Cysteine mutants and methods for detecting ligand binding to biological molecules

#The drug discovery process usually beings with massive functional screening of compound libraries to identify modest affinity leads (Kd˜1 to 10 μM) 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 the results of such screens are ambiguous and difficult to interpret. Still in other cases, the assay conditions for high throughput screening are such that they are prone to artifacts. As a result, alternative methods for ligand discovery are needed that to not necessarily rely on functional assays. The present invention provides such methods.

#The present invention relates generally to variants of target biological molecules (“TBMs”) and to methods of making and using the same to identify ligands of TBMs. More specifically, the invention relates to individual variant TBMs and sets of variant TBMs, each of which represents a modified version of a protein of interest where a thiol has been introduced at or near a site of interest. Ligands of TBMs are identified in part through the formation of a covalent bond between a potential ligand and a reactive thiol on the TBM.

#FIG. 1 schematically illustrates one embodiment of the tethering method wherein the target is a protein and the covalent bond is a disulfide. A thiol-containing protein is reacted with a plurality of ligand candidates. A ligand candidate that possesses an inherent binding affinity for the target is identified and a ligand is made comprising the identified binding determinant (represented by the circle).

#FIG. 2 is a representative example of a tethering experiment. FIG. 2A is the deconvoluted mass spectrum of the reaction of thymidylate synthase (“TS”) with a pool of 10 different ligand candidates with little or no binding affinity for TS. FIG. 2B is the deconvoluted mass spectrum of the reaction of TS with a pool of 10 different ligand candidates where one of the ligand candidates possesses an inherent binding affinity to the enzyme.

#FIG. 3 shows three illustrative examples of the distribution pattern of the residues that are each mutated to a cysteine. FIG. 3A is an example where the residues are distributed about a single site of interest. The structure is of the core domain of HIV integrase with the portion comprising the site of interest shaded in dark gray. FIG. 3B is an example where the residues are distributed about two sites of interest. The structure is of the human interleukin-1 receptor with the portions comprising the two sites of interested shaded in dark gray. FIG. 3C is an example where the residues are distributed throughout the surface of a protein. The structure is the trimeric structure of human TNF-α.

#The present invention relates generally to variants of target biological molecules (“TBMs”) and to methods of making and using the same to identify ligands of TBMs.

#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.

#Definitions