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

Publication 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

  • 891
    Page views
  • 177
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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

Further reading

    1. Structural Biology and Molecular Biophysics
    Abhilash Padavannil, Anjaneyulu Murari ... James A Letts
    Research Article

    Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway ‘deactive’ state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.

    1. Structural Biology and Molecular Biophysics
    Elena Farah Lehmann, Márton Liziczai ... Cristina Manatschal
    Research Article

    A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe2+ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter.