Targeting the Aurora-A/TPX2 Interaction

The mitotic serine/threonine kinase Aurora-A and its partner protein TPX2 are both overexpressed in many different cancers. It has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for the activation and localisation of Aurora-A during mitosis, thereby ensuring proper cell division. Disruption of the Aurora-A/TPX2 interface is therefore a potential target for novel anti-cancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. This study focuses on further characterisation of the Aurora-A/TPX2 interaction and the application of multiple approaches to developing inhibitors of Aurora-A and its complex with TPX2. We identify three key hot-spot sites within the interaction. We also reveal a previously unknown functional role within the complex for the first six residues of TPX2 and the tolerance of its flexible linker region to mutation and shortening. We show that TPX2 binds to phosphorylated Aurora-A with an order of magnitude greater affinity and provide evidence for our model of how the phosphorylation state of Aurora-A is ‘signaled’ to TPX2. To aid ongoing drug discovery efforts to inhibit Aurora-A, we show the crystal structures of several ATP-competitive inhibitors bound to Aurora-A and explain the structural mechanisms behind their high selectivity for Aurora-A. We show an alternative approach to allosterically inhibiting Aurora-A; through the use of a hydrocarbon-stapled peptidomimetic of TPX2. This stapled peptide binds to Aurora-A with a greater affinity than its wild- type, recombinantly expressed analogue and activates Aurora-A autophosphorylation to the same extent. Finally, we describe our work to develop a small molecule inhibitor of the Aurora-A/TPX2 interaction using fragment-based drug design. Following a high-throughput X-ray crystallography- based fragment screen we identified many fragment hits that bound within the Aurora-A/TPX2 binding interface. Our top fragments show inhibition of Aurora- A kinase activity, inhibition and activation of Aurora-A autophosphorylation and weaken the affinity between Aurora-A and TPX2 in competition assays

McIntyre_Strauss_2017_evolution_cytotypes

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X‑ray Crystal Structure of <i>rac-</i>[Ru(phen)₂dppz]²⁺ with d(ATGCAT)₂ Shows Enantiomer Orientations and Water Ordering

We report an atomic resolution X-ray crystal structure containing both enantiomers of <i>rac-</i>[Ru­(phen)₂dppz]²⁺ with the d­(ATGCAT)₂ DNA duplex (phen = phenanthroline; dppz = dipyridophenazine). The first example of any enantiomeric pair crystallized with a DNA duplex shows different orientations of the Λ and Δ binding sites, separated by a clearly defined structured water monolayer. Job plots show that the same species is present in solution. Each enantiomer is bound at a TG/CA step and shows intercalation from the minor groove. One water molecule is directly located on one phenazine N atom in the Δ-enantiomer only

X‑ray Crystal Structure of <i>rac-</i>[Ru(phen)₂dppz]²⁺ with d(ATGCAT)₂ Shows Enantiomer Orientations and Water Ordering

We report an atomic resolution X-ray crystal structure containing both enantiomers of <i>rac-</i>[Ru­(phen)₂dppz]²⁺ with the d­(ATGCAT)₂ DNA duplex (phen = phenanthroline; dppz = dipyridophenazine). The first example of any enantiomeric pair crystallized with a DNA duplex shows different orientations of the Λ and Δ binding sites, separated by a clearly defined structured water monolayer. Job plots show that the same species is present in solution. Each enantiomer is bound at a TG/CA step and shows intercalation from the minor groove. One water molecule is directly located on one phenazine N atom in the Δ-enantiomer only

Patterns of richness by data type of California freshwater species.

<p>Maps show the number of native freshwater species when summarized by: (A) observational data recorded after 1980; (B) observational data recorded before 1980 or observations of extirpated populations; and (C) data that includes range maps, historical range maps, modeled habitat, professional judgment, critical habitat designations, and management area designations. Spatial data with an unknown observation date or unknown type are not included in any panel. The black lines on the maps represent the major hydrologic regions in the study area.</p

Taxonomic grouping and conservation status of freshwater taxa native to California.

<p>Percentage of freshwater species by taxonomic groups that are considered vulnerable (at risk of extinction) in California watersheds, “Insects and other invertebrates” includes the classes Arachnida, Branchiopoda, Insecta and Polychaeta.</p

Study area.

<p>The extent of the study area in California and the major hydrologic regions it contains. Inset shows the location of California in North America. Shaded relief is from “The National Map” by the U.S. Geological Survey.</p

Location of hotspots.

<p>Comparison of the location of hotspot watersheds (top 5% by richness) for A) listed species with all non-listed species, and B) vulnerable but non-listed species.</p

Patterns of richness and vulnerability of freshwater species endemic to California, watersheds.

<p>Maps of (A) the number of endemic freshwater species in each HUC12 watershed (includes current, historic, range and modeled data). The range of endemic species richness is shown in quintiles, therefore the darkest blue is the top 20% of species richness, the lightest blue the lowest 20%.; (; (B) percentage of endemic species considered vulnerable in each HUC12 watershed; and (C) percentage of endemic species in each HUC12 watershed that are listed as endangered or threatened under state or federal ESA lists. Maps in panels B and C share the legend on the right of the figure. The black lines on the maps represent the major hydrologic regions in the study area.</p

Patterns of freshwater species richness by taxonomic group.

<p>Maps show richness of: (A) fishes; (B) herpetofauna; (C) birds; (D) mollusks/crustaceans; (E) insects and other invertebrates; (F) plants.</p