Teaming up synthetic chemistry and histochemistry for activity screening in galectin-directed inhibitor design

A hallmark of endogenous lectins is their ability to select a few distinct glycoconjugates as counterreceptors for functional pairing from the natural abundance of cellular glycoproteins and glycolipids. As a consequence, assays to assess inhibition of lectin binding should necessarily come as close as possible to the physiological situation, to characterize an impact of a synthetic compound on biorelevant binding with pharmaceutical perspective. We here introduce in a proof-of-principle manner work with sections of paraffin-embedded tissue (jejunum, epididymis) and labeled adhesion/growth-regulatory galectins, harboring one (galectin-1 and galectin-3) or two (galectin-8) types of lectin domain. Six pairs of synthetic lactosides from tailoring of the headgroup (3'-O-sulfation) and the aglycone (beta-methyl to aromatic S- and O-linked extensions) as well as three bi- to tetravalent glycoclusters were used as test compounds. Varying extents of reduction in staining intensity by synthetic compounds relative to unsubstituted/free lactose proved the applicability and sensitivity of the method. Flanking cytofluorimetric assays on lectin binding to native cells gave similar grading, excluding a major impact of tissue fixation. The experiments revealed cell/tissue binding of galectin-8 preferentially via one domain, depending on the cell type so that the effect of an inhibitor in a certain context cannot be extrapolated to other cells/tissues. Moreover, the work with the other galectins attests that this assay enables comprehensive analysis of the galectin network in serial tissue sections to determine overlaps and regional differences in inhibitory profiles.R.R. thanks the Natural Sciences and Engineering Research Council of Canada (NSERC) for a Canadian Research Chair for financial support. P.V.M. thanks Science Foundation Ireland for financial support (08/SRC/B1393 and 12/IA/1398), the latter being co-funded under the European Regional Development Fund under Grant No. 14/SP/2710. H.-J.G. thanks the excellence program of the Ludwig-Maximilians-University Munich, the Verein zur Förderung des biologisch-technologischen Fortschritts in der Medizin e.V. (Heidelberg, Germany) and the EC (for ITN network funding; GLYCOPHARM) for generous support and also Drs. B. Friday, G. Ippans and A. Leddoz for inspiring discussions.peer-reviewe