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Methods for identifying compounds that modulate enzymatic activities by employing covalently bonded target-extender complexes with ligand candidates

Methods for identifying compounds that modulate enzymatic activities by employing covalently bonded target-extender complexes with ligand candidates description/claims

This is a continuation of application Ser. No. 10/374,499, filed Feb. 25, 2003, the entire disclosure of which is hereby expressly incorporated by reference.

The drug discovery process usually begins with massive functional screening of compound libraries to identify modest affinity leads for subsequent medicinal chemistry optimization. However, not all targets of interest are amenable to such screening. In some cases, an assay that is amenable to high throughput screening is not available. In other cases, the target can have multiple binding modes such that any result from such screens is ambiguous and difficult to interpret. Still in other cases, the assay conditions for high throughput assays are such that they are prone to artifacts. As a result, alternative methods for ligand discovery are needed that do not necessarily rely on functional screens.

FIG. 1 is a schematic illustration of one embodiment of the tethering method.

FIG. 2 is a schematic illustration of another embodiment of the tethering method.

FIG. 3 is one embodiment of an alignment of the first 298 residues of PTP-1B (SEQ ID NO: 7) and the corresponding residues for TC-PTP (SEQ ID NO: 8) and LAR (SEQ ID NO: 9). All three PTPs are human versions of the enzymes.

FIG. 4 is the mass spectrum of the R47C mutant of PTP-1B that is modified with an extender comprising a first functionality, a cleaveable linker with a latent second functionality and a phosphotyrosine mimetic. FIG. 4A is the spectrum for the R47 mutant of PTP-1B. FIG. 4B is the specturm for the R47 mutant of PTP-1B that has been modified with the extender wherein a covalent bond is formed between the thiol (on residue 47 and the first functionality). This complex is referred to as the PTP-1B-extender complex. FIG. 4C is the spectrum wherein the cleavable linker is cleaved thereby exposing the second functionality (in this case a thiol) and releasing the phosphotyrosine mimetic. This complex is referred to as the modified PTP-1B-extender complex.

The present invention relates to the use of “tethering” to identify compounds that modulate enzymatic activity.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. References, such as Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

In one aspect of the present invention, compounds are provided. Unless explicitly or implicitly indicated otherwise, these compounds can be in the form of an individual enantiomer, diasteromer, geometric isomer, or mixtures thereof. In the case of compounds containing double bonds, these double bonds can be either Z or E or a mixture thereof, unless otherwise indicated.

The definition of terms used herein include:

The term “aliphatic” or “unsubstituted aliphatic” refers to a straight, branched, cyclic, or polycyclic hydrocarbon and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

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