of cysteine proteases in crude cell lysates (23). Both techniques use only very small quantities of proteins as well as whole cell lysates, which permit the screening of small molecules across the soluble proteome.

Catalytic RNAs are also being used for small molecule screening to identify protein-protein disruptors. A novel screen described last year employs protein-dependent RNA catalysts (ribozymes) that can monitor protein-protein, protein-RNA, and protein-small molecule interactions in real time using changes in fluorescence as a readout (24). Ribozymes have been engineered with fluorescent tags and nucleic acid-protein binding domains. Binding of a protein target to the reporter ribozyme alters the ribozyme's self-cleavage activity, which can be monitored by changes in the amount of fluorescence detected. In this way, a library of antibiotics was screened by monitoring fluorescence changes to inhibit the interaction between the HIV-1 Rev protein and Rev-binding element (RBE). An inhibitor was identified which attenuated HIV-1 replication in cells. In a second example, it was possible to monitor interactions between the blood-clotting factor thrombin and its protein partners. The approach appears to be applicable to many targets, and particularly suitable for protein-protein interactions.

A novel application of FTIR to study protein-protein interactions has recently been reported. This approach combines FTIR with principle components analysis and protein titration experiments to identify association-induced changes in protein structure (25). The system studied was the complex formation between bacterial monooxygenase Cytochrome P450BM-3 Heme Domain and Flavin Mononucleotide (FMN) Reductase Domain. The solution phase secondary structure for each protein was established and compared both with the crystal structure and the solution-phase changes in structure on formation of the complex. Indeed, the structures for the BMD-FMND complex were different in solution relative to the X-ray.

New Chemical Approaches to Small Molecule Disruptors of Protein-Protein Interactions - A number of approaches to evaluate the ability of small molecules to disrupt protein-protein interactions have emerged recently from diversity oriented synthesis to designed and targeted systems. They are discussed in a thorough recent review (26).

Recently, a synthetic molecule (2) has been designed to display a large, functionalized and variable interaction exterior which bound to the surface of chymotrypsin and disrupted its interaction with some of its protein inhibitors (PI). In the case of chymotrypsin - soybean trypsin inhibitor, the mechanism appears to involve the formation of an initial ternary complex with time-dependent displacement of the PI. This represents the first example of a synthetic agent that blocks the interaction of a protease and its PI. As such, it is an unusual illustration of a strategy which seeks to prevent natural association of proteins by blocking large surface areas of that interaction.

This type of synthetic design has been adapted to a combinatorial approach (27). The approach is to generate libraries of compounds with two or more binding groups separated by variable linkers which can interact with one or more protein targets. In this case, 600 symmetrical dimeric structures with three diversity subunits were used to identify a prototypical inhibitor (3) of the association of MMP2 with integrin avf}3 in an in vitro binding assay. Interestingly, the inhibitor did not directly inhibit MMP2 activity or disrupt the binding of integrin avp3 to its natural extracellular matrix target, vitronectin, but bound integrin a^ in a dose-dependent manner. Such a mechanism suggests that this agent and others that are similar may have both direct and indirect effects mediated by perturbations of smaller, more specific protein-protein interactions than that of 2. Compound 3 possesses in vivo activity (suppression of angiogenesis in a chick CAM model and near complete reduction of solid tumors in xenograph models), making it an impressive example of small molecule intervention at protein-protein interaction sites (28). The design of solution-phase combinational libraries for the modulation of both protein-protein and proteinDNA interactions has been successfully generalized (29).

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