Chemical and Structural Biology of Mechanoenzymes
Chemical and structural biology of mechanoenzymes
We develop and use chemical biology approaches to study mechanoenzymes, proteins that can harness the energy from ATP hydrolysis to transport cellular cargoes, unfold proteins for degradation or regulate the assembly dynamics of macromolecular complexes. A major focus of our efforts is the discovery of cell permeable chemical inhibitors of mechanoenzymes. As these chemical probes can inhibit target proteins on the timescale of minutes in cells, they can be powerful tools to analyze dynamic cellular processes. Importantly, these inhibitors can also provide valuable starting points for therapeutic development.
We have also developed and used: (i) DrugTargetSeqR, a method that can uncover recurring mutations that confer chemotype-specific resistance, to analyze on-target activity of chemical inhibitors and chemotherapeutics in cellular contexts. (ii) iCLASPI, a photo-crosslinking-based chemical proteomics method to profile direct protein-protein interactions in living cells.
Current projects:
To discover chemical inhibitors of mechanoenzymes we have used cell-based high-throughput screens to identify ciliobrevins and ribozinoindoles, cell-permeable chemical inhibitors of dynein, a microtubule-based AAA motor protein, and Mdn1, a ~540 kdal AAA protein required for ribosome assembly, respectively.
Our more recent efforts have focused on the design of chemical inhibitors by leveraging the extensive structural data available for mechanoenzymes. In particular, we have developed an approach, which we named RADD (resistance analysis during design), that involves generating multiple alleles of a target protein with engineered point mutations that do not disrupt biochemical activity. Testing compounds against these target protein alleles, along with computational analyses, rapidly leads to robust models for inhibitor-target binding, without the need for structural studies of inhibitor-protein complexes using X-ray crystallography or Cryo-EM.
RADD models can guide the design of potent and selective drug-like chemical inhibitors. Importantly, RADD can also help anticipate resistance-conferring mutations in the target and thereby lead to strategies that address drug resistance, a major challenge in developing molecularly targeted therapeutics. We have used RADD to design spastazoline, a cell-permeable chemical inhibitor of spastin, a microtubule-severing AAA+ protein that coordinates membrane and cytoskeleton dynamics.
Eukaryotes have ~100 AAA mechanoenzymes that play important roles in many essential cellular processes, including DNA replication, protein degradation, and intracellular transport. We recently used RADD to design ASPIR-1, a chemical inhibitor that can covalently bind and selectively inhibit a AAA protein with an engineered point mutation. This ‘chemical genetics’ strategy can be used to probe the function of AAA proteins in cells and to test hypotheses for developing therapeutics that target AAA proteins.
Recent publications:
1. Cupido, T., Pisa, R., Kelley, M.E., Kapoor, T.M. (2019), Designing a chemical inhibitor of the AAA protein spastin using active site mutations. Nature Chemical Biology, 15, 444-52. PMC6558985.
2. Cupido, T., Jones, N.H., Grasso, M.J., Pisa, R., Kapoor, T.M. (2021) A chemical genetics approach to examine the functions of AAA proteins. Nature Structural and Molecular Biology, 28, 388-97. PMC33782614.
For the mechanoenzymes for which we have discovered chemical inhibitors we have employed X-ray crystallography and Cryo-EM to develop structural models. Our ongoing work builds on the following studies:
(i) Midasin (Mdn1), an essential ATPase required for the assembly of the 60S subunit of the ribosome. We reported the first structures, including one in the presence of the chemical inhibitor we discovered, of this unusually large (~5000aa) protein. Comparisons of Cryo-EM structures suggests a model for Mdn1’s mechanochemical cycle and how it can remodel ribosome precursors.
(ii) Spastin, a microtubule severing AAA+ protein, bound to spastazoline analogs. These data indicated that our models derived using RADD are robust.
(iii) Dyneins, AAA proteins that transport cellular cargoes along microtubules. Guided by analyses of the chemical structures ciliobrevins, chemical inhibitors of dynein discovered using cell-based screens, we designed dynapyrazoles, new dynein inhibitors with improved potencies and chemical properties. More recently, we determined the structure of a dynapyrazole analog bound to dynein. Our studies reveal how chemical inhibitors can disrupt the allosteric communication across the different ATPase sites in dynein.
Recent publications:
1. Chen, Z., Suzuki, H., Kobayashi, Y., Wang, A.C., DiMaio, F., Kawashima, S.A., Walz, T., Kapoor, T.M. (2018), Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis. Cell, 175, 822-34. PMC5775914.
2. Santarossa, C.C., Mickolajczyk, K.J., Steinman, J.B., Urnavicius, L., Chen, N., Hirata, Y., Fukase, Y., Coudray, N., Ekiert, D.C., Bhabha, G., Kapoor, T.M. (2020), Targeting Allostery in the Dynein Motor Domain with Small Molecule Inhibitors. Cell Chemical Biology, 28, 1-14. PMC34015309. bioRxiv 2020.09.22.308700; doi: https://doi.org/10.1016/j.chembiol.2021.04.024.