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From vibrations to function: Spectroscopic detection and quantification of π-π stacking in drug-responsive protein
Narangerel Altangerel1, Esther J Ocola1, Benjamin W Neuman1
1Texas A&M University, College Station, TX 77843, USA.
Science Advances
|April 8, 2026
Summary
A new spectroscopic method, TRIP, directly measures aromatic π-π stacking in proteins. This technique precisely quantifies interactions within the SARS-CoV-2 main protease (Mpro), aiding in the design of effective antiviral drugs.
Area of Science:
- Biophysics
- Structural Biology
- Drug Discovery
Background:
- Aromatic π-π stacking is crucial for protein structure and function.
- Quantifying these interactions in biological settings is difficult.
- The SARS-CoV-2 main protease (Mpro) is a key drug target.
Purpose of the Study:
- To develop and validate a method for direct, label-free quantification of aromatic π-π interactions.
- To investigate π-π stacking in the SARS-CoV-2 Mpro dimer.
- To correlate π-π stacking strength with drug efficacy.
Main Methods:
- Thermostable Raman interaction profiling (TRIP) spectroscopy.
- Analysis of phenylalanine benzene ring breathing (BRB) modes.
- Density functional theory (DFT) calculations.
- Biochemical assays and cell-based antiviral efficacy tests.
Main Results:
- TRIP successfully detected and quantified π-π stacking in Mpro.
- BRB spectral changes correlated with Mpro dimerization and ligand binding.
- Potent inhibitors (MPI8, nirmatrelvir) showed stronger π-π stacking signals.
- Spectroscopic data correlated with IC50 values and antiviral activity.
Conclusions:
- TRIP is a robust tool for probing π-π stacking in native-like protein environments.
- This method can guide the design of drugs targeting aromatic protein-protein interfaces.
- Understanding π-π stacking is vital for developing effective therapeutics.
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