<sup>19</sup>F NMR viewed through two different lenses: ligand-observed and protein-observed<sup>19</sup>F NMR applications for fragment-based drug discovery

Buchholz, Caroline R.; Pomerantz, William C.

doi:10.1039/d1cb00085c

Cited by 40 publications

(56 citation statements)

References 162 publications

(339 reference statements)

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“…In addition, the bio-orthogonality of fluorine atoms facilitates their use for biomolecular and biological samples, especially in the context of “exchange-based” small molecule screening against biomolecular targets inside membrane-less organelles. 28,31,32 The efficacy of drug candidates against biomolecular targets crucially relies on their pharmacokinetic properties ensuring their bio-availability. 33 Similar to the case of biomolecules located inside membrane-bound organelles, drugs have to be able to efficiently enter the phase separated membrane-less compartment.…”

Section: Resultsmentioning

confidence: 99%

Dynamical component exchange in a model phase separating system: an NMR-based approach

Pantoja

Zweckstetter

Rezaei‐Ghaleh

2022

Phys. Chem. Chem. Phys.

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Section: Resultsmentioning

confidence: 99%

Dynamical component exchange in a model phase separating system: an NMR-based approach

Pantoja

Zweckstetter

Rezaei‐Ghaleh

2022

Phys. Chem. Chem. Phys.

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“…At the same time nonspecific binding events can be differentiated. Thereby, new drug candidates and corresponding binding sites and affinities can be identified (Buchholz and Pomerantz 2021 ).…”

Section: Resultsmentioning

confidence: 99%

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags

et al. 2022

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Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.

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“…The lack of biological fluorines greatly simplifies the analysis of 19 F NMR spectra of protein-ligand mixtures when compared to 1 H NMR spectra. 23 However, the reversible affinities of these compounds to proteins lacking the M72C mutation were unconfirmed.…”

Section: Resultsmentioning

confidence: 99%

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy

Peacock

Shokat

2022

Preprint

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While there has been recent success in the development of KRasG12C inhibitors, unmet needs for selective inhibitors and tool compounds targeting the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by 19F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRasG12D. These differences in selectivity, combined with the high resolution of 19F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.

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¹⁹F NMR viewed through two different lenses: ligand-observed and protein-observed¹⁹F NMR applications for fragment-based drug discovery

Abstract: 19F NMR has emerged as a powerful tool in drug discovery, particularly in fragment-based screens. The favorable magnetic resonance properties of the fluorine-19 nucleus, the general absence of fluorine in...

Cited by 40 publications

References 162 publications

Dynamical component exchange in a model phase separating system: an NMR-based approach

Dynamical component exchange in a model phase separating system: an NMR-based approach

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy

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19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

Abstract: 19F NMR has emerged as a powerful tool in drug discovery, particularly in fragment-based screens. The favorable magnetic resonance properties of the fluorine-19 nucleus, the general absence of fluorine in...

Cited by 40 publications

References 162 publications

Dynamical component exchange in a model phase separating system: an NMR-based approach

Dynamical component exchange in a model phase separating system: an NMR-based approach

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy

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¹⁹F NMR viewed through two different lenses: ligand-observed and protein-observed¹⁹F NMR applications for fragment-based drug discovery