Targets for Protein-Protein Intervention - Recently available methods for probing, detecting and identifying specific functional activities of proteins have allowed evaluation of opportunities where intervention may lead to the understanding of fundamental processes in diseases as well as to drug therapy. For instance, apoptosis, an essential process in both normal development and in the disease state, is initiated and regulated by protein-protein interactions. The use of small molecule ligands targeting the Bcl-2 or Bcl-xL systems as well as the interactions of lAPs with caspases and Smac/DIABLO have been highlighted (30).
Examples in the past year of new protein-protein targets which have been the subject of small molecule disruption include the dimerization of the strongly oncogenic Myc transcription factor with Max, a basic helix-loop-helix leucine zipper protein (31). The library of small molecules utilized in this study emerged from a rigid bicyclic core resembling an Arg-Gly-Asp (RGD) mimic with substituents inspired by recent advances which identified nonclassical peptide-like compounds as high affinity ligands for integrin avp3 receptors. From this peptidomimetic library, candidate inhibitors 4 and 5 emerged as inhibitors of this dimerization in FRET, Elisa and EMSA assays. While their in vitro inhibition was measured in the 20uM range, these compounds also interfered with Myc-induced oncogenic transformation in chicken embryo fibroblast cultures.
An exhaustive and unbiased yeast two hybrid screen has been carried out to identify interaction partners of two human Raf kinase isoforms, A-Raf and C-Raf. Using the N-terminal regulatory domain as bait, 20 different proteins that were isolated in this screen included three which were previously found : Ha-Ras, R-Ras, and 14-3-3 proteins (32). In addition, another 17 new Raf-interacting proteins were also found, and these were not detected by high throughput two-hybrid systems which use multiple 'baits' that compete with each other. The novel interactors included TOPK/PBK kinase (a signalosome component) and two new putative protein phosphatases. The cysteine-rich zinc binding domain (CRD) within the N-terminal domain was found to interact with all 20 proteins.
The yeast two-hybrid system has been a widely used tool to identify proteinprotein interactions; however, its utility is not applicable to all proteins. One class which meets this limitation is that of membrane bound proteins, which do not enter the nucleus of a cell, a requirement for the two-hybrid system. Therefore, membrane proteins, are not available for study using this method in its current format. A new two-hybrid system has now been developed which makes it possible to detect protein partners that interact with membrane proteins, and this technology has been adapted into a high-throughput screening format (33). The method utilizes the properties of ubiquitin, a small, conserved protein which is used to tag the N-termini of proteins for proteosomal degradation. Ubiquitin can be split into N-terminal (Nub) and C-terminal (Cub) halves, which can reconstitute spontaneously to from a 'split ubiquitin' that is recognized by ubiquitin-speclfic proteases (UBPs). A reporter protein can be fused to the Cub moiety, which can itself be fused to a membrane protein. When such a modified membrane protein (or a cDNA library) interacts with another protein which has been fused to the Nub moiety, this interaction promotes the reconstitution of 'split ubiquitin'. This reassembly event triggers a proteolysis that releases the reporter protein, which enters the nucleus and activates reporter genes. Such a membrane-based two-hybrid system enables detection of interactions between membrane and cytosolic proteins, as well as weak and transient proteinprotein interactions in vivo and in situ. Small molecule inhibitors of these interactions identified in this cell-based manner may be less likely to carry cytotoxicity liabilities.
Viral replication and assembly in host cells involves the formation of macromolecular structures and enzyme complexes, both of which inherently constitute protein-protein interactions. Examples of viral assemblies include HSV, HIV, HPV, RSV and their associated proteins, all of which are targets for drug discovery. Recent developments in antiviral chemotherapy based on these proteinprotein interaction targets have been summarized (34). A common theme in the o o
Boc review is the development of peptides and peptidomimetics which mimic one of the faces within the viral protein-protein complexes to generate requisite specificity.
Novel Small Molecule Inhibitors - The opportunities and challenges of small molecules as inhibitors of protein-protein targets have been outlined (35). Proteinprotein interfaces are typically large, flat surfaces which make it difficult for small molecules to access. However, by examining the effect of alanine scanning on binding, it has been demonstrated that only a small set of "hot spot" residues at protein interfaces contribute significantly to the binding free energy of human growth hormone to its receptor (36). This may be a general feature of other protein-protein interactions.
This understanding has been applied towards drug discovery by use of a fragment assembly process called tethering, a site directed screening method for rapid identification of low-affinity fragments that bind to specific sites on a target protein (37). These fragments with affinity for the target are then connected to yield more potent molecules. By this methodology, a potent (IC50 = 60nM) small molecule inhibitor (8) of the IL-2 / IL-2Ra was recently identified (38). The medicinal chemistry efforts initiated on compound 6 (a known low micromolar binder of IL-2) using structure-based design approaches generated a novel lead series exemplified by 7. Tethering methods which used 10 individual cysteine mutations of IL-2 to screen a library of 7000 disulfide-containing fragments focused on one region of the mutants. This region selected a defined set of ca. ten structurally related small aromatic carboxylic acid fragments. The SAR of aromatic carboxylic acids which emerged form this study led to 8.
A recent example of a natural product inhibitor of a protein-protein interaction is UCS15A (9), a small molecule non-kinase inhibitor of Src signal transduction (39). Src tyrosine kinase is important in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. As Src-signal transduction defects are implicated in a number of disease states, this protein could be a target for small molecules that either block the kinase activity or the interactions between Src and other signaling molecules.
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