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

Abstract

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]

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

“…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).…”

“…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).…”

“…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.…”

Design of Tracers in Fluorescence Polarization Assay for Extensive Application in Small Molecule Drug Discovery

“…On the basis of this probe, Zhang's team continued to develop more suitable inhibitors with a comprehensive method�in situ inhibitor synthesis and screening (ISISS)�in 2022. 117 In this new method, FP assay using tracer 30 was combined with a biorthogonal in situ reaction to detect various fragment combinations, which provides an efficient method for identifying and optimizing small molecule inhibitors. Based on the structural characteristics of the PHD2 active site and the biorthogonal acylhydrazone formation reaction, two series of potential building blocks for ISISS identification of PHD inhibitors were confirmed.…”

“…Due to the research above, it is a fact that FP assay which employs tracer 30 has the potential to monitor in real time the ligand–PHD2 dynamic interaction and screen Fe­(II)-binding PHD2 inhibitors. On the basis of this probe, Zhang’s team continued to develop more suitable inhibitors with a comprehensive method in situ inhibitor synthesis and screening (ISISS)in 2022 . In this new method, FP assay using tracer 30 was combined with a biorthogonal in situ reaction to detect various fragment combinations, which provides an efficient method for identifying and optimizing small molecule inhibitors.…”