An emerging paradigm to develop analytical methods based on immobilized transmembrane proteins and its applications in drug discovery

Li, Qian; Yin, Guowei; Wang, Jing; Li, Linkang; Liang, Qi; Zhao, Xue; Chen, Yuanyuan; Zheng, Xiaohui; Zhao, Xinfeng

doi:10.1016/j.trac.2022.116728

Cited by 17 publications

(8 citation statements)

References 127 publications

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“…The way to synthesize immobilized protein is a debated and critical factor needing to be considered when HPAC is applied in drug–protein binding analysis using immobilized protein as the stationary phase. , This raises the question as to what extent the stationary phase models the behavior of the original protein since immobilization proves to have an influence on the protein activity. In most cases, it induces loss of protein functionality by disordered orientation, denaturation, and changes of solubility .…”

Section: Resultsmentioning

confidence: 99%

Affinity Chromatographic Method for Determining Drug–Protein Interaction with Enhanced Speed Than Typical Frontal Analysis

Ou,

Qiao,

et al. 2023

Langmuir

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Revealing drug−protein interaction is highly important to select a drug candidate with improved drug-like properties in the early stages of drug discovery. This highlights the urgent need to develop assays that enable the analysis of drug−protein interaction with high speed. Herein, this purpose was realized by the development of an affinity chromatographic method with a two-fold higher speed than typical assays like frontal analysis and zonal elution. The method involved synthesis of a stationary phase by immobilizing poly(ADPribose) polymerase-1 (PARP 1 ) onto macroporous silica gel through a one-step bioorthogonal reaction, characterization of mutual displacement interaction of two canonical drugs to the immobilized PARP 1 , determination of the interaction between three (iniparib, rucaparib, and olaparib) drugs and the protein, and validation of these parameters by typical frontal analysis. The numbers of binding sites on the column were (2.85 ± 0.05) × 10 −7 , (1.89 ± 0.71) × 10 −6 , and (1.49 ± 0.06) × 10 −7 M for iniparib, rucaparib, and olaparib, respectively. On these sites, the association constants of the three drugs to the protein were (9.85 ± 0.56) × 10 4 , (2.85 ± 0.34) × 10 4 , and (1.07 ± 0.35) × 10 5 M −1 . The determined parameters presented a good agreement with the calculation by typical frontal analyses, which indicated that the current continuous competitive frontal analysis method was reliable for determining drug−protein interaction. Application of the methods was achieved by screening tubeimosides I and II as the bioactive compounds against breast cancer in Bolbostemma paniculatum. Their mechanism may be the interference of DNA repair via down-regulating PARP 1 and meiotic recombination 11 expressions, thus leading to oncogene mutations and death of cancer cells. The method was high speed since it allowed simultaneous determination of binding parameters between two drugs and a protein with a smaller number of experiments to be performed. Such a feature made the method an attractive alternative for high-speed analysis of drug−protein interaction or the other bindings in a binary system.

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Section: Resultsmentioning

confidence: 99%

Affinity Chromatographic Method for Determining Drug–Protein Interaction with Enhanced Speed Than Typical Frontal Analysis

Ou,

Qiao,

et al. 2023

Langmuir

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“…Monoclonal antibodies have an N-type glycan chain attached to enzyme immobilization, which is critical for the transition to a bio-based circular economy and suffers from precision issues that lead to the disruption of enzyme structure and loss of activity. [76,77] Current methods include covalent immobilization with cross-linkers, which is imprecise, and immobilization on supports; these methods suffer from leaching and activity dilution. Recombinant DNA and bio-orthogonal chemistry offer more precise immobilization and facilitate enzyme production, purification, and immobilization in a single step.…”

Section: Protein Immobilizationmentioning

confidence: 99%

Lipoic Acid Ligase A‐Mediated Ligation: Mechanism, Applications, and Emerging Innovations in Bioconjugation

Yamazaki,

Matsuda

2023

ChemistrySelect

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This review outlines the latest findings on bioconjugation using lipoic acid ligase A (LplA), a key enzyme that allows the introduction of new functional groups into biomolecules. LplA functions via a characteristic mechanism of covalently binding orthologous lipoic acid derivatives in vivo to a specific 13 amino acid sequence called the LplA acceptor peptide (LAP). This unique functionality has enabled a wide range of applications, including cell‐surface modification, protein immobilization, fluorescent labeling of antibodies. Recently, the modification of proteins without LAP sequences has been demonstrated, suggesting their potential for future biopharmaceutical production. Although several review articles on enzyme ligation have been published, research on LplA remains limited. This review attempts to fill this gap by introducing LplA based on its mechanism, applications, and recent research reports.

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“…Several modern techniques have been used to examine drug–protein interactions, including surface plasmon resonance [ 6 ], graph neural networks [ 7 ], multispectroscopic analysis [ 8 ], molecular docking and molecular dynamics simulations [ 9 , 10 ], and chromatographic methods [ 11 ]. Among these methodologies, quantitative affinity chromatography is extremely powerful.…”

Section: Introductionmentioning

confidence: 99%

“…Among these methodologies, quantitative affinity chromatography is extremely powerful. This method combines chromatographic separation and the specific binding capacity of the receptor protein [ 11 , 12 ], the advantages of which not only provide good specificity but also address the principle of drug–receptor interactions efficiently [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Immobilization of M3 Muscarinic Receptor to Rapidly Analyze Drug—Protein Interactions and Bioactive Components in a Natural Plant

Fan

Huang

Zhang

et al. 2023

IJMS

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Despite its increasing application in pursing potential ligands, the capacity of receptor affinity chromatography is greatly challenged as most current research studies lack a comprehensive characterization of the ligand–receptor interaction, particularly when simultaneously determining their binding thermodynamics and kinetics. This work developed an immobilized M3 muscarinic receptor (M3R) affinity column by fixing M3R on amino polystyrene microspheres via the interaction of a 6-chlorohexanoic acid linker with haloalkane dehalogenase. The efficiency of the immobilized M3R was tested by characterizing the binding thermodynamics and kinetics of three known drugs to immobilized M3R using a frontal analysis and the peak profiling method, as well as by analyzing the bioactive compounds in Daturae Flos (DF) extract. The data showed that the immobilized M3R demonstrated good specificity, stability, and competence for analyzing drug–protein interactions. The association constants of (−)-scopolamine hydrochloride, atropine sulfate, and pilocarpine to M3R were determined to be (2.39 ± 0.03) × 104, (3.71 ± 0.03) × 104, and (2.73 ± 0.04) × 104 M−1, respectively, with dissociation rate constants of 27.47 ± 0.65, 14.28 ± 0.17, and 10.70 ± 0.35 min−1, respectively. Hyoscyamine and scopolamine were verified as the bioactive compounds that bind to M3R in the DF extract. Our results suggest that the immobilized M3R method was capable of determining drug–protein binding parameters and probing specific ligands in a natural plant, thus enhancing the effectiveness of receptor affinity chromatography in diverse stages of drug discovery.

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An emerging paradigm to develop analytical methods based on immobilized transmembrane proteins and its applications in drug discovery

Cited by 17 publications

References 127 publications

Affinity Chromatographic Method for Determining Drug–Protein Interaction with Enhanced Speed Than Typical Frontal Analysis

Affinity Chromatographic Method for Determining Drug–Protein Interaction with Enhanced Speed Than Typical Frontal Analysis

Lipoic Acid Ligase A‐Mediated Ligation: Mechanism, Applications, and Emerging Innovations in Bioconjugation

Immobilization of M3 Muscarinic Receptor to Rapidly Analyze Drug—Protein Interactions and Bioactive Components in a Natural Plant

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