Protein-Protein Interactions in Drug Discovery

March 1, 2005 (Vol. 25, No. 5)

Another Piece of the Complex Drug Discovery Pocess Is Explored

Protein-protein interactions are at the center of almost every cellular process from cell motility to DNA replication. Among other things, protein-protein interactions allow a cell to see the surrounding milieu, talk to the neighboring cells, and respond to extracellular signals.

Protein-protein interactions can be stable or transient. Stable protein interactions are required for the construction of macromolecular structures such as cytoskeleton, the nuclear scaffold, and the mitotic spindle. Relatively smaller structures such as nuclear pores, centrosomes, and kinetochores also depend on protein-protein interactions.

Transient protein-protein interactions are involved in the regulation of fundamental cellular processes by carrying out enzyme-substrate reactions such as phosphorylation, dephosphorylation, and glycosylation.

Another example of protein-protein interactions is the oligomerization of cell surface receptors upon binding to a specific ligand. Thus, signal transduction pathways are comprised of large multiprotein complexes that transmit the signal from the outside of the cell to the cell nucleus.

Potential Clues

Understanding protein-protein interactions provides us with clues to help elucidate the function of a known or novel protein and the role it plays in a known pathway. Understanding protein-protein interactions is akin to solving a jigsaw puzzle, albeit on a molecular level.

Just as in solving a puzzle, scientists attempt to find the missing pieces by looking for partners of proteins whose functions are already known. Discovering novel partners provides leads into the unknown reaches of the cellular processes.

Alterations in protein-protein interactions perturb the normal sequence of events in the cell and contribute to diseases such as cancer. Thus, understanding the normal pattern of protein-protein interactions can lead to the development of drugs to fight the underlying cause of the diseases.

In an era of molecularly targeted therapies for cancer, there is increasing interest in defining and understanding of signal transduction pathways, says Jay Boniface, Ph.D., senior director, protein science, Prolexys Pharmaceuticals (www.prolexys.com). For instance, growth factor pathways have been known for a while but are not fully understood.

There are two main reasons to study protein-protein interactions at the cellular level, according to Dr. Boniface. One is that proteins that bind to known members of a known pathway are potential new members of the pathway and, as such, potential drug targets. The other reason is to identify the target(s) of existing drugs and new lead molecules. This process of “chemi-proteomics” uses protein interaction discovery to elucidate the mechanism of action of small molecule agents.

Tools Available

There are several technologies available to study the interactions of proteins. Prolexys Pharmaceuticals uses both a proprietary system based on the yeast two-hybrid system (HyNet) and a method based on mass spectrometry (HySpec), says Dr. Boniface.

Using the HyNet process, Prolexys Pharmaceuticals has created the Prolexys Human Interactome Database, a human protein interaction map useful for identifying proteins that can be targeted for therapeutic and diagnostic applications.

Prolexys has also applied its directed Hynet and HySpec strategies to achieve in-depth analysis of proteomes in areas of therapeutic interest to the company and its partners. Based on this analysis, Prolexys has identified and validated novel, druggable targets in oncology and cardiovascular disease and has identified small molecule compounds that act at these targets.

The yeast two-hybrid system is one of the most popular techniques available for identifying protein-protein interactions. The two-hybrid system exploits the fact that the DNA binding domain (DB) of a yeast transcription factor requires physical, but not necessarily covalent, association with its activating domain (AD) to activate transcription.

Advantages

In general, the genes for two fusion proteins, one containing the DNA-binding domain with protein X of interest and the other having the activation domain with protein Y of interest, are co-transfected into an appropriate yeast strain. If proteins X and Y physically interact with each other, this interaction brings DB and AD of the transcription factor together and results in the transcription of a reporter gene.

An advantage of this system is that it requires only the cDNA, full length or partial, of the genes of interest. This system can also be used to screen libraries for potential partners of a known protein. The two-hybrid system amplifies even weak interactions and is thus very sensitive.

The best argument in favor of this system is the speed with which several signal transduction pathways have been elucidated by using this method. The yeast two-hybrid system can also be utilized to identify inhibitors of protein-protein interactions.

