine whether a candidate drug, or class of candidate drugs, is a CYP450 substrate and / or whether the candidate drug, or class of candidate drugs, is a CYP450 inhibitor, and related methods for selecting a candidate drug, and for formulating and administering that drug, having determined that the drug will not be metabolized by at least one CYP450 enzyme and / or that the drug will not act as an inhibitor of at least one CYP450 enzyme, and, thus, having determined that the drug will not, respectively, be too efficiently metabolized by a CYP 450 enzyme and / or elicit an unfavorable drug-drug interaction. Methods of selecting the candidate drug of the present invention may be by conventional methods or may be part of high or ultra high throughput screening of libraries of drug candidates.

Problems solved by technology

Efficient metabolism of a candidate drug by a CYP450 enzyme may lead to poor pharmacokinetic properties, while drug candidates that act as potent inhibitors of a CYP450 enzyme can cause undesirable drug-drug interactions when administered with another drug that interacts with the same CYP450.

Consequently, such earlier-available, reliable CYP450 pharmokinetic information may result in greatly reduced drug development costs and / or increased profits from earlier market entrance.

However, even the most recently developed fluorogenic CYP450 substrates either have relatively poor kinetics, or the enzymatic products do not have the desired physical and optical properties to allow reduction of the amount of enzyme needed to levels that would make large scale screening affordable and feasible.

More specifically, these fluorogenic CYP450 substrates exhibit relatively poor turnover rates, poor aqueous solubility, low extinction coefficients and quantum yields, and / or weak fluorescence of the resultant phenolic dye.

Finally, most of these fluorogenic CYP450 substrates are not specific for the CYP450 isozyme they are meant to detect, and therefore cannot be used for measurement in human liver microsomal preparations, a preferred analytical method that avoids potential artifacts caused by the alternative method of using an insect cell microsomal preparation.

Images