Small GTPases comprise a family of highly relevant targets in chemical biology and medicinal chemistry research and have been considered "undruggable" due to the persisting lack of effective synthetic modulators and suitable binding pockets. As molecular switches, small GTPases control a multitude of pivotal cellular functions, and their dysregulation is associated with many human diseases such as various forms of cancer. Rab-GTPases represent the largest subfamily of small GTPases and are master regulators of vesicular transport interacting with various proteins via flat and extensive protein-protein interactions (PPIs). The only reported synthetic inhibitor of a PPI involving an activated Rab GTPase is the hydrocarbon stapled peptide StRIP3. However, this macrocyclic peptide shows low proteolytic stability and cell permeability. Here, we report the design of a bioavailable StRIP3 analogue that harbors two hydrophobic cross-links and exhibits increased binding affinity, combined with robust cellular uptake and extremely high proteolytic stability. Localization experiments reveal that this double-stapled peptide and its target protein Rab8a accumulate in the same cellular compartments. The reported approach offers a strategy for the implementation of biostability into conformationally constrained peptides while supporting cellular uptake and target affinity, thereby conveying drug-like properties.

Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.

The imino Diels-Alder reaction is an efficient method for the synthesis of aza-heterocycles. While different stereo- and enantioselective inverse-electron-demand imino Diels-Alder (IEDIDA) reactions have been reported before, IEDIDA reactions including electron-deficient dienes are unprecedented. The first enantioselective IEDIDA reaction between electron-poor chromone dienes and cyclic imines, catalyzed by zinc/binol complexes is described. The novel reaction provides a facile entry to a natural product inspired collection of ring-fused quinolizines including a potent modulator of mitosis.