Proteins are made up of amino acids, and they are the basic building blocks of life. Peptides are a result of amino acids, which have been coded by the genes to form peptides, which then form the various types of proteins known today. Also, proteins have a crucial role to play in a variety of biological processes, such as transportation of molecules, immune reactions, catalytic reactions, and even signal transduction between cells.
Additionally, vital biological processes that happen at the cell level that impact our health directly, such as DNA transcription, replication, translation, and signal transduction, all depend on certain specific functions of proteins. The regulation of all these biological activities, is a result of complex protein processes, which in most cases, are driven by protein-protein interactions – PPIs. Protein-protein interaction at the cell level, usually follows a complicated network that is commonly referred to as “interactome.” This network has an immensely vital role to play in a variety of physiological and biological processes, including influencing processes, such as cell proliferation, apoptosis, differentiation, growth, and transduction.
Consequently, the aberrant of protein-protein interaction, is interlinked with a variety of human diseases, including conditions such as neurodegenerative diseases, various forms of cancer, and a myriad of infectious diseases. However, it should be noted that most of the time, the classic drug targets are usually enzymes, ions, and receptors. Protein-protein interactions have thus provided a new potential pathway in therapeutic drug targeting. Recently, protein-protein interactions have been receiving a lot of attention, as a viable avenue for dealing with the development of novel treatment of refractory diseases. Also, its regulation is currently being viewed as a viable approach to drug discovery.
Top challenges that come with the discovery of PPIs modulators
The main focus of the classic small molecule drug discovery, has always been based on protein-ligand interactions, featuring receptors, enzymes, and ion channels. The main reason behind this, is because such proteins are normally seen to have properly defined ligand-binding sites, which allows for easy interactions with the small molecules. It is, however, considered quite a challenge; the PPIs modulate through small molecules, and for a long time, PPIs have always been viewed as undruggable targets.
Current estimations put the total number of PPIs within the human interactome to be about 650,000. In as much as there are many protein complexes compared to enzymes and receptors, it has always been a challenge to design small molecules, with the ability to effectively bind themselves to protein-protein interfaces. To begin with, protein-protein interactions, usually take place on the interface of a specific domain, and for such an interaction to be successful, such a domain must have two identical or two different proteins, which must be in contact. Now, this is not usually a very common scenario. Secondly, it has been observed that the protein-protein interaction interfaces, are usually flat in nature, and may sometimes have grooves or pockets.
With such a structure or design, it becomes exceedingly difficult for any of the designed small molecules, to effectively bind to them. Thirdly, most of the amino acid residues that are usually involved with protein-protein interactions, are neither discontinuous nor continuous, in their protein structure. The problem with this, is that it normally leads to high-affinity binding, between the proteins, which in turn, makes it more challenging for small molecules to have an equally high-affinity interaction. The fourth reason, is that when you compare the traditional drug target enzymes or receptor with the PPIs, you will notice that the PPIs don’t have small molecular glands, which can be used for reference. Also, when PPIs are compared to small molecule compounds, the compounds that tend to be effective on PPIs, have been noticed to have higher molecular weights. This, in turn, makes it a huge challenge to meet criteria, such as Lipinski's rule of 5.
The current approaches used in the discovery of PPI modulators
The greatest challenge when it comes to targeting PPIs, is their unique interface. The conventional protein targets, usually have well-defined binding pockets, but for PPIs, they tend to have a relatively flat surface. As such, the classical chemistry methods have proven to be less effective, when it comes to the design and identification of PPI modulators. Consequently, there is a great need to come up with more effective approaches, to make screening of PPI modulators successful. Currently, there are various strategies being explored, with the major ones being the following:
High-throughput screening – HTS, is a solid and trusted method, which has been used for several years in the discovery of classic drug targets. It has also been effective in the identification of various compounds, with the capability of targeting the hotspots of PPI interfaces. With the particular nature of the PPI interface, it may not be a good idea to use the compound library that has been in use for years to screen conventional drug targets, for screening PPI modulators.
It is of immense importance that there be a wide compound library that portrays an incredible chemical diversity, which will then make it easy to narrow down on the PPI target matches. However, through high-throughput screening, it has been observed that the approach could be effective in identifying molecules at the very initial stages. For instance, it has been used successfully to screen out inhibitors for MDM2/p53 interactions.
Fragment-based drug discovery
With Fragment-based drug discovery – FBDD, the aim of the approach, is to pick out molecular fragments obtained from fragment libraries. When FBDD is compared to HTS, it has been observed that FBDD tends to be a more effective approach, when dealing with PPIs modulator designs. The reason for this, is because the PPI interface usually portrays discontinuous hot spots. With this method, processes such as Nuclear Magnetic Resonance, surface plasmon resonance, and X-ray crystallography, as well as mass spectroscopy, can be used in discovering and validating the fragment hits.
After the successful identification of the fragment, processes such as fragment linking, fragment optimization, and fragment self-assembly, can then be deployed to obtain the hits. Through NMR and X-ray crystallography, it is possible to get the structural information necessary for optimizing the fragment hits. As such, FBDD is not the best approach for targets that don’t have well-defined structures.
Due to the fact that most of the PPIs don’t have endogenous small molecule ligands, it is not easy to come up with a rational design of the accompanying PPI modulators. But, through the information obtained from the hot spots, it is possible to come up with a basis for the rational design of PPI modulators. Currently, two design strategies for the structure-based design of PPI modulators are being pursued. The first strategy relies on the structure of the hot spot. With the help of bioisosterism and de novo design approaches, it is now possible to come up with novel small molecule modulators. A good example of this, is the development of VHL PPI inhibitors, where Hyp546 was identified as the crucial amino acid, and with the use of de novo design targeting, it was possible to obtain the associated inhibitor.
The second approach for structure-based design, is peptidomimetic design. This design mainly uses computer modeling, as well as phage display, for the simulation of the secondary structure of the key peptides in PPIs. Additionally, with the use of stable alpha-helix structures formed by key peptides, it has been possible to synthesize small molecules, as well as binding peptides. The alpha-helix structure happens to be the most commonly identified secondary structure for the protein-protein interactions. Presently, it has been possible to successfully develop many PPI modulators, based on the alpha-helix structure.
The virtual screening method, is based on the use of professional software for screening out hits that may be obtained from libraries of compounds. One of the greatest challenges when it comes to the development of PPI modulators, is the identification of disease-related, as well as druggable PPIs, from the hundreds of thousands present in the available pool. Through a process such as virtual screening, it has been possible to quickly and effectively locate the binding site. This is usually achieved by analyzing the protein surface.
With virtual screening, you would be correct to classify it as either a ligand-based approach, or a structure-based approach. With the ligand-based approach, the goal is to always screen compounds that may meet the requirements of the built pharmacophore model. The structure-based approach, on the other hand, is the complete opposite of this. This is because the structure-based approach primarily relies on the structural information obtained from the target protein.
This simply means that if the structural information can’t be obtained from the protein, then the process of virtual screening won’t be successful. It is pleasing to note that virtual screening has been successfully applied in the design, as well as the development of a variety of PPI modulators, including modulators such as MDM2/p53 and Ubc13/Uevl, among others. This is a good sign that this approach could potentially be used in the identification of many more modulators in the future.
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