Fukase, Y. | bioRxiv Science

bioRxiv2020

Targeting Allostery in the Dynein Motor Domain with Small Molecule Inhibitors

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dyneins unusually large size, four distinct nucleotide-binding sites, and the existence of closely-related isoforms with different functions, pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with single molecule experiments and mutagenesis studies, indicate that the compounds likely inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dyneins main catalytic site in the AAA1 domain. A double Walker B mutant in AAA3 and AAA4 is an inactive enzyme, suggesting that inhibiting these regulatory sites can have a similar effect to inhibiting AAA1. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dyneins AAA domains.

biochemistry↗

bioRxiv2019

Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations

The integrin V{beta}3 receptor has been implicated in several important diseases, but no V{beta}3 antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit V{beta}3-mediated cell adhesion to V{beta}3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the V{beta}3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitides enhancement of tumor growth in vivo.\n\nSignificance StatementV{beta}3 is a potential therapeutic target for several important human diseases, but there are currently no V{beta}3 antagonists approved for human therapy. Current candidates are primarily based on the Arg-Gly-Asp (RGD) motif and act as partial agonists in that they induce V{beta}3 to undergo a conformational change that converts it into a high-affinity ligand-binding state. We have used structure-guided design to produce pure small-molecule V{beta}3 antagonists that do not induce the conformational change as judged by protein crystallography, electron microscopy, and receptor priming. These compounds inhibit V{beta}3-mediated bone resorption in vitro, but unlike the partial agonist cilengitide, do not enhance angiogenesis at low doses, a property that correlates with low-dose cilengitides enhancement of tumor growth in vivo. These pure V{beta}3 antagonists can help define V{beta}3s role in animal models. If they demonstrate benefits over partial agonists in these model systems, they may be appropriate to consider for human therapy.

pharmacology and toxicology↗