Asymmetric Allosteric Coupling Drives Paradoxical Activation of BRAF by Type II Kinase Inhibitors

Core Concepts

Asymmetric allosteric coupling between inhibitor binding and BRAF dimerization, where binding of the first inhibitor molecule has a much stronger effect than the second, selectively induces catalytically active partially occupied BRAF dimers, leading to paradoxical activation of the MAPK pathway.

Abstract

The content describes a comprehensive biophysical and biochemical analysis of how different classes of RAF kinase inhibitors, specifically type I and type II inhibitors, modulate BRAF dimerization and activation through distinct allosteric mechanisms.
Key highlights:

Type II inhibitors exhibit asymmetric allosteric coupling, where binding of the first inhibitor molecule to the BRAF dimer promotes dimerization much more strongly than binding of the second molecule. In contrast, type I inhibitors show more symmetric allosteric coupling.
This asymmetric allosteric coupling by type II inhibitors selectively induces partially occupied BRAF dimers with one subunit bound to inhibitor and one subunit catalytically active. These partially occupied dimers are highly active and account for the paradoxical activation of the MAPK pathway observed with type II inhibitors.
In contrast, type I inhibitors lack sufficient allosteric asymmetry to induce paradoxical activation of BRAF homodimers, but can still activate the MAPK pathway through induction of BRAF-CRAF heterodimers.
NMR experiments reveal that the BRAF dimer is not locked in a symmetric αC-in conformation, but rather dynamically samples multiple conformational states, providing a structural basis for the observed allosteric asymmetry.
The degree of allosteric asymmetry, represented by the ratio of the allosteric coupling factors α and β, quantitatively predicts the amplitude of paradoxical activation for different RAF inhibitors.

Stats

The BRAF dimer has a baseline dimerization affinity (KDdimer) of 62.4 ± 2.9 μM.
Type II RAF inhibitors have allosteric coupling factors α as large as 104 and β as small as 102, indicating highly asymmetric coupling.
Type I RAF inhibitors have similar α and β values, indicating more symmetric allosteric coupling.
The catalytic turnover rate of partially occupied BRAF BBD dimers is (3.2 ± 0.3) x 10-3 s-1, similar to that of fully active BRAF dimers.

Quotes

"Paradoxical activation by αC-in inhibitors is linked to their ability to induce BRAF dimers, but the molecular mechanisms triggering this activation remain elusive."
"Remarkably, despite binding to both subunits of the BRAF dimer in x-ray structures and being reportedly equipotent for both subunits, these αC-in inhibitors still induce paradoxical activation of MAPK/ERK signaling in cells."
"The failure of the FDA-approved BRAF inhibitors to block BRAF dimers is attributed to negative allostery, in which inhibitors preferentially bind the inactive αC-out state of BRAF and are unable to bind to the active αC-in state adopted by BRAF dimers."

Key Insights Distilled From

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

by Rasmussen,D.... at www.biorxiv.org 04-19-2023

https://www.biorxiv.org/content/10.1101/2023.04.18.536450v2

Deeper Inquiries

How do the distinct conformational states of the BRAF dimer observed by NMR relate to the functional asymmetry and differential inhibitor binding affinities between the two subunits

The distinct conformational states of the BRAF dimer observed by NMR, where one subunit is locked in the αC-in state (αC-innarrow) and the other subunit dynamically samples multiple conformations (αC-inbroad), provide insights into the functional asymmetry and differential inhibitor binding affinities between the two subunits. The static αC-innarrow state represents a stable conformation where the αC-helix is rigid and locked in the active state, while the dynamic αC-inbroad state indicates conformational flexibility and exchange between different substates. This dynamic heterogeneity in the BRAF dimer allows one subunit to explore various conformations, potentially influencing its interaction with inhibitors and other binding partners. The presence of these distinct conformational states suggests that the BRAF dimer is not symmetrically locked in the αC-in state, highlighting functional asymmetry between the subunits. This asymmetry may contribute to differential inhibitor binding affinities, where one subunit may have a higher affinity for inhibitors than the other, leading to asymmetric allosteric coupling and selective induction of partially occupied dimers.

What structural features of type II inhibitors versus type I inhibitors underlie the observed differences in allosteric coupling asymmetry, and how could this information guide the design of next-generation RAF inhibitors that avoid paradoxical activation

The structural features of type II inhibitors, such as belvarafenib and ponatinib, compared to type I inhibitors, like GDC0879, underlie the observed differences in allosteric coupling asymmetry. Type II inhibitors reach further into the active site of BRAF, triggering additional conformational changes in the catalytic DFG motif and activation loop, which are not achieved by type I inhibitors. These structural differences likely unlock the allosteric coupling asymmetry that defines the activating potential of type II inhibitors. Specifically, type II inhibitors induce an asymmetric coupling mechanism where the binding of the first inhibitor molecule promotes dimerization much more strongly than the binding of the second inhibitor molecule. This asymmetry results in higher affinity for the first subunit of the dimer than for the second subunit, leading to the selective induction of BRAF dimers with only one subunit bound to the inhibitor. To guide the design of next-generation RAF inhibitors that avoid paradoxical activation, understanding the structural determinants of allosteric coupling asymmetry is crucial. Future inhibitors could be designed to target specific conformations or binding sites within the BRAF dimer to achieve more balanced and symmetric allosteric effects, minimizing paradoxical activation while maintaining therapeutic efficacy.

Given the central role of BRAF dimerization in both normal signaling and drug resistance, how might the insights from this study be leveraged to develop alternative therapeutic strategies that target BRAF dimers more effectively

The insights from this study on the central role of BRAF dimerization in both normal signaling and drug resistance can be leveraged to develop alternative therapeutic strategies that target BRAF dimers more effectively. One approach could involve designing inhibitors that specifically target the dynamic conformational states of the BRAF dimer, aiming to stabilize or disrupt these states to modulate signaling pathways. By understanding the functional asymmetry and differential inhibitor binding affinities between the subunits of the BRAF dimer, novel inhibitors could be developed to selectively target the subunit with higher affinity, avoiding paradoxical activation. Additionally, the development of combination therapies that target multiple points in the RAF-MEK-ERK pathway, including both monomeric and dimeric forms of BRAF, could provide more comprehensive and effective treatment strategies for cancers driven by dysregulated MAPK signaling. Overall, leveraging the mechanistic insights from this study to design precision therapies that target BRAF dimers with improved selectivity and efficacy could lead to better outcomes for patients with RAF-driven cancers.