Combinatorial approaches to drug discovery
The concept of rational drug design drew emphasis away from the traditional random screening approach to drug discovery. However, the evelopment of techniques capable of generating large numbers of novel synthetic chemicals (combinatorial libraries), coupled with high-throughput screening methods, has generated fresh interest in the random screening approach.
Libraries can be generated relatively inexpensively and in a short time period. While many larger pharmaceutical companies maintain libraries of several hundred thousand natural and synthetic compounds, combinatorial chemistry can generate millions of compounds with relative ease. Peptides, for example, play various regulatory roles in the body, and several enjoy therapeutic application. Synthetic peptide libraries, containing peptides displaying millions of amino acid sequence combinations, can now be generated by combinatorial chemistry with minimal requirement for expensive equipment. Two approaches can be followed to generate a combinatorial peptide library: ‘split synthesis’ and ‘T-bag synthesis’.
The split level approach, for example, results in the creation of a large peptide library in which the peptides are grown on small synthetic beads. Peptides grown on any single bead will all be of identical amino acid sequences. Individual amino acids can be coupled to the growing chain by straightforward solid phase peptide synthesis techniques. In the split-level approach, a pool of beads are equally distributed into separate reaction vessels, each containing a single amino acid in solution. After chemical coupling to the beads, the beads are recovered, pooled and randomly distributed into the reaction vessels once more. This cycle can be repeated several times to extend the peptide chain.
The combinatorial approach is characterized not only by rapid synthesis of vast peptide libraries but also by rapid screening of these libraries (i.e. screening of the library to locate any peptides capable of binding to a ligand of interest — perhaps an enzyme or a hormone receptor). The soluble ligand is first labelled with an easily visible tag, often a fluorescent tag. This is then added to the beads. Binding of the ligand to a particular peptide will effectively result in staining of the bead to which the peptide is attached. This bead can then be physically separated from the other beads, e.g. using a microforceps. The isolated bead is then washed in 8 M guanidine hydrochloride (to remove the screening ligand) and the sequence of the attached peptide is then elucidated using a microsequencer. Typically, a bead will have 50–200 pmol of peptide attached, while the lower limit of sensitivity of most microsequencers is of the order of 5 pmol. Once its amino acid sequence is elucidated, it can be synthesized in large quantities for further study.
A library containing several million beads can be screened in a single afternoon. Furthermore, the library is reusable, as it may be washed in 8 M guanidine hydrochloride and then re-screened using a different probe. This split synthesis approach displays the ability to generate peptide libraries of incredible variety, variety tha t can be further expanded by incorporation of, for example, D -amino acids or rarely occurring amino acids.
Overall, therefore, various approaches may be adopted in the quest to discover new drugs. The approach generally adopted in biopharmacentical discovery differs from most other approaches in that biopharmaceuticals are produced naturally in the body. Discovery of a biopharmaceutical product, therefore, becomes a function of an increased knowledge of how the body itself functions. After its initial discovery, the physicochemical and biological characteristics of the potential drug can then be studied.