Novel glycomimetic inhibitors and proteolysis-targeting chimeras for human galectins

Sammanfattning: Galectins are a family of soluble proteins that bind β-D-galactopyranoside-containing glycoconjugates through their conserved carbohydrate-recognition domains. Galectins have emerged as promising drug targets due to their involvement in various pathological conditions, such as tumor growth and metastasis, autoimmune and inflammatory diseases, as well as metabolic disorders.This first part of the thesis describes the design, synthesis, and evaluation of novel glycomimetic inhibitors of human galectin-8, which plays an essential role in pathological lymphangiogenesis, immune system modulation, bone remodeling, and is upregulated in several cancers. The structure-based design of inhibitors of galectin-8 N-terminal domain (galectin-8N) identified a benzimidazole-galactoside with a Kd of 1.8 µM for galectin-8N and 3-fold selectivity over galectin-3, and higher selectivity over the other human galectins. Molecular dynamics simulation showed that the benzimidazole-galactoside binds the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Furthermore, the subconjunctival injection of the benzimidazole-galactoside reduced the severity of bacterial keratitis caused by Pseudomonas aeruginosa in a mouse model, providing the first evidence that galectin-8 inhibitors can be effective in an actual disease model.Subsequently, we designed and synthesized a set of C-3 substituted D-galactal derivatives, which led to the discovery of a D-galactal-benzimidazole hybrid with a Kd of 48 µM for galectin-8N and 15-fold selectivity over galectin-3, and higher selectivity over the other human galectins. X-ray structural analysis of the D-galactal-benzimidazole hybrid in complex with galectin-8N followed by molecular dynamics simulation and quantum mechanical calculations showed that the high affinity of the compound for galectin-8N is probably due to the orbital overlap between the LUMO of Arg45 with the electron-rich HOMOs of the olefin and O4 of the D-galactal. A functional assay of the D-galactal-benzimidazole hybrid and the abovementioned benzimidazole-galactoside showed that both compounds reduced the secretion of the proinflammatory interleukin-6 (IL-6) and IL-8 in a dose-dependent manner. Attachment of a p-chlorophenyl moiety at C4 of the benzimidazole of the D-galactal-benzimidazole hybrid resulted in the discovery of the most potent selective galectin-8N inhibitor to date with a Kd of 2.9 µM for galectin-8N and 50-fold selectivity over galectin-3, and even higher selectivity over the other human galectins. X-ray structural analysis revealed that the high affinity of the compound for galectin-8N is probably due to the interaction of the p-chlorophenyl moiety with Arg59 and/or Tyr141 via cation-π stacking and/or π-π stacking, respectively. This compound represents a promising starting point for the design of ligands that bind galectin-8N with higher affinity and selectivity.Finally, we designed and synthesized two proteolysis-targeting chimeras (PROTACs) for human galectins to investigate whether galectins are amenable to targeted protein degradation. Although both compounds displayed nanomolar affinities for galectin-3, they failed to induce galectin-3 degradation in JIMT-1 and MDA-MB-231 breast cancer cell lines at a concentration of 25 µM. This lack of effect can be either due to the higher topological polar surface area of the compounds or the hook effect caused by the high concentration used in the assay.