TABLE 1.

Biophysical methods for ligand discovery targeting PPI.

Technique	Acronyms	Method	Pros	Cons
Fluorescence Polarization	FP	It uses a fluorescent ligand that changes polarization when interacts with/dissociates from the protein target. It is carried out in a competitive inhibition mode, with a labeled truncated protein containing “hot-spots”.	Simple; low cost; low volumes; suitable for HTS	Introduction of fluorophore tags; non-native binding properties
Amplified Luminescent Proximity Homogeneous Assay Screen	AlphaScreen	Chemiluminescent technology that uses a donor bead (DB) and an acceptor bead (AB), with emission signal at interaction (<200 nm). It is carried out in a competitive inhibition mode	Sensitive; label-free proteins; suitable for HTS; relatively expensive	It needs a specialized plate reader; it can generate false positive
Fluorescence Resonance Energy Transfer	FRET	It uses nonradiative energy transfer between an excited probe (donor), and an accepted probe (acceptor) at interaction (<10 nm). The presence of a PPI dissociating ligand alters the emission wavelength. Time-resolved FRET introduces lanthanide ions as donor to limit signal contaminations	Sensitive; low-cost; low volumes; used with a range of protein sizes	Each interacting protein partner need to be fused with a fluorescent protein; findings can be altered by fluorescence ligand interference
Differential Scanning Fluorimetry (Thermal Shift Assay)	DSF (TSA)	It uses the biding of hydrophobic fluorescent dye to hydrophobic regions of protein targets. The presence of a ligand stabilizing (destabilizing) the PPI increases (decreases) the melting temperature	Simple; Low cost; label-free proteins; immobilization free; suitable for HTS; it can be done in common real-time PCR machines	It is incompatible with low solubility compounds
Nuclear Magnetic Resonance	NMR	NMR experiments identify binding events either by looking at the resonance signals of the ligand or the protein. It can detect non-specific ligand binding. Methods include approaches of Water-LOGSI, Saturation Transfer Difference, Spin Labelling, Inverse NOE pumping	Very sensitive and valuable; label free; immobilization free; it provides epitope mapping, binding affinity (from pM to mM) kinetics and thermodynamics	It is incompatible with low solubility of compounds and targets; it requires high protein concentration; 2D NMR mapping requires labeled proteins; expensive equipment
Surface Plasmon Resonance	SPR	Processes that alter the local refractive index (ligand or protein adsorption) onto the biosensor layer (with an immobilized partner) can be monitored in a surface sensitive fashion by recording the shift of the resonance minimum	Very sensitive; label free; it provides affinity (from nM to mM) kinetics and thermodynamics of protein-protein association/dissociation and ligand binding; a gold standard of PPI, suitable for HTS	It requires immobilization of a binding partner; generally, it requires a positive reference ligand to limit false negatives
Isothermal Titration Calorimetry	ITC	It measures directly the enthalpic energy contribution associated with the binding reaction of two components, and the associated interaction free energy by titration	Sensitive; label free; immobilization free; it directly measures all thermodynamics parameters	It requires high amount of both ligand and protein; expensive equipment
Mass Spectrometry	MS	It detects ligands using irreversible binding compounds/fragments, and approaches of disulfide tethering on targets containing both native and introduced cysteine residues	Very sensitive; label free. It provides epitope mapping; suitable for HTS	It is incompatible with not irreversible ligands; expensive equipment
X-ray Crystallography (Diffraction)	XRD	The electron density map obtained from X-ray diffraction directly yields a high-resolution picture of the ligand–protein complex, providing atomic level insights into the physical chemistry of complex formation	Very powerful technique for studying and validating protein-protein/ligand interactions at atomic resolution. Complex structures can be generated very rapidly. It gives key initial components for molecular dynamics and structure- or fragment-based drug design	It needs high amount of sample and known protein crystallization conditions. Complex structures tend to be more problematic to interpret unambiguously at low-resolution (>3 Å)
Single particle Cryo-Electron Microscopy	CryoEM	The Coulomb potential map can be used to determine at near-atomic resolution the structure of biological macromolecules and large protein complexes that are not accessible to X-ray crystallographic analysis	Powerful technique for studying and validation protein-protein and protein-ligand interactions at near atomic resolution. The reconstruction of various intermediate states can help to understand the dynamics of a complex system	Sample preparation often requires a great deal of optimization. The resolution is often limited to 3–4 Å. Each data collection spans the course of several hours or days, making the throughput for cryo-EM much slower than XRD.