Role of proteomics in Drug Discovery

The term ‘proteomics’ indicates PROTEins expressed by a genOME and is the systematic analysis of protein profiles of tissues. The term ‘proteome’ refers to all proteins produced by a species, much as the genome is the entire set of genes. Proteomics parallels the related field of genomics.Proteomics is  important for drug discovery because many of the pharmaceutically important regulation systems operate through proteins (i.e., post-translationally). Major drugs act by binding to proteins. For example a ‘protease inhibitor’ drug is designed to disable the protease enzyme (which is a protein) that allows a particular virus to reproduce. A drug with the right shape can latch on to the surface of the protease protein and keep it from doing its job. If the protease is disabled, the virus cannot reproduce itself, so the damage it can inflict is limited. The role of proteomics in drug development can be termed ‘pharmacoproteomics’ and is an important component of personalised medicine.
1. Proteins as Drug Targets
The majority of drug targets are proteins that are encoded by genes expressed within tissues affected by a disease. It is estimated that there are approximately 10 000 different enzymes, more than 2000 different G-protein-coupled receptors, 200 different ion channels and 100 different nuclear hormone receptors encoded in the human genome. These proteins are key components of the pathways involved in disease and, therefore, are likely to be a rich source of new drug targets.
Proven drug targets share certain other characteristics, which can only be identified by understanding their expression levels in cells and cannot be determined by their gene sequences alone. Drug targets are (1) often expressed primarily in specific tissues, allowing for selectivity of pharmacological action and reducing the potential for adverse side-effects and (2) generally expressed at low abundance in the cells of the relevant organ. An effective target discovery system would therefore allow the detection of genes that encode for proteins expressed in specific tissues at low abundance, thereby permitting the rapid identification of proteins, which are likely to be targets for therapeutic and diagnostic development. Some proteomic technologies that are useful for drug discovery are described briefly.

2. Protein Expression Mapping by 2D Gel Electrophoresis
2D gel electrophoresis (2DGE) and image analysis may be used for the quantitative study of global changes in protein expression in tissues, cells or body fluids. This method has the advantages of direct determination of protein abundance and detection of post-translational modifications such as glycosylation or phosphorylation, which result in a shift in mobility; mass spectrometry may be used for the subsequent characterisation of proteins of interest. Because thousands of proteins are imaged in one experiment, a picture of the protein profile of the sample at a given point in time is obtained, allowing comparative proteome analysis. Protein expression changes may give clues to the role of certain proteins in disease and some of the identified proteins map to known genetic loci of a disease.
3. Liquid Chromatography-based Drug Discovery
Liquid chromatography (LC) shifts the high-resolution separation from front-end 2DGE to back end LC–mass spectrometry (MS). Proteins are kept in solution so that a higher percentage of the sample is analysed using affinity chromatography and having access to the entirely accurately assembled genome of the organism under study. Protein mixture may be digested to analyse at the peptide level. This high-efficiency proteomics technology is applied to small-molecule drug discovery. Instead of separating individual proteins using 2DGE, complex mixture analysis can start with proteins pooled after partial fractionation by multi-dimensional LC, which involves serial protein separations over a variety of chromatographic matrices. The complex mixtures are treated with trypsin and the resulting peptides are separated by LC and measured by MS. This approach may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.
4. Matrix-assisted Laser Desorption/Ionisation Mass Spectrometry
Among several proteomic technologies used in drug discovery, matrixassisted laser desorption/ionisation (MALDI)-MS and its variants, and related techniques, play an important role. MALDI time-of-flight (TOF) MS targets, when uniformly precoated with a thin film of matrix/ nitrocellulose, make the sample preparation straightforward and permit the enrichment and analysis of proteins at low levels in proteomics samples. In general, the sensitivity for proteins and peptides can be enhanced 10–50 times compared with traditional MALDI sample preparation techniques. Tissue imaging mass spectrometry (IMS) by MALDI and ion trap MS with higher order MS scanning functions have been used for localisation of dosed drug or metabolite in tissues.
Laser capture microscopy (LCM) is used to obtain related samples from tissue for analyses by standard MALDI-MS and HPLC-MS. IMS by MALDI ion trap MS has proved sensitive, specific and highly amenable to the image analysis of traditional small molecule drug candidates directly in tissue.
A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by MALDI-IMS with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-dihydroxybenzoic acid matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI-MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample.
5. Protein–Protein Interactions
Protein interactions can be monitored in vivo over the course of the cell cycle, drug treatments or other environmental stimuli. The development of green fluorescent protein derivatives has provided the opportunity to study protein–protein interactions in living cells. The structural organisation of macromolecular protein complexes, which may contain scores of protein interactions and may be difficult to study in vitro, can be analysed. Techniques to study protein–protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, and also the development and optimisation of pharmaceuticals for modulating protein–protein interactions.
Protein–protein interaction networks are also called ‘interactome’ networks. The interactome is a map of all interactions that take place in an org anism between all proteins, in all cells, all tissues, at all ages and inresponse to all possible environmental conditions. The ability to find links between sets of proteins involved in different genetic disorders offers a novel approach for more rapidly identifying new candidate genes involved in human diseases. Pharmaceutical investigators use interaction data to prioritise potential drug targets as these networks help the companies to weed out proteins that have several interaction, some of which are irrelevant to the target.
6. Use of Proteomic Technologies for Important Drug Targets

Protein kinases are encoded by more than 2000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are regulated by the reversible phosphorylation of proteins on serine, threonine and tyrosine residues. At least 30% of all proteins contain covalently bound phosphate. A novel method to determine if drugs and drug targets are effective in combating disease is by identifying the key regulatory protein ‘switches’’ (phosphorylation sites) inside human cells. This allows the identification and characterisation of changes in the chemical modification of proteins that may arise in response to drug treatment. It can be used to identify novel targets in disease, to compare the effects of different drug candidates and to develop assays that can be used throughout preclinical and clinical development. Kinomics, omic for kinome (the kinase complement of the human genome), is a useful tool for identifying protein kinases that play an important role in disease. It also assists in the drug optimisation process. Kinomics can be used to understand both the mechanism of action and the specificity of potential drugs. This knowledge forms a crucial base from which to develop potent and selective compounds with minimal side-effects. Protein kinases are important drug targets in human cancers, inflammation and metabolic diseases.
Proteomic technologies have been used for the study of G-protein coupled receptors (GPCRs), which are an important class of drug targets that exist as proteins on the surface membranes of all cells. GPCRs are a superfamily of proteins accounting for approximately 1% of the human genome and are associated with a wide range of therapeutic categories, including asthma, inflammation, obesity, cancer, cardiovascular, metabolic, gastrointestinal and neurological diseases. Purified multiple GPCRs in a functional form can be used for the identification of tight binding ligands. There are estimated to be B2000 GPCRs within the human body with potential availability as drug discovery targets. GPCRs have historically been valuable drug targets, but to date there are only approximately 100 well-characterised GPCRs with known ligands, several of which are currently targets of commercial drugs. Approximately 60% of all currently available prescription drugs interact with these receptors.