Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors

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    This elegant study presents important findings into how small molecules that were originally developed to inhibit the oncogenic kinase, BRAF, instead trigger activation of this kinase target. Compelling and comprehensive evidence supports a new allosteric model to explain the paradoxical activation. This rigorous work will be of great interest to biochemists, structural biologists, and those working on strategies to inhibit kinases in the context of human disease.

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Abstract

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

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  1. eLife assessment

    This elegant study presents important findings into how small molecules that were originally developed to inhibit the oncogenic kinase, BRAF, instead trigger activation of this kinase target. Compelling and comprehensive evidence supports a new allosteric model to explain the paradoxical activation. This rigorous work will be of great interest to biochemists, structural biologists, and those working on strategies to inhibit kinases in the context of human disease.

  2. Reviewer #1 (Public Review):

    Summary:

    The authors quantitatively describe the complex binding equilibria of BRAF and its inhibitors resulting in some cases in the paradoxical activation of BRAF dimer when bound to ATP competitive inhibitors. The authors use a biophysical tour de force involving FRET binding assays, NMR, kinase activity assays, and DEER spectroscopy.

    Strengths:

    The strengths of the study are the beautifully conducted assays that allow for a thorough characterization of the allostery in this complex system. Additionally, the use of F-NMR and DEER spectroscopy provides important insights into the details of the process.

    The resulting model for binding of inhibitors and dimerization (Figure 4) is very helpful.

    Weaknesses:

    This is a complex system and its communication is inherently challenging. It might be of interest to the broader readership to understand the implications of the model for drug development and therapy.

  3. Reviewer #2 (Public Review):

    Summary:

    This manuscript uses FRET, 19F-NMR, and DEER/EPR solution measurements to examine the allosteric effects of a panel of BRAF inhibitors (BRAFi). These include first-generation aC-out BRAFi, and more recent Type I and Type II aC-in inhibitors. Intermolecular FRET measurements quantify Kd for BRAF dimerization and inhibitor binding to the first and second subunits. Distinct patterns are found between aC-in BRAFi, where Type I BRAFi binds equally well to the first and second subunits within dimeric BRAF. In contrast, Type II BRAFi shows stronger affinity for the first subunit and weaker affinity for the second subunit, an effect named "allosteric asymmetry". Allosteric asymmetry has the potential for Type II inhibitors to promote dimerization while favoring occupancy of only one subunit (BBD form), leading to the enrichment of an active dimer.

    Measurements of in vitro BRAF kinase activity correlate amazingly well with the calculated amounts of the half-site-inhibited BBD forms with Type II inhibitors. This suggests that the allosteric asymmetry mechanism explains paradoxical activation by this class of inhibitors. DEER/EPR measurements further examine the positioning of helix aC. They show systematic outward movement of aC with Type II inhibitors, relative to the aC-in state with Type I inhibitors, and further show that helix aC adopts multiple states and is therefore dynamic in apo BRAF. This makes a strong case that negative cooperativity between sites in the BRAF dimer can account for paradoxical kinase activation by Type II inhibitors by creating a half-site-occupied homodimer, BBD. In contrast, Type I inhibitors and aC-out inhibitors do not fit this model, and are therefore proposed to be explained by previously proposed models involving negative allostery between subunits in BRAF-CRAF heterodimers, RAS priming, and transactivation.

    Strengths:

    This study integrates orthogonal spectroscopic and kinetic strategies to characterize BRAF dynamics and determine how it impacts inhibitor allostery. The unique combination of approaches presented in this study represents a road map for future work in the important area of protein kinase dynamics. The work represents a worthy contribution not only to the field of BRAF regulation but to protein kinases in general.

    Weaknesses:

    Some questions remain regarding the proposed model for Type II inhibitors and its comparison to Type I and aC-out inhibitors that would be useful to clarify. Specifically, it would be helpful to address whether the activation of BRAF by Type II inhibitors, while strongly correlated with BBD model predictions in vitro, also depends on CRAF via BRAF-CRAF in cells and therefore overlaps with the mechanisms of paradoxical activation by Type I and aC-out inhibitors.