Fragment-based screening by protein-detected NMR spectroscopy

Kerber, Paul J.; Nuñez, Raymundo; Jensen, Davin R.; Zhou, Angela L.; Peterson, Francis C.; Hill, R. Blake; Volkman, Brian F.; Smith, Brian C.

doi:10.1016/bs.mie.2023.06.018

“…In addition, comparing similar compounds with different affinities is essential to build a structure–activity relationship rationale, in complement or not, of 3D protein–ligand complex structures. 55 The reported structures of PIN1 in complexes with compounds 1 and 2 ( Figure S4A,B ) 43 confirm that the STD and photo-CIDNP signal quenching is specific to each proton regarding its environment in the binding pocket. More specifically, the strongest signal quenching is observed for the proton H 4 of both compounds, which inserts into the proton-rich hydrophobic core of PIN1’s binding site (L122, M130, F134).…”

Section: Discussionmentioning

confidence: 63%

“…In addition, comparing similar compounds with different affinities is essential to build a structure–activity relationship rationale, in complement or not, of 3D protein–ligand complex structures . The reported structures of PIN1 in complexes with compounds 1 and 2 (Figure S4A,B) confirm that the STD and photo-CIDNP signal quenching is specific to each proton regarding its environment in the binding pocket.…”

Section: Discussionmentioning

confidence: 93%

Rapid Protein–Ligand Affinity Determination by Photoinduced Hyperpolarized NMR

Bütikofer,

Stadler,

Kadavath

et al. 2024

J. Am. Chem. Soc.

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The binding affinity determination of protein−ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5−15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNPpolarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein−ligand affinities in the micro-to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.

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“…To exemplify the performances of the fiber-coupled cryogenic probe, we recorded the most emblematic experiment of bioNMR, namely a [ 15 N, 1 H]-hetero single quantum coherence (HSQC) (Bodenhausen and Ruben, 1980) which is often used in fragment screening (Kerber et al, 2023) or for affinity determination. (Williamson, 2013) The [ 15 N, 1 H]-HSQC of the KRAS G13D mutant was recorded in the absence and presence of optical fiber (Figure 2C), and we examined the peak shape by extracting the row and column slices (slice command in Topspin®).…”

Section: Resultsmentioning

confidence: 99%

An automated NMR platform with light-coupled cryogenic probes to detect low micromolar samples

Wüster,

Gebbers,

Renn

et al. 2024

Preprint

1

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Abstract. Recent advances in NMR fragment screening use sample illumination to boost NMR sensitivity, reduce measurement time to a few seconds, and reduce sample concentration to a few micromolars. Nevertheless, the absence of a fully automated solution to measure several hundreds of samples with photoinduced hyperpolarization limits the large-scale applicability of the method. We present a setup to couple an optical fiber with a cryogenic probe using the flow-cell accessory port. This setup is compatible with commercially available autosamplers, enabling the fully automated measurement of several hundreds of samples per day.

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Light-coupled cryogenic probes to detect low-micromolar samples and allow for an automated NMR platform

Wüster,

Gebbers,

Renn

et al. 2024

Magn. Reson.

0

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Abstract. Recent advances in NMR fragment screening use sample illumination to boost NMR sensitivity, reduce measurement time to a few seconds, and reduce sample concentration to a few micromolars. Nevertheless, the absence of a fully automated solution to measure several hundreds of samples with photoinduced hyperpolarization limits the large-scale applicability of the method. We present a setup to couple an optical fiber with a cryogenic probe using the flow-cell accessory port. This setup is compatible with commercially available autosamplers, enabling the fully automated measurement of several hundreds of samples per day.

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Fragment-based screening by protein-detected NMR spectroscopy

Cited by 3 publications

References 37 publications

Rapid Protein–Ligand Affinity Determination by Photoinduced Hyperpolarized NMR

Rapid Protein–Ligand Affinity Determination by Photoinduced Hyperpolarized NMR

An automated NMR platform with light-coupled cryogenic probes to detect low micromolar samples

Light-coupled cryogenic probes to detect low-micromolar samples and allow for an automated NMR platform

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