Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS

  1. Metehan Ilter
  2. Ramazan Kaşmer
  3. Farzaneh Jalalypour
  4. Canan Atilgan
  5. Ozan Topcu
  6. Nihal Karakaş  Is a corresponding author
  7. Ozge Sensoy  Is a corresponding author
  1. Istanbul Medipol University, Turkey
  2. Sabanci University, Turkey

Abstract

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

Data availability

Simulated data used to generate the figures in the commentary are available online (https://osf.io/z2y5s/?view_only=070ebb995ba945bb9aac40d5979bd508).

Article and author information

Author details

  1. Metehan Ilter

    1Graduate School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey
    Competing interests
    The authors declare that no competing interests exist.
  2. Ramazan Kaşmer

    Institute for Health Sciences and Technologies, Istanbul Medipol University, Istanbul, Turkey
    Competing interests
    The authors declare that no competing interests exist.
  3. Farzaneh Jalalypour

    Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
    Competing interests
    The authors declare that no competing interests exist.
  4. Canan Atilgan

    Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0557-6044
  5. Ozan Topcu

    Institute for Health Sciences and Technologies, Istanbul Medipol University, Istanbul, Turkey
    Competing interests
    The authors declare that no competing interests exist.
  6. Nihal Karakaş

    Institute for Health Sciences and Technologies, Istanbul Medipol University, Istanbul, Turkey
    For correspondence
    nkarakas@medipol.edu.tr
    Competing interests
    The authors declare that no competing interests exist.
  7. Ozge Sensoy

    Institute for Health Sciences and Technologies, Istanbul Medipol University, Istanbul, Turkey
    For correspondence
    osensoy@medipol.edu.tr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5950-3436

Funding

Health Institute of Turkey (3561/2019-TA-02)

  • Metehan Ilter
  • Ozan Topcu
  • Ozge Sensoy

The Scientific and Technological Research Council of Turkey (116F229)

  • Farzaneh Jalalypour
  • Canan Atilgan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Qiang Cui, Boston University, United States

Version history

  1. Preprint posted: April 24, 2022 (view preprint)
  2. Received: April 25, 2022
  3. Accepted: November 30, 2022
  4. Accepted Manuscript published: December 2, 2022 (version 1)
  5. Accepted Manuscript updated: December 5, 2022 (version 2)
  6. Version of Record published: December 19, 2022 (version 3)

Copyright

© 2022, Ilter et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,437
    views
  • 257
    downloads
  • 4
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Metehan Ilter
  2. Ramazan Kaşmer
  3. Farzaneh Jalalypour
  4. Canan Atilgan
  5. Ozan Topcu
  6. Nihal Karakaş
  7. Ozge Sensoy
(2022)
Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS
eLife 11:e79747.
https://doi.org/10.7554/eLife.79747

Share this article

https://doi.org/10.7554/eLife.79747

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Thomas RM Germe, Natassja G Bush ... Anthony Maxwell
    Research Article

    DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.