Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder

Donohue, Elizabeth; Khorsand, Sina; Mercado, Gabriel; Varney, Kristen M.; Wilder, Paul T.; Yu, Wenbo; MacKerell, Alexander D.; Alexander, Patrick; Van, Que N.; Moree, Ben; Stephen, Andrew; Weber, David J.; Salafsky, Joshua; McCormick, Frank

doi:10.1073/pnas.1905516116

Cited by 19 publications

(19 citation statements)

References 41 publications

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“…Second-harmonic generation (SHG) is another biophysical screening method that has recently been used in combination with fragment libraries. [27,73,74] The method requires labeling the target molecule with a dye, immobilizing it, and then radiating it with 800 nm red light (Figure 15). The second-harmonic generation causes two incoming photons to be absorbed by the dye and then emit 400 nm light, and the intensity of the emitted light is dependent on the position of the dye relative to the surface normal.…”

Section: Second-harmonic Generationmentioning

confidence: 99%

“…The method is comparable to SPR, but where SPR only provides binding information such as affinity, SHG provides both binding information and conformational data, making it especially useful for screening of fragments as they have inherently low affinity but can still induce conformational changes upon binding. [73] The method has also been applied with success using small molecules against RNA targets including hairpins and riboswitches by Birman et al [28] The authors optimized the assay for the theophylline aptamer which binds theophylline, a molecule that fits within most of the RO3 fragment guidelines. Even though the group used small molecules to screen against the aptamer, it seems likely that this method could be a valuable tool in FBDD against RNA.…”

Section: Second-harmonic Generationmentioning

confidence: 99%

“…Two photons are absorbed and then emitted as one 400 nm blue photon, where the intensity depends on the dye's angle of orientation (θ) relative to the surface normal z. [73] Adapted from Ref. [73] by Donohue et al with permission from National Academy of Sciences, copyright 2019.…”

Section: Cryogenic Electron Microscopymentioning

confidence: 99%

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Fragment‐Based Drug Discovery for RNA Targets

et al. 2021

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Rapid development within the fields of both fragment-based drug discovery (FBDD) and medicinal targeting of RNA provides possibilities for combining technologies and methods in novel ways. This review provides an overview of fragment-based screening (FBS) against RNA targets, including a discussion of the most recently used screening and hit validation methods such as NMR spectroscopy, X-ray crystallography, and virtual screening methods. A discussion of fragment library design based on research from small-molecule RNA binders provides an overview on both the currently limited guidelines within RNA-targeting fragment library design, and future possibilities. Finally, future perspectives are provided on screening and hit validation methods not yet used in combination with both fragment screening and RNA targets.

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Section: Second-harmonic Generationmentioning

confidence: 99%

Section: Second-harmonic Generationmentioning

confidence: 99%

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Fragment‐Based Drug Discovery for RNA Targets

et al. 2021

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“…For instance, the small molecule Cmpd2 binds to a pocket between K-Ras and the membrane, stabilizing K-Ras in an occluded orientation [64]. Dedicated technologies such as the second harmonic generation (SHG)-based screening method are sensitive to the orientation of a protein and may therefore enable specific drug screening campaigns for modulators of the Ras/RAF conformation in the future [88] (Table 1). Table 1.…”

Section: What Do We Know About the Activation Of Raf By Ras On The Mementioning

confidence: 99%

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again

Abankwa

Gorfe

2020

Biomolecules

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Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation is plausibly linked to Ras-nanoclustering and -dimerization. We then look at how these mechanistic features could cooperate in the engagement and activation of RAF by Ras. Moreover, we show how this structural information can be integrated with microscopy data that provide nanoscale resolution in cell biological experiments. Synthesizing the available data, we propose to distinguish between two types of Ras nanoclusters, an active, immobile RAF-dependent type and an inactive/neutral membrane anchor-dependent. We conclude that it is possible that Ras reorientation enables dynamic Ras dimerization while the whole Ras/RAF complex transits into an active state. These transient di/oligomer interfaces of Ras may be amenable to pharmacological intervention. We close by highlighting a number of open questions including whether all effectors form active nanoclusters and whether there is an isoform specific composition of Ras nanocluster.

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“…A number of mutations with subtle MAPK effects have now been mechanistically linked to the above Ras-RAF activation scenario [56,64,81] binds to a pocket between K-Ras and the membrane, stabilizing K-Ras in an occluded orientation [81]. Dedicated technologies such as the second harmonic generation (SHG)-based screening method are sensitive to the orientation of a protein and may therefore enable specific drug screening campaigns for modulators of the Ras/ RAF conformation in the future [86] ( Table 1). [7] prohibitin inhibitors [87], salinomycin [88] Ras orientation RASopathies and cancer; mutations in Ras switch III [56,64] Cmpd 2 [81] Ras dimerization ?…”

Section: What Do We Know About the Activation Of Raf By Ras On The Mementioning

confidence: 99%

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again

Abankwa¹,

Gorfe²

2020

Preprint

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Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we will review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanocluster. We will discuss how G-domain reorientation is plausibly linked to Ras-nanoclustering and -dimerization. We will then look at how these mechanistic features could cooperate in the engagement and activation of RAF by Ras. Moreover, we will show how this structural information can be integrated with microscopy data that provide nanoscale resolution in cell biological experiments. Synthesizing the available data, we propose to distinguish between two types of Ras nanoclusters, an active, immobile RAF-dependent type and an inactive/ neutral membrane anchor-dependent. We conclude that it is possible that Ras reorientation enables dynamic Ras dimerization, while the whole Ras/ RAF complex transits into an active state. These transient di/oligomer interfaces of Ras may be amenable to pharmacological intervention. We close by highlighting a number of open questions including, whether all effectors form active nanoclusters and whether there is an isoform specific composition of Ras nanocluster.

show abstract

Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder

Cited by 19 publications

References 41 publications

Fragment‐Based Drug Discovery for RNA Targets

Fragment‐Based Drug Discovery for RNA Targets

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again

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