Solid-phase synthesis of double-headed epoxysuccinyl activity-based probes for selective targeting of papain family cysteine proteases.

The completion of genome sequencing of several organisms, including man, has provided global information on gene regulation. Emphasis has now shifted to understanding protein function. To facilitate functional studies, small molecules that label subsets of enzymatic proteins have been developed as a means to simplify complex proteomes and allow bulk profiling of enzyme activity. These activity-based probes (ABPs) combine tags for visualization or purification with moieties that covalently attach to the active sites of enzymatic targets in an activity-dependant manner. By using this approach, a specific protein or protein family can be readily monitored in complex protein mixtures, in intact cells, and even in vivo. Furthermore, enzyme-class-specific probes can be used to develop screens for small-molecule inhibitors that can be used in functional studies. 5] Therefore, methods that will facilitate the development of novel ABPs have great value for advancing the use of this technology. Proteolysis controls a number of essential biological processes ranging from cell division to cell death. Amongst the cysteine proteases, the lysosomal cathepsins play important roles in a number of human diseases. However, the members of this family display similar substrate preferences, thus making the development of tools to study individual cathepsins a challenging task. The natural product E-64 (1; Scheme 1) inhibits cysteine proteases by covalent attachment to the active-site sulfhydryl nucleophile. This reagent contains a leucine residue that mimics the critical P2 residue of a substrate and therefore binds efficiently in the S2 specificity pocket of virtually all cysteine cathepsins. As a result this reagent is a broad-spectrum inhibitor and activity probe. Recently, our laboratory has reported peptide-based ABPs based on the E-64 scaffold. One of the first ABPs reported with the epoxide moiety, DCG-04 (2) has found widespread use for a number of functional studies of the cysteine cathepsins. However, this general probe makes use of a single peptide piece that makes contacts with only one side of the protease active site. A recent crystal structure of a double-headed epoxide inhibitor showed that the entire structure binds along the active-site cleft, with contact made on both sides of the reactive cysteine nucleophile. Thus, we reasoned that development of a method that would allow facile attachment of diverse peptide sequences on both sides of the epoxide moiety would facilitate the synthesis of probes with increased selectivity compared to the general DCG-04 probe. Here we report our preliminary results involving the solid-phase synthesis of cathepsin B-specific ABPs. In the past, selective inhibitors for both cathepsins B and L have been reported. Most of these compounds, such as CA074 (3), have taken advantage of diversity elements on both sides of the epoxide. In fact, virtually all of the selectivity for cathepsin B has derived from specific interactions with residues on the occluding loop found only in this protease. This unique specific interaction has been used to generate small molecules that target cathepsin B. However, the previously reported inhibitors and probes were obtained by time-consuming solution-phase synthesis, thus limiting the extent of structure– activity studies that could be performed. In order to efficiently synthesize double-headed epoxysuccinyl probes, we needed to design a solid-phase synthesis protocol that would allow peptide synthesis to be carried out followed by attachment of the epoxide group and then further elongation of the probe. Our initial probe design was based on the broad-spectrum ABP DCG-04 (2) containing the dipeptide of CA-074 (3). Scheme 2 outlines the basic strategy which makes use of standard solid-phase peptide chemistry followed by capping of the N terminus with the epoxysuccinyl synthon by using the activated nitrophenyl ester 4. The epoxysuccinyl ester 5 can then be hydrolyzed on resin thereby allowing elongation of the probe on the other side of the epoxide moiety. The use of a PEG-based Rink resin permitted the use of alcohols as a solvent system during this ester-bond cleavage. Thus, hydrolysis of ethyl ester 5 was achieved by treatment with a [a] Dr. S. H. L. Verhelst, Dr. M. Bogyo Departments of Pathology, and Microbiology and Immunology Stanford University School of Medicine 300 Pasteur Drive, Stanford, CA 94305 (USA) Fax: (+1)650-725-7424 E-mail : mbogyo@stanford.edu Scheme 1. Epoxysuccinyl-based cysteine protease inhibitors E-64 (1), CA-074 (3), and activity-based probe DCG-04 (2).