Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX

Human carbonic anhydrase IX (CA IX)
is overexpressed in a number
of solid tumors and is considered to be a marker for cellular hypoxia
that it is not produced in most normal tissues. CA IX contributes
to the acidification of the extracellular matrix, which, in turn,
favors tumor growth and metastasis. Therefore, CA IX is considered
to be a promising anti-cancer drug target. However, the ability to
specifically target CA IX is challenging due to the fact that the
human genome encodes 15 different carbonic anhydrase isoforms that
have a high degree of homology. Furthermore, structure-based drug
design of CA IX inhibitors so far has been largely unsuccessful due
to technical difficulties regarding the expression and crystallization
of the enzyme. Currently, only one baculovirus-produced CA IX structure
in complex with a nonspecific CA inhibitor, acetazolamide, is available
in Protein Data Bank. We have developed an efficient system for the
production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily
crystallized in the presence of inhibitors, as we have demonstrated
for several 2-thiophene-sulfonamide compounds. We have also observed
significant differences in the binding mode of chemically identical
compounds to CA IX and CA II, which can be further exploited in the
design of CA IX-specific inhibitors