Probing the Small-Molecule Inhibition of an Anticancer Therapeutic Protein-Protein Interaction Using a Solid-State Nanopore
Authors: Kim Ki-Bum , Chae Hongsik , Kwak Dong-Kyu , Lee Mi-Kyung , Ha Ji-Hyang , Goyal Gaurav , Kim Min Jun , Chi Seung-Wook 
Published: February 2020

Targeting protein–protein interactions (PPIs) for therapeutic interventions has been an attractive strategy in drug discovery. Because drug development for enzyme targets has been limited by the difficulties in achieving sufficient specificity, PPI inhibitors with extremely high specificity have recently attracted considerable attention. Despite advances in various techniques, such 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; ii) the low sensitivity of SPR for detection of small-molecule binding to proteins; and iii) the requirement for fluorophore labeling in FP. Hence, there is a need to develop a robust HTS methodology to facilitate the discovery of small molecule drugs against PPI targets.

Nanopore sensors have several unique advantages compared with more conventional techniques, including single-molecule resolution and ultrasensitivity, label-free and real-time measurements, and high-throughput detection. Although nanopores have been used to characterize the biophysical properties of diverse biomolecules, including DNA, RNA, and proteins, they have never been applied to the screening of small-molecule drugs such as PPI inhibitors.

Blocking the interaction between mouse double minute 2 (MDM2) and p53 transactivation domain (p53TAD) has been an attractive strategy for cancer therapy because it can restore p53 function, resulting in cancer cell apoptosis. Using this therapeutic strategy, many p53TAD-mimetic lead compounds have been identified for cancer treatment. Among them, Nutlin-3 is one of the most potent MDM2 antagonists and acts as a competitive inhibitor of the MDM2/p53TAD interaction (Kd=0.1 μM). Nutlin-3 structurally mimics the 15-residue a-helical p53TAD peptide that binds to MDM2 (Kd=0.6μM).

To probe the MDM2/p53TAD interaction and its small molecule inhibition using solid-state nanopores, we fabricated ~10–15 nm-sized nanopores in low-pressure chemical vapor deposition (LPCVD) SiNx membranes transferred to the Pyrex substrates. The structure of the N-terminal p53TAD binding domain of MDM2 (PDB code: 1YCR) showed dimensions of 3.1 nm × 3.5 nm × 4.2 nm. At the applied voltage of -175 mV across the nanopore, the positively charged MDM2 domain (residues 3–109, MW=12.3 kDa, net charge at pH 7.4=+2.9e, pI=9.02) was electrophoretically driven from one chamber toward the negative electrode in the other chamber. The passage of MDM2 through the nanopore gave rise to a temporary reduction in the ionic current, leading to the detection of MDM2 translocation events at the single-molecule level.

In contrast to the vigorous translocation of free MDM2, the number of translocation events for the MDM2/GST-p53TAD complex through the nanopore was dramatically reduced to a negligible level. Upon complex formation, the net charge of the proteins at pH 7.4 changes from +2.9e (in free MDM2) to -13.7e (in the MDM2/GST-p53TAD complex) owing to charge masking of MDM2 by negatively charged GST-p53TAD. As a result, the overall negatively charged protein complex could not translocate through the nanopore at the applied negative voltage.

We measured the nanopore translocation of the MDM2/GST-p53TAD complex in the presence of Nutlin-3. After the addition of Nutlin-3 to the protein complex, the translocation of free MDM2 was almost recovered, which indicated that the disruption of the MDM2/GST-p53TAD interaction by Nutlin-3 liberated MDM2 from the GST-p53TAD-bound complex.

To test whether the MDM2/GST-p53TAD interaction is indeed specifically inhibited by Nutlin-3, we repeated the nanopore experiment with a negative control, ABT-737, which is an inhibitor of Bcl-2 family proteins and does not bind to MDM2. Unlike Nutlin-3, ABT-737 could not recover the translocation of MDM2, confirming that Nutlin-3 specifically blocked the interaction between MDM2 and GST-p53TAD.

We believe that this nanopore-based drug screening platform will provide a remarkable improvement over current technological limitations in drug discovery at protein-protein interfaces.