Designing Allele-Specific Inhibitors of Spastin, a Microtubule-Severing AAA Protein

Abstract

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.

Who Needs a Contractile Actomyosin Ring? The Plethora of Alternative Ways to Divide a Protozoan Parasite

“…Aside from protein kinases, sphingosine derivatives and inhibitors of sphingolipid biosynthetic enzymes have potential against Giardia (Sonda et al, 2008; Stefanic et al, 2010) and T. brucei (Fridberg et al, 2008; Jones et al, 2015), while repurposing of human PDE inhibitors shows promise in T. brucei (de Koning et al, 2012; Ochiana et al, 2015). In the future, it may also be possible to repurpose inhibitors of human Plk1 (Gutteridge et al, 2016), Hippo pathway inhibitors (Qiao et al, 2019) or enzymes such as katanin/spastin as they are developed (Pisa et al, 2019). However, currently, further work is required to refine existing cytokinesis inhibitors before they will be clinically useful.…”

AAA ATPases as therapeutic targets: Structure, functions, and small-molecule inhibitors

“…The electron-withdrawing property and the unfavored angle (~112 o ) between F•••C=O may decrease the binding affinity of compound 30 to WT spastin (Figure 12C). However, the N527A, -S, and -T mutant constructs are inhibited ~15-100 fold less potently than N527C, which is likely due to favorable stereoelectronic interactions of the fluorine atom with the Cys527 residue [143]. Furthermore, two similar inhibitors (compound 31 and 32, Figure 12A) were identified by the RADD (resistance analysis during design) involved in engineering point mutations in the target to generate active alleles.…”

Analyzing Resistance to Design Selective Chemical Inhibitors for AAA Proteins

“…We used the selected inhibitor-spastin binding model to design modifications of the core scaffold and generated spastazoline, a potent and selective inhibitor of spastin (Cupido et al, 2019). Structural models we generated by X-ray crystallography confirmed the predicted binding models (Pisa et al, 2019). However, it remains unclear if our approach, which we now name RADD (for resistance analysis during design), can be used to identify binding site interactions of inhibitors based on different chemical scaffolds and if the target-binding modes we predict are accurate.…”

“…To further characterize binding of compound 1 to spas-tin alleles, we used differential scanning fluorimetry. For these assays we focused on a truncated construct including only spastin's AAA domain (aa: 445-758, Drosophila melanogaster spastin, hereafter, spastin-AAA; Figures S1B and S1C) that we had characterized (Pisa et al, 2019). A dose-dependent increase in the melting temperature of spastin-AAA constructs in presence of compound 1 was observed .…”

“…In most cases, the inhibitor binding sites have been mapped to the AAA domain, the core enzymatic module of AAA proteins (Wendler et al, 2012), either in the active site (Anderson et al, 2015;Cupido et al, 2019;Magnaghi et al, 2013) or in an allosteric site (Magnaghi et al, 2013;Banerjee et al, 2016;Pohler et al, 2018). Structural models for a few inhibitor-bound AAA proteins are also now available (Banerjee et al, 2016;Boyaci et al, 2016;Pisa et al, 2019;Tang et al, 2019). However, for many AAA proteins the key inhibitor-target interactions needed for the design of selective chemical inhibitors are not known.…”