Probing the Small‐Molecule Inhibition of an Anticancer Therapeutic Protein‐Protein Interaction Using a Solid‐State Nanopore

Abstract

Nanopore sensing is an emerging technology for the single-molecule-based detection of various biomolecules.I n this study,w ep robed the anticancer therapeutic p53 transactivation domain (p53TAD)/MDM2 interaction and its inhibition with as mall-molecule MDM2 antagonist, Nutlin-3, using low-noise solid-state nanopores.A lthough the translocation of positively charged MDM2 through an anopore was detected at the applied negative voltage,t his MDM2 translocation was almost completely blocked upon formation of the MDM2/GST-p53TAD complex owing to charge conversion. In combination with NMR data, the nanopore measurements showed that the addition of Nutlin-3 rescued MDM2 translocation, indicating that Nutlin-3 disrupted the MDM2/GST-p53TAD complex, thereby releasing MDM2. Taken together, our results reveal that solid-state nanopores can be av aluable platform for the ultrasensitive,p icomole-scale screening of small-molecule drugs against protein-protein interaction (PPI) targets. Targetingprotein-proteininteractions(PPIs)fortherapeuticinterventions has been an attractive strategy in drug discovery. [1,2] Because drug development for enzyme targets has been limited by the difficulties in achieving sufficient specificity,P PI inhibitors with extremely high specificity have recently attracted considerable attention. Despite advances in various techniques,s uch as nuclear magnetic resonance (NMR), surface plasma resonance (SPR), and fluorescence polarization (FP), the high-throughput screening (HTS) of small-molecule PPI inhibitors is highly challenging owing to several critical limitations:i )the large amount (mg quantities) of sample required for NMR; [3] ii)the low sensitivity of SPR for detection of small-molecule binding to proteins; [4,5] and iii)the requirement for fluorophore labeling in FP.H ence,t here is an eed to develop ar obust HTS methodology to facilitate the discovery of smallmolecule drugs against PPI targets.Nanopore sensing is an emerging single-molecule technique for biomolecule analysis.W henv oltage is applied across ananoscale pore,the translocation of acharged analyte through the nanopore transiently blocks the ionic current, which is characterized by dwell-time and current amplitude. Nanopore sensors have several unique advantages compared with more conventional techniques,including single-molecule resolution and ultrasensitivity,label-free and real-time measurements,a nd high-throughput detection. [6][7][8][9][10][11][12] Although nanopores have been used to characterize the biophysical properties of diverse biomolecules,i ncluding DNA, RNA, and proteins, [9][10][11][12][13][14][15][16] they have never been applied to the screening of small-molecule drugs such as PPI inhibitors.Despite having much lower resolution and sensitivity, solid-state nanopores have advantages over biological protein nanopores,including high stability,awide range of pore sizes, and controllable surface properties. [17,18] However,t he problem of excess noise,which corresponds to afew tens of pA to 100 pA, [19,20] remains ...

Discrimination of α-Thrombin and γ-Thrombin Using Aptamer-Functionalized Nanopore Sensing

“…Protein nanopores such as α-hemolysin were first used for single-stranded DNA and RNA sensing, peptide structure sensing and analysis, , and single-nucleotide discrimination . Nowadays, nanopore-based biosensors are widely used for detection and analysis of single biomolecules such as nucleic acids, proteins, and small molecules . The most popular application of nanopore sensing is DNA sequencing, which holds great promise in the field of single-molecule sensing for clinical applications .…”

“…7 Nowadays, nanopore-based biosensors are widely used for detection and analysis of single biomolecules such as nucleic acids, 8 proteins, 9 and small molecules. 10 The most popular application of nanopore sensing is DNA sequencing, 8 which holds great promise in the field of single-molecule sensing for clinical applications. 11 Moreover, in the field of proteomics, nanopore biosensors are once again presented as analytical tools with unprecedented single-molecule sensitivity.…”

Single‐Molecule Measurement of Carbonic Anhydrase in Cation Coordinated Environment Using MspA Nanopore†

“…[ 18‐19 ] This work provides a new path to de novo design of protein channels with customized transmembrane traffic functions. Besides, relying on the ionic transport, nanopore electro measurement not only exhibits excellent performance for protein profiling, [ 20‐22 ] but also offers opportunities to illustrate phenomena of protein conformational changes, [ 23 ] protein‐protein interaction, [ 24‐25 ] and explore enzyme dynamics in real‐time manner. [ 26‐27 ] Interestingly, nanopore is also possible at this stage, to directly identify the detailed sequence changes taking place during a biosynthesis of a protein, by enzymatic modifications (post‐translational modifications), [ 28‐29 ] and decoding (sequencing).…”

Label-free Detection of PD-1 Antibody and Antigen Immunoreaction Using Nano-Sensors

“…纳米孔 Freedman 小 组 [29] 分别检测了人体免疫缺陷病毒包膜表面的糖蛋白 gp120 单体及其与特异性抗体的结合物并模拟了人体内 的多组分环境. Kwak 小组 [30] 应用固态纳米孔探究了 (p53TAD)/MDM2 相互作用, 并体外模拟了 MDM2 抑制 剂 Nutlin-3 对(p53TAD)/MDM2 信号通路的阻断作用. 除了抗原抗体结合之后的被动检测, 还有一种主动检测 法, Ying 等 [31]…”