Some disadvantages of the yeast two-hybrid system include the requirement for post-translational modifications for some interactions that might not happen in yeast, the presence of mammalian proteins that might be toxic in yeast, inadequate representation while screening libraries, and the appearance of false positives.

The Prolexys HyNet system makes use of plasmid libraries encoding human proteins fused to either of the two domains to create bait and prey hybrid proteins. Bait proteins are fused to the BD, and this hybrid binds directly to the promoter of the reporter genes.

In contrast, the prey proteins are fused to the AD. These hybrids are recruited to the promoter only if the human protein portions of the bait-and-prey proteins bind each other. Libraries of bait-and-prey proteins are created in haploid yeast of opposite mating types. Mating between the haploids results in diploids, which contain both bait-and-prey protein as well as the reporter genes.

If bait-and-prey interact, a complex is formed that localizes the AD to the promoter of the reporter gene resulting in expression of the reporter. Yeast colonies only grow if the reporter gene is expressed, so the resulting colonies all represent fruitful protein-protein interactions between bait and prey.

The Prolexys HySpec process is a directed process of protein expression, purification, and high throughput analysis of protein-protein interactions based on pull down experiments and mass spectrometry. Multiprotein complexes (MPCs) are formed in vitro by incubating purified tagged bait proteins with mammalian cell extracts under conditions that allow protein interactions to take place.

After a series of washes to remove nonspecific contaminants, tandem affinity purification (TAP) is used to isolate the MPCs. In addition, MPCs can also be isolated directly from human cells expressing the tagged protein bait, again by using TAP. The individual proteins of the MPCs are identified by making use of the power of mass spectrometry.

This technique can be modified to identify the interacting partners of drugs and drug candidates by using these small molecule agents as the bait. Identification of the drug can lead to an understanding of the molcular mechanisms of action and the design of protein complexes (targets) associated with newer and better drugs, remarks Dr. Boniface.

Another high throughput method to identify interacting proteins is to use protein arrays. ProteinOne (www.proteinone. com) has developed a protein array for identifying interacting partners to a known protein, says Neil Sharma, senior scientist.

The array contains 3040 immobilized proteins and can be used to screen for binding to the protein of interest. The ProteinOne array offers proteins that are highly pure and functionally active. One advantage of this system is that several variants of the protein of interest can be generated and immobilized on the array and be used as bait to study protein-protein interactions and the effects of the mutations on the interactions.

The limitations of the system include the availability of only about 200 mammalian proteins in a purified and functionally active state. The detection system used is radioactive and other substitutes are being currently researched.

Chromatography Arrays

Ciphergen Biosystems (www. ciphergen.com) provides preactivated chemistry-based arrays that carry either epoxy or carbonyl diimidazole groups on them. Both of these groups help couple purified proteins to the arrays since any protein with an amine group can form a covalent bond with the epoxy or the carbonyl diimidazole groups.

This reactive chemistry-based array is ideal for building customized arrays with a choice of proteins such as antibodies, streptavidin-bound oligos,and small molecule drugs with free amine groups, says Jennifer Cannon, Ph.D., marketing manager.

Cell or tissue extracts, plasma, serum, or other mixtures of homogenized proteins, can be used as the source of protein partners. After the capture of interacting proteins on the arrays, detection is performed using mass spectrometry. An energy-absorbing matrix is added to the array and when the arrays are hit with nitrogen laser, interacting proteins can be identified as peaks.

One of the advantages of this system is the high resolution, which can resolve proteins that differ by just one amino acid. Another advantage is the requirement for only small amounts of sample to be loaded on the array.

Some of the technologies, including the yeast two-hybrid system, have been around for more than 15 years and given rise to newer and improved assays. Some of these include the mammalian two-hybrid system for use with mammalian host cells, and the yeast three-hybrid systems.

The three-hybrid, or tribrid, system employs dimeric small molecule ligands that serve to dimerize protein ligand-binding domains and reconstitute functional transcriptional activation of reporter genes.

Other assays, such as protein arrays coupled with the power of mass spectrometry, are becoming popular and, with improvements in the availability of purified and active proteins, can be used to complement in vivo systems such as the yeast two-hybrid system.