Cryptic binding sites in protein-ligand complexes

• Binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. Some pockets or concave site can already exist in a ligand-free structure of a protein.
• Sometimes, a binding site is flat in the absence of a ligand and only forms in the presence of a ligand (i.e., induced fit) or only opens transiently for short periods of time (i.e., conformational selection); such binding sites are called cryptic sites.
• Cryptic pockets: not easily detectable in the APO state, requires conformational changes to become apparent, and requires a ligand to be detected. These sites can be important for drug discovery because they can provide previously undescribed pockets and thus enable targeting of proteins that would otherwise be considered undruggable.
• Conventional approaches for cryptic site discovery: experimentally by fragment-based ligand discovery and computationally by long molecular dynamics simulations, fragment docking, identifications of small molecule binding hot-spots. Limitations: time consuming, expensive and not always successful.
• Therefore, there is a need for an accurate, automated, and efficient method to predict the location of cryptic pockets in a given ligand-free protein structure. 
• Advantages for detection of cryptic sites: a cryptic site may be the only suitable binding site on the target protein; for example, when activation is required and thus the active site cannot be targeted, or active site ligands need to be avoided due to adverse off-target effects.
• To address a number of questions: what are the sequence, structure, and dynamics attributes of a cryptic site, especially in comparison to binding pockets? Can they be accurately, automatically, and efficiently predicted? How common are cryptic sites? Are they common enough to significantly expand the druggable proteome? Can we predict cryptic sites in specific proteins of clinical significance?  For this Cryptosite was developed.
• Cryptosite: Construction of a set of structurally defined apo–holo pairs with cryptic sites; characterized the cryptic sites in terms of their sequence, structure, and dynamics attributes. Findings: cryptic sites tend to be as conserved in evolution as traditional binding pockets but are less hydrophobic and more flexible. They predicted cryptic sites in the entire structurally characterized human proteome (11,201 structures, covering 23% of all residues in the proteome). CryptoSite increases the size of the potentially “druggable” human proteome from ~ 40% to ~ 78% of disease-associated proteins. 

Recently, Sun et. al. published a study on Structure based analysis of cryptic site opening. (reference below)

AIM: To consider a set of proteins with validated cryptic sites and to study whether the sites remain always cryptic without ligand binding, or whether pockets already form in some of the structures.

General workflow of the study:

Conclusions from the study:

• The binding of a ligand molecule is often accompanied by conformational changes of the protein. This is definitely the case if the binding site is cryptic, and thus is not detectable in the unliganded protein.
• Since the binding proceeds from the free energy minimum of the separate target protein to the free energy minimum of the receptor-ligand complex, the distinction is kinetic rather than thermodynamic. 
• However, the free energy landscape of the protein determines the pathway of the association. In fact, the unbound state is always an ensemble of conformations.
• If conformations without the pocket formed are at deep free energy minima, the probability of pocket formation without ligand binding is small. 
• On the other extreme, if the landscape includes minima leading to conformations with pockets formed, the binding site is most likely cryptic only in a certain fraction of the conformational ensemble.
• MD is increasingly considered a valuable tool to characterize conformational ensembles of macromolecules. Major strengths: it provides both thermodynamic and kinetic information
• The study has revealed that (A) few proteins have even approximately “genuine” cryptic pockets that are unlikely to form without ligand binding; (B) proteins on the other extreme, with spontaneously opening and closing cryptic sites, are also rare; (C) The largest group includes proteins that, under some conditions, have a cryptic pocket with very low druggability but easily form a more druggable pocket if the conditions change. 
• In most proteins the pocket formation is impacted by mutations or off-site.
• X-ray structures of proteins provide ample information on cryptic site opening. 
• Biased MD and druggability scores confirm results from the X-ray structures
• Detection, comparison and analysis of binding pockets are pivotal to structure based drug design.

Useful links: 

• Sun, Zhuyezi, et al. "Structure-based analysis of cryptic-site opening." Structure 28.2 (2020): 223-235.
• Cimermancic P, Weinkam P, Rettenmaier TJ, et al. CryptoSite: Expanding the Druggable Proteome by Characterization and Prediction of Cryptic Binding Sites. J Mol Biol. 2016;428(4):709-719. doi:10.1016/j.jmb.2016.01.029
• Oleinikovas, Vladimiras, et al. "Understanding cryptic pocket formation in protein targets by enhanced sampling simulations." Journal of the American Chemical Society 138.43 (2016): 14257-14263.