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.

Drug resistance is a challenge in anticancer therapy, particularly with targeted therapeutics and cytotoxic compounds. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug sensitivity prior to treatment. We therefore isolated clonal cell lines that were either sensitive or resistant to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features typically different between resistant and sensitive clones. These features were compiled to generate a morphological signature of bortezomib resistance, which correctly predicted the bortezomib treatment response in seven of ten cell lines not included in the training dataset. This signature of resistance was specific to bortezomib over other drugs targeting the ubiquitin-proteasome system. Our results provide evidence that intrinsic morphological features of drug resistance exist and establish a framework for their identification.