Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo–electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical “active” conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.

Download Full-text

Dynamics of human protein kinase Aurora A linked to drug selectivity

eLife ◽

10.7554/elife.36656 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 17

Author(s):

Warintra Pitsawong ◽

Vanessa Buosi ◽

Renee Otten ◽

Roman V Agafonov ◽

Adelajda Zorba ◽

...

Keyword(s):

Protein Dynamics ◽

Conformational Changes ◽

Drug Targets ◽

Active Sites ◽

Kinase Inhibitors ◽

Bound State ◽

Drug Binding ◽

Aurora A ◽

Drug Selectivity ◽

Approved Drugs

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.

Download Full-text

Erlotinib binds both inactive and active conformations of the EGFR tyrosine kinase domain

Biochemical Journal ◽

10.1042/bj20121513 ◽

2012 ◽

Vol 448 (3) ◽

pp. 417-423 ◽

Cited By ~ 81

Author(s):

Jin H. Park ◽

Yingting Liu ◽

Mark A. Lemmon ◽

Ravi Radhakrishnan

Keyword(s):

Epidermal Growth Factor Receptor ◽

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Active Conformation ◽

Receptor Signalling ◽

Epidermal Growth ◽

Egfr Mutated

Erlotinib and gefitinib, tyrosine kinase inhibitors used to block EGFR (epidermal growth factor receptor) signalling in cancer, are thought to bind only the active conformation of the EGFR-TKD (tyrosine kinase domain). Through parallel computational and crystallographic studies, we show in the present study that erlotinib also binds the inactive EGFR-TKD conformation, which may have significant implications for its use in EGFR-mutated cancers.

Download Full-text

Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation

Nature Communications ◽

10.1038/s41467-021-21087-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chuan Hong ◽

Noel J. Byrne ◽

Beata Zamlynny ◽

Srivanya Tummala ◽

Li Xiao ◽

...

Keyword(s):

Small Molecule ◽

Receptor Activation ◽

Terminal Segment ◽

Active State ◽

Active Conformation ◽

Cryo Electron Microscopy ◽

Current Therapies ◽

Carboxy Terminal ◽

Peptide Agonist

AbstractNarcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain’s ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

Download Full-text

Dynamics of human protein kinases linked to drug selectivity

10.1101/286310 ◽

2018 ◽

Author(s):

Warintra Pitsawong ◽

Vanessa Buosi ◽

Renee Otten ◽

Roman V. Agafonov ◽

Adelajda Zorba ◽

...

Keyword(s):

Protein Dynamics ◽

Protein Kinases ◽

Conformational Changes ◽

Drug Targets ◽

Active Sites ◽

Kinase Inhibitors ◽

Drug Binding ◽

Inhibition Mechanism ◽

Aurora A ◽

Drug Selectivity

AbstractProtein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

Download Full-text

Crystal structure of an Aurora-A mutant that mimics Aurora-B bound to MLN8054: insights into selectivity and drug design

Biochemical Journal ◽

10.1042/bj20091530 ◽

2010 ◽

Vol 427 (1) ◽

pp. 19-28 ◽

Cited By ~ 60

Author(s):

Charlotte A. Dodson ◽

Magda Kosmopoulou ◽

Mark W. Richards ◽

Butrus Atrash ◽

Vassilios Bavetsias ◽

...

Keyword(s):

Active Site ◽

Active Sites ◽

Rational Design ◽

Kinase Inhibitors ◽

Protein Kinase Inhibitors ◽

Structural Basis ◽

Cancer Drug ◽

Aurora B ◽

Aurora A ◽

Point Mutant

The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple-point mutant of Aurora-A (AurAx3) which mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the T217E substitution, which we have confirmed using a single-point mutant. The activation loop of Aurora-A in the AurAx3–MLN8054 complex exhibits an unusual conformation in which Asp274 and Phe275 side chains point into the interior of the protein. There is to our knowledge no documented precedent for this conformation, which we have termed DFG-up. The sequence requirements of the DFG-up conformation suggest that it might be accessible to only a fraction of kinases. MLN8054 thus circumvents the problem of highly homologous active sites. Binding of MLN8054 to Aurora-A switches the character of a pocket within the active site from polar to a hydrophobic pocket, similar to what is observed in the structure of Aurora-A bound to a compound that induces DFG-out. We propose that targeting this pocket may be a productive route in the design of selective kinase inhibitors and describe the structural basis for the rational design of these compounds.

Download Full-text

Inhibitory-κB Kinase (IKK) α and Nuclear Factor-κB (NFκB)-Inducing Kinase (NIK) as Anti-Cancer Drug Targets

Cells ◽

10.3390/cells7100176 ◽

2018 ◽

Vol 7 (10) ◽

pp. 176 ◽

Cited By ~ 18

Author(s):

Andrew Paul ◽

Joanne Edwards ◽

Christopher Pepper ◽

Simon Mackay

Keyword(s):

Drug Targets ◽

Kinase Inhibitors ◽

Nuclear Factor Κb ◽

Tumour Microenvironment ◽

Pharmacological Intervention ◽

Cancer Drug ◽

Nuclear Factor ◽

Transcriptional Responses ◽

Cancer Hallmarks ◽

Mutational Status

The cellular kinases inhibitory-κB kinase (IKK) α and Nuclear Factor-κB (NF-κB)-inducing kinase (NIK) are well recognised as key central regulators and drivers of the non-canonical NF-κB cascade and as such dictate the initiation and development of defined transcriptional responses associated with the liberation of p52-RelB and p52-p52 NF-κB dimer complexes. Whilst these kinases and downstream NF-κB complexes transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes, they also play a key role in the pathogenesis of a number of inflammatory-based conditions and diverse cancer types, which for the latter may be a result of background mutational status. IKKα and NIK, therefore, represent attractive targets for pharmacological intervention. Here, specifically in the cancer setting, we reflect on the potential pathophysiological role(s) of each of these kinases, their associated downstream signalling outcomes and the stimulatory and mutational mechanisms leading to their increased activation. We also consider the downstream coordination of transcriptional events and phenotypic outcomes illustrative of key cancer ‘Hallmarks’ that are now increasingly perceived to be due to the coordinated recruitment of both NF-κB-dependent as well as NF-κB–independent signalling. Furthermore, as these kinases regulate the transition from hormone-dependent to hormone-independent growth in defined tumour subsets, potential tumour reactivation and major cytokine and chemokine species that may have significant bearing upon tumour-stromal communication and tumour microenvironment it reiterates their potential to be drug targets. Therefore, with the emergence of small molecule kinase inhibitors targeting each of these kinases, we consider medicinal chemistry efforts to date and those evolving that may contribute to the development of viable pharmacological intervention strategies to target a variety of tumour types.

Download Full-text