A small-molecule probe for monitoring binding to prolyl hydroxylase domain 2 by fluorescence polarisation

Li, Zhihong; Zhen, Shuai; Su, Kaijun; Tumber, Anthony; Yu, Quanwei; Dong, Ying; McDonough, Michael A.; Schofield, Christopher J.; Zhang, Xiaojin

doi:10.1039/d0cc06353c

Cited by 9 publications

(10 citation statements)

References 26 publications

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“…All the target compounds mentioned were assayed for their affinities to PHD2 protein based on the FP assay reported previously. , PHD2 protein (181–426), which was similar activity to the full-length PHD2 protein, was from Nanjing Zoombio Biotechnology. The experiments were performed in 384-well, flat-bottom, black assay plates (#3575, Corning) with a final volume of 60 μL containing a 5 nM small molecule probe (CAS registry number: 2319641-91-1) previously developed by our group, , 50 nM PHD2 protein (181-426), inhibitors with different concentrations, 10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% Tween-20, and less than 1% DMSO. The polarization signal was measured from the top of the well with a SPARK Multi-Mode Microplate Reader (Tecan) with polarized filters and optical modules for fluorescein (λ ex = 485 ± 25 nM, λ em = 535 ± 25 nM).…”

Section: Methodsmentioning

confidence: 99%

“…46,47 PHD2 protein (181−426), which was similar activity to the full-length PHD2 protein, was from Nanjing Zoombio Biotechnology. The experiments were performed in 384-well, flat-bottom, black assay plates (#3575, Corning) with a final volume of 60 μL containing a 5 nM small molecule probe (CAS registry number: 2319641-91-1) previously developed by our group, 46,47 50 nM PHD2 protein (181-426), inhibitors with different concentrations, 10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% Tween-20, and less than 1% DMSO. The polarization signal was measured from the top of the well with a SPARK Multi-Mode Microplate Reader (Tecan) with polarized filters and optical modules for fluorescein (λ ex = 485 ± 25 nM, λ em = 535 ± 25 nM).…”

Section: Methyl (5-(benzyloxy)-1′-(4-chlorobenzoyl)-1′2′3′6′tetrahydr...mentioning

confidence: 99%

“…In this novel preferred conformation-guided drug design, the dihedral angles between the pyridine and piperidinyl-containing linkers were explored to match the desired docking conformation in PHD2 with the lowest energy conformation of the inhibitors. An affinity-based FP assay, reported previously using a small-molecule probe, , was utilized for structure–activity relationship (SAR) optimization in this research. As expected, the inhibitors with lower-energy dihedral angles of the desired docking conformation showed preferable affinity.…”

Section: Introductionmentioning

confidence: 99%

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Preferred Conformation-Guided Discovery of Potent and Orally Active HIF Prolyl Hydroxylase 2 Inhibitors for the Treatment of Anemia

Zhang

Sun

et al. 2023

J. Med. Chem.

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In this work, we discovered a novel series of prolyl hydroxylase 2 (PHD2) inhibitors with improved metabolic properties based on a preferred conformation-guided drug design strategy. Piperidinyl-containing linkers with preferred metabolic stability were designed to match the dihedral angle of the desired docking conformation in the PHD2 binding site with the lowest energy conformation. Based on the piperidinyl-containing linkers, a series of PHD2 inhibitors with high PHD2 affinity and favorable druggability were obtained. Remarkably, compound 22, with an IC 50 of 22.53 nM toward PHD2, significantly stabilized hypoxia-inducible factor α (HIFα) and upregulated the expression of erythropoietin (EPO). Furthermore, oral administration of 22 dose-dependently stimulated erythropoiesis in vivo. Preliminary preclinical studies showed that 22 has good pharmacokinetic properties and an excellent safety profile, even at 10 times the efficacious dose (200 mg/kg). Taken together, these results indicate that 22 is a promising candidate for anemia treatment.

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Section: Methodsmentioning

confidence: 99%

Section: Methyl (5-(benzyloxy)-1′-(4-chlorobenzoyl)-1′2′3′6′tetrahydr...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Preferred Conformation-Guided Discovery of Potent and Orally Active HIF Prolyl Hydroxylase 2 Inhibitors for the Treatment of Anemia

Zhang

Sun

et al. 2023

J. Med. Chem.

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“…We applied the FP‐based ISISS strategy to discovery of in vivo active inhibitors of human prolyl hydroxylase 2 (PHD2), a target for treatment of chronic kidney disease (CKD) related anemia. The ISISS method employs a fluorescein‐labeled probe, which was prepared by linking fluorescein isothiocyanate (FITC) and a potent PHD2 inhibitor (the probe structure is shown in Figure S2) and which monitors binding of competitive ligands using a low concentration of human PHD2 (20 nM) via FP analysis (Figure S2) [10] . PHD catalysis negatively regulates biosynthesis of erythropoietin, hence PHD inhibitors promote hemoglobin (Hb) production and erythropoiesis [11] .…”

Section: Figurementioning

confidence: 99%

In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery

Zhen

et al. 2022

Angewandte Chemie

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Target‐directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high‐throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl‐hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone‐based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.

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“…The ISISS method employs a fluorescein-labeled probe, which was prepared by linking fluorescein isothiocyanate (FITC) and a potent PHD2 inhibitor (the probe structure is shown in Figure S2) and which monitors binding of competitive ligands using a low concentration of human PHD2 (20 nM) via FP analysis (Figure S2). [10] PHD catalysis negatively regulates biosynthesis of erythropoietin, hence PHD inhibitors promote hemoglobin (Hb) production and erythropoiesis. [11] PHD2 inhibitors have the potential to revolutionize the treatment of anemia, with the first-in-class PHD2 inhibitor, Roxadustat, now approved for clinical use.…”

mentioning

confidence: 99%

In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery

Zhen

et al. 2022

Angew Chem Int Ed

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Target-directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high-throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl-hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone-based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.The efficient discovery of small-molecule modulators of biomacromolecule function remains a limiting step in target validation and drug discovery. [1] In the conventional procedure for hit compound discovery, iterative cycles of "designsynthesis-screen" are carried out until a potent candidate for further research is obtained. Target-directed dynamic combinatorial chemistry (tdDCC) has been developed as an alternative method for hit identification. [2] tdDCC involves the target-based selection of hits from dynamic mixtures of reversibly reacting compounds (Figure S1). [3] tdDCC can enable the spatially resolved identification of compounds that bind strongly to a defined target protein. [4]

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A small-molecule probe for monitoring binding to prolyl hydroxylase domain 2 by fluorescence polarisation

Abstract: Inhibition of the dioxygen sensing hypoxia-inducible factor prolyl hydroxylases has potential therapeutic benefit for diseases, including anaemia. We describe the discovery of a small-molecule probe useful for monitoring binding to...

Cited by 9 publications

References 26 publications

Preferred Conformation-Guided Discovery of Potent and Orally Active HIF Prolyl Hydroxylase 2 Inhibitors for the Treatment of Anemia

Preferred Conformation-Guided Discovery of Potent and Orally Active HIF Prolyl Hydroxylase 2 Inhibitors for the Treatment of Anemia

In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery

In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery

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