Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states

Lake, Eric W; Muretta, Joseph M.; Thompson, Andrew R.; Rasmussen, Damien M.; Majumdar, Abir; Faber, Erik B.; Ruff, Emily F.; Thomas, David D.; Levinson, Nicholas M.

doi:10.1073/pnas.1811158115

Cited by 56 publications

(58 citation statements)

References 70 publications

(104 reference statements)

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“…Likewise, activation loop dynamics have been suggested by simulations of the Fyn tyrosine kinase domain (31). However, to date, only Aurora A kinase has been found to undergo activation loop motions in its phosphorylated form (32)(33)(34)(35). Here, time-resolved Förster resonance energy transfer and single-molecule fluorescence spectroscopic measurements show interconversion of the phosphorylated activation loop between active and inactive conformations.…”

Section: Discussionmentioning

confidence: 73%

“…Here, time-resolved Förster resonance energy transfer and single-molecule fluorescence spectroscopic measurements show interconversion of the phosphorylated activation loop between active and inactive conformations. These are coupled with exchange between DFG-in and DFG-out conformations and can be shifted by Type II inhibitors (34,35). The characteristics of ERK2 differ from those of Aurora A in that dynamic motions of the activation loop coupled with global exchange in the active site involve movements distinct from DFG backbone rotation.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Pegram

Liddle

Xiao

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.

show abstract

Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Pegram

Liddle

Xiao

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Since we have found that these treatments do not affect endogenous AURKA levels, nor AURKA-Venus levels in intact cells, we assumed that the reduced AURKA levels seen in IF reflected loss of AURKA in the fixation step, that could be a consequence of reduced interaction with the mitotic spindle. Indeed, MLN8237 and TPX2 may compete with each other for AURKA binding 50 (see Discussion). We measured pole-pole distances in this experiment and found them reduced by PROTAC-D treatment.…”

Section: Resultsmentioning

confidence: 94%

“…Given the complex conformational and spatial regulation of AURKA 15,17,56 we tested for conformationspecific targeting of the kinase using different versions of AURKA-Venus. The conformational dynamics of AURKA are strongly constrained through interaction with TPX2 50,57,58 , which favours the so-called 'DFG-In' active confirmation and it has been suggested that different functional pools possess distinct conformational properties that will modulate interactions with inhibitors 50 . In this study, alisertib/MLN8237 was shown to be a 'Type 1' inhibitor that promotes the inactive DFG-Out state, and TPX2 to oppose DFG-Out inducers, exhibiting negative cooperativity on binding with MLN8237 50 .…”

Section: Discussionmentioning

confidence: 99%

Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool

Wang

Abdelbaki

Ascanelli

et al. 2020

Preprint

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Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (referred to as ‘PROTACs’, for PROteolysis Targeting Chimeras) that recruit a ubiquitin ligase to the target of interest, leading to ubiquitination of the target and its destruction via the ubiquitin-proteasome system. A number of PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate the centrosome- and chromatin-based microtubule spindle assembly pathways. In interphase cells we find that PROTAC-mediated clearance of non-centrosomal AURKA, and not PROTAC-mediated inhibition of its activity, efficiently modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential accessibility of subcellular pools of substrate, governed by substrate conformation or localization in compartments more or less accessible to PROTAC action, a phenomenon not previously described for this new class of drugs.

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“…The low energetic barrier allows Vertex-11e to trap the active conformation of the activation loop, and SCH772984 to disrupt it. (35,36). Here, crystal structures of the unphosphorylated ERK2 double mutant revealed that Type II inhibitors bound to a DFG-out conformer.…”

Section: Dfg-in State Unlike What Is Observed In Structures Of Kinasmentioning

confidence: 85%

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Pegram

Liddle

Xiao

et al. 2019

Preprint

View full text Add to dashboard Cite

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dualphosphorylation induces global motions involving exchange between two states, "L'' and "R".We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L⇌R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L⇌R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.

show abstract

Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states

Cited by 56 publications

References 70 publications

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

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