The impact of molecular biology on drug discovery.

The discipline of molecular biology has become increasingly important in recent times for the process of drug discovery. We describe the impact of molecular biology across the whole process of drug discovery and development, including (i) the identification and validation of new drug targets, (ii) the development of molecular screens to find new candidate drugs, and (iii) the generation of safety data and competences leading to enhanced clinical efficacy. We also speculate on emerging developments in drug discovery where it seems likely that molecular biology will play an even more vital role in the generation of future therapies.

Translation elongation factor-3 (EF-3): an evolving eukaryotic ribosomal protein?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, and Pneumocystis carinii). Computer-assisted analysis of the predicted S. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an amino-terminal region showing significant similarity to the E. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal "accuracy center," which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of the E. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes.

Translation elongation factor 3: a fungus-specific translation factor?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), was first described in the yeast Saccharomyces cerevisiae and has subsequently been identified in a wide range of fungal species including Candida albicans and Schizosaccharomyces pombe. EF-3 exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome, but which are essential for translation elongation. Recent studies on the structure of EF-3 from several fungal species have shown that it consists of a repeated domain, with each domain containing the expected putative ATP- and GTP-binding motifs. Overall, EF-3 shows striking amino acid similarity to members of the ATP-binding Cassette (ABC) family of membrane-associated transport proteins although EF-3 is not itself directly membrane-associated. Regions of the EF-3 polypeptide also show structural homology with other translation-associated factors including aminoacyl-tRNA synthetases and the Escherichia coli ribosomal protein S5. While the precise role of EF-3 in the translation elongation cycle remains to be defined, recent evidence suggests that it may be involved in optimizing accuracy during mRNA decoding at the ribosomal A site. Furthermore, the essential nature of EF-3 with respect to the fungal cell indicates that it may be an effective antifungal target. Its apparently ubiquitous occurrence throughout the fungal kingdom also suggests that it may be a useful fungal taxonomic marker.

Saccharomyces cerevisiae strains that overexpress heterologous proteins.

We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae. These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media. Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.

The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences.

We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae. Studies with five KEX2 processed leader sequences, namely the S. cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described. We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product. In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product. Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.