DeLaBarre, Byron; Wright, Gerald D.; Berghuis, Albert. M. McMaster University, Hamilton, ON, CANADA
We are attempting to apply protein crystallography towards the design of antifungal agents. Fungal pathogens have become a serious problem because of their acquired resistance to existing antifungal agents (1). Agriculture, where fungi can cause crop losses both before and after the harvest, would benefit greatly from an improved fungicide. The worldwide sales of antifungal agents is estimated to be close to $5 billion US (2). Fungal pathenogens are also an important problem for people with compromised immune systems such as AIDS victims, burn patients, and chemotherapy subjects.
The approach we are using is known as structure based drug design (3); it provides the researcher with the a priori knowledge necessary for a rational search for drug compounds. Structure based drug design requires a thorough understanding of the underlying biochemistry of the pathology. An important contribution to this understanding is the determination of the three dimensional structure of the biological molecule with which the drug molecule interacts; such information is obtained by the technique of X-ray crystallography.
An excellent target for structure based drug design is the altered amino acid metabolism of fungi. The pathway shown generates essential amino acids for the fungus, but has no corresponding pathway in animals (animals obtain the required amino acids through their diet). We have selected the homoserine dehydrogenase (HSD) enzyme as a subject for structural determination. HSD was chosen for two reasons:
(1) The protein can be obtained in large quantities - an overexpression vector and a purification scheme have already been established.
(2) It is known that 2-amino-4-oxo-5-hydroxypentanoic acid inhibits fungal growth by interacting with HSD (4).
We have crystallized the HSD protein as plates which diffract to 2.5 Angstrom resolution. The enzyme has been cocrystallized with the NAD+ cofactor and also with an NAD+ analogue and the substrate L-homoserine. Data collection for these crystals is currently underway.