Targeting the CRD F‐face of human galectin‐3 and allosterically modulating glycan binding by angiostatic PTX008 and a structurally optimized derivative

12 p.-8 fig.Calix[4]arene PTX008 is an angiostatic agent that inhibits tumor growth in mice by binding to galectin‐1, a β‐galactoside‐binding lectin. To assess the affinity profile of PTX008 for galectins, we used 15N,1H HSQC NMR spectroscopy to show that PTX008 also binds to galectin‐3 (Gal‐3), albeit more weakly. We identified the contact site for PTX008 on the F‐face of the Gal‐3 carbohydrate recognition domain. STD NMR revealed that the hydrophobic phenyl ring crown of the calixarene is the binding epitope. With this information, we performed molecular modeling of the complex to assist in improving the rather low affinity of PTX008 for Gal‐3. By removing the N‐dimethyl alkyl chain amide groups, we produced PTX013 whose reduced alkyl chain length and polar character led to an approximately eightfold stronger binding than PTX008. PTX013 also binds Gal‐1 more strongly than PTX008, whereas neither interacts strongly, if at all, with Gal‐7. In addition, PTX013, like PTX008, is an allosteric inhibitor of galectin binding to the canonical ligand lactose. This study broadens the scope for galectin targeting by calixarene‐based compounds and opens the perspective for selective galectin blocking.K.H.M. is grateful to the Ludwigs-Maximillians-Universität (LMU) Center for Advanced Study, as well as to the Alexander von Humboldt Stiftung, for financial support during his sabbatical stay at LMU in Munich, Germany. K.H.M. also holds a van der Laar Visiting Professorship in Structural Biology at Maastricht University,Netherlands. K.H.M. acknowledges that NMR instrumentation was provided with funds from the National Science Foundation(BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical Foundation.Peer reviewe