Label-free flow cytometry-based enzyme inhibitor identification

Nickelsen, Anna; Jose, Joachim

doi:10.1016/j.aca.2021.338826

Cited by 3 publications

(3 citation statements)

References 34 publications

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“…The ion channel properties such as voltage dependence, activation and closure kinetics are altered by the binding of endogenous cyclic nucleotides cAMP or cGMP to the cyclic nucleotide binding domain (CNBD) [ 53 ]. Here we analyse the impact of eight different amino acids within the CNBD on ligand binding using the method of Autodisplay [ 54 ] and flow cytometry [ 55 ]. The native HCN4 C-Linker-CNBD and the mutants were separately displayed as fusion proteins on the surface of E. coli cells.…”

Section: Early Career Researcher Presentationsmentioning

confidence: 99%

30th Annual GP2A Medicinal Chemistry Conference

et al. 2023

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Section: Early Career Researcher Presentationsmentioning

confidence: 99%

30th Annual GP2A Medicinal Chemistry Conference

et al. 2023

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“…When proteins are exposed on the cell surface, radioligands, and fluorescent probes, − can often be used to analyze affinities of small molecules. However, for intracellular proteins, which comprise ∼86% of the proteome, quantitative binding studies of living cells typically require sophisticated microscopy approaches, , which may have limited generality, or fusion of expressed proteins to protein or peptide tags. , One widely used approach is NanoBRET, where small molecules are linked to fluorophores that accept energy from nanoluciferase.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells

Yin,

Zhao,

Rane

et al. 2023

J. Am. Chem. Soc.

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The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (K d ) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular K d values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular K i values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular K i values matched known biochemical K i values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.

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“…For this purpose, the HCN4 C-Linker-CNBD ( Figure 1 B) was displayed on the surface of E. coli . Fluorescent cAMP derivative 8-Fluo-cAMP ( Figure 1 A) was used to determine ligand binding to the CNBD by single-cell analysis via flow cytometry [ 42 , 43 ]. The assay conditions were rectified with regard to equilibrium state conditions, ligand depletion and non-specific ligand binding.…”

Section: Introductionmentioning

confidence: 99%

A Novel Flow Cytometry-Based Assay for the Identification of HCN4 CNBD Ligands

2023

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Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are promising therapeutic targets because of their association with the genesis of several diseases. The identification of selective compounds that alter cAMP-induced ion channel modulation by binding to the cyclic nucleotide-binding domain (CNBD) will facilitate HCN channel-specific drug development. In this study, a fast and protein purification-free ligand-binding approach with a surface-displayed HCN4 C-Linker-CNBD on E. coli is presented. 8-Fluo-cAMP ligand binding was monitored by single-cell analysis via flow cytometry, and a Kd-value of 173 ± 46 nM was determined. The Kd value was confirmed by ligand depletion analysis and equilibrium state measurements. Applying increasing concentrations of cAMP led to a concentration-dependent decrease in fluorescence intensity, indicating a displacement of 8-Fluo-cAMP. A Ki-value of 8.5 ± 2 µM was determined. The linear relationship of IC50 values obtained for cAMP as a function of ligand concentration confirmed the competitive binding mode: IC50: 13 ± 2 µM/16 ± 3 µM/23 ± 1 µM/27 ± 1 µM for 50 nM/150 nM/250 nM/500 nM 8-Fluo-cAMP. A similar competitive mode of binding was confirmed for 7-CH-cAMP, and an IC50 value of 230 ± 41 nM and a Ki of 159 ± 29 nM were determined. Two established drugs were tested in the assay. Ivabradine, an approved HCN channel pore blocker and gabapentin, is known to bind to HCN4 channels in preference to other isoforms with an unknown mode of action. As expected, ivabradine had no impact on ligand binding. In addition, gabapentin had no influence on 8-Fluo-cAMP’s binding to HCN4-CNBD. This is the first indication that gabapentin is not interacting with this part of the HCN4 channel. The ligand-binding assay as described can be used to determine binding constants for ligands such as cAMP and derivatives. It could also be applied for the identification of new ligands binding to the HCN4-CNBD.

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Label-free flow cytometry-based enzyme inhibitor identification

Cited by 3 publications

References 34 publications

30th Annual GP2A Medicinal Chemistry Conference

30th Annual GP2A Medicinal Chemistry Conference

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells

A Novel Flow Cytometry-Based Assay for the Identification of HCN4 CNBD Ligands

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