Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport. Currently, we lack chemical inhibitors to probe spastin function in such dynamic cellular processes. To design a chemical inhibitor of spastin we tested selected heterocyclic-scaffolds against wildtype protein and constructs with engineered mutations in the nucleotide-binding site that do not substantially disrupt ATPase activity. These data, along with computational docking, guided improvements in compound potency and selectivity and led to spastazoline, a pyrazolyl-pyrrolopyrimidine-based cell-permeable probe for spastin. These studies also identified spastazoline resistance-conferring point mutations in spastin. Spastazoline, along with matched inhibitor-sensitive and inhibitor-resistant cell lines we generated, were used in parallel experiments to dissect spastin-specific phenotypes in dividing cells. Together, our findings suggest how chemical probes for AAA proteins, along with inhibitor resistance-conferring mutations, can be designed and used to dissect dynamic cellular processes.

Without affecting the overall 3D structure, amide‐to‐ester backbone substitution (or ester scan) exerts a pronounced influence on the conformational equilibrium of the RGD cyclopeptide cilengitide and its derivatives (see figure; RGD=Arg‐Gly‐Asp). The appropriate substitution, which stabilized the receptor‐complementary conformations, improved the biological activity of this integrin antagonist.

Doppelfunktion: Die Titelverbindung, ein bereits identifizierter wirksamer Hemmstoff des Hedgehog‐Signalwegs, der embryonischen Zellen notwendige Informationen für die Entwicklung liefert, moduliert die Genexpression von Hedgehog‐Targets, indem sie Mikrotubuli depolymerisiert. Auf diesem Weg wurde eine doppelte Steuerungsfunktion des Zytoskeletts nachgewiesen (siehe Bild).

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The bump-hole approach is a powerful chemical biology strategy to specifically probe the functions of closely related proteins. However, for many protein families, such as the ATPases associated with diverse cellular activities (AAA), we lack structural data for inhibitor-protein complexes to design allele-specific chemical probes. Here we report the X-ray structure of a pyrazolylaminoquinazoline-based inhibitor bound to spastin, a microtubule-severing AAA protein, and characterize the residues involved in inhibitor binding. We show that an inhibitor analogue with a single-atom hydrogen-to-fluorine modification can selectively target a spastin allele with an engineered cysteine mutation in its active site. We also report an X-ray structure of the fluoro analogue bound to the spastin mutant. Furthermore, analyses of other mutant alleles suggest how the stereoelectronics of the fluorine-cysteine interaction, rather than sterics alone, contribute to the inhibitor-allele selectivity. This approach could be used to design allele-specific probes for studying cellular functions of spastin isoforms. Our data also suggest how tuning stereoelectronics can lead to specific inhibitor-allele pairs for the AAA superfamily.

Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

An efficient and simple two-step procedure for the formation of hydroxy-Freidinger lactams is presented. The methodology allows assembly of the cyclic threonine motif (cThr) in solution and on solid support during conventional peptide synthesis.