Designing a chemical inhibitor for the AAA protein spastin using active site mutations

Abstract

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.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

“…Targeting viral helicases in general has been challenging ( 54 , 55 ). However, recent successes in targeting different ATPases ( 56 , 57 , 58 ) suggests that selective and potent inhibitors of nsp13 could be developed. The bulk and single-molecule assay platforms reported here may be leveraged in future drug discovery efforts and in detailed mechanistic investigation of nsp13 inhibition.…”

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

“…Steady-state ATPase activity of nsp13 was determined using an NADH-coupled assay as described previously ( Cupido, et. al.…”

Chemical strategies to overcome resistance against targeted anticancer therapeutics

“…Furthermore, testing diaminotriazole-based scaffolds, which are chemically unrelated to spastazoline, against spastin mutant alleles revealed how even minor modifications of the inhibitors can lead to distinct binding modes in the active site 94 . Together, these studies indicate that analyses of resistance can be valuable for the inhibitor design process 94,95 . RADD can be especially useful for evaluating unoptimized compounds (e.g., screening hits) as models of inhibitor-target interactions for these compounds are often not readily available.…”

“…Therefore, identifying key residues and inhibitorprotein contacts can be useful not only for anticipating resistance and validating targets of inhibitors in cells 93 , but also for optimizing inhibitor potency and specificity. An approach termed resistance analysis during design (RADD) 94 has recently been used for the design of spastazoline, a cell-permeable, potent and selective chemical probe of spastin 95 . In particular, RADD involves engineering biochemically silent mutations in the target protein and testing compounds against them.…”