Proteomics & Drug Development

Drug development is generally based around the desire to upregulate or downregulate a specific activity implicated in disease pathogenesis or in treatment-associated side-effects. Most drugs exert their effects on proteins. The strategy of working forward from the gene has been used: a specific genetic lesion is identified and the resultant changes in protein structure, function, or expression are elucidated, so that a drug to counteract or correct such aberrations can be rationally designed. The identification on bioactivity grounds of a protein that  is pivotal in a biological process has led to the specific design of  drugs to manipulate these properties. 

The challenge in proteomics-based approaches still lies in identifying the target molecules.Only a few thousand human genes are likely to be suitable targets, and with any single company only able to work on a few hundred, selection is of key importance. Cell-mapping proteomics has a more defined goal of studying protein-protein interactions by systematically characterising the components of protein complexes and building up a map of cellular pathways and interactions that may be important either in a disease process or in the mechanism of action of a drug. By use of specific antibodies or artificially introduced tags, specific proteins can be isolated and any associated proteins can be identified rapidly by mass spectrometry. Targeting of analysis to multiprotein complexes may reveal likely functions of specific proteins more rapidly and indicate appropriate biological studies.

In addition to target selection, equally important in progress to clinical use are target validation and toxicity studies. 

Some of the studies of protein expression in relation to genes that have shown an impact on drug discovery are as follows:

Identification and functional analysis of secreted proteins is being carried out at Genetics Institute. The cloning and expression of genes encoding this subset of proteins has enabled study of these factors in functional assays. Data from such studies will be important for the identification of novel protein therapeutics and targets.

CD-tagging (Jarvik et al. 1996) provides a unique means to investigate each member of the proteome and follow its various activities at the molecular and cellular levels. This technology is being developed by Sequel Genetics. With CD-tagging, a special CD-cassette is inserted into the cell's genome. When the insertion occurs in the proper orientation in an intron in an expressed gene, the result is the addition of a unique guest exon to the mRNA and the addition of a unique guest peptide to the encoded protein. 
CD-tagging has the following features:
§ Intron-rich genomes such as that of the human are preferred targets.
§ Tagged genes, transcripts and proteins generally retain normal function
§ High throughput.
§ Genes and proteins are identified and analyzed in their natural cellular environment
§ Tagged genes and cDNAs are readily amplified and sequenced.
§ Tagged proteins are localized and tracked at the cellular and subcellular levels.
§ Tagged proteins are purified directly from tagged cells for biochemical analysis.
§ Tissue specificity for transcript and protein expression is readily assessed.
§ Gene function is assessed via modulation of gene function in vivo.
§ Transgenic organisms carrying knockout mutations are rapidly created using proprietary ancillary technology.

To facilitate detection of tagged proteins, the system presently uses two kinds of tags -epitope tags recognized by existing high titer antibodies, and naturally fluorescent GFP tags. Not only can tagged proteins be observed and studied in vivo with these tags, but they can also be readily affinity-purified for biochemical examination or for use in biochemical or functional assays. Further, once a tagged cell of interest has been identified, analysis of the gene that is tagged is straightforward, since one can use the unique tag sequences in the mRNA and DNA to recover and sequence the DNA or RNA.

Although CD-tagging is superficially similar to a number of other tagging methods, it has a number of distinct and unique advantages. Most importantly, the CD-tagged gene is generally regulated normally, since the tag is inserted into the natural gene that retains its own regulatory elements. This is in distinct contrast to standard cDNA-based methods in which epitope-tagged proteins are expressed from heterologous promoter, typically strong promoters of viral origin, with attendant loss of all natural regulation. And the tagging methods that generally do preserve normal regulation, in particular gene trapping and enhancer trapping, have severe limitations at the protein level. In the case of gene trapping, a fusion is produced that lacks much of the sequence of the native protein; not surprisingly, protein function is usually severely compromised. With enhancer traps the situation is even worse, since the reporter and the target gene are expressed separately and so there is no direct means to detect the target gene product. CD-tagged proteins, in contrast, can be observed or purified on the basis of their tags, and at the same time all of the native protein sequence is retained. Although in some cases the tag may interrupt a functional domain of the protein and alter its normal activity or localization, in general the tagged protein retains appropriate localization potential and biological activity.

Transcription-aided drug design (TADD) is in development at the department of pharmacology of the University of Pennsylvania. The aim is to assess mRNA or protein level changes which result from a disease state in specific subclasses of cells. An assay has been developed to detect relative levels of phosphorylated versus non-phosphorylated Tau protein (abnormal in Alzheimer's disease) in single live neurons and is being extended to quantify protein interactions. This assay has been extended to assess multiple transcription factors to characterize the ability of the cells to transcriptionally respond to specific modulators. The aim is to design drugs to revert abnormal function to normal by manipulating the system using mRNA levels as a guide.

References

Proteomics- a major new technology for the drug discovery process

Proteomics: New tools for a new era

Will genomics revolutionize antimicrobial drug discovery

Is the development of a new tuberculosis vaccine possible