“Hotspot” is one of those extremely versatile words, similar to “model” and “buffer”, which can mean a variety of things depending on context. According to Merriam-Webster, a hotspot is “a place of more than usual interest, activity, or popularity”. This is the most general definition of the concept I could find in a quick search, and the one I find closest in spirit to the way hotspots are perceived in a structural biology context. What this blog post is definitely not about are hotspots as “areas of political, military, or civil unrest” (my experience with them has so far been mostly peaceful), or anything to do with geology, WiFi connections, or forest fires.
However, even within the context of structural biology and structure-based drug design, the word “hotspot” has multiple meanings. In this blog post, I will try to summarise the main ones I have come across, the (sometimes subtle) differences between them, and provide a few useful papers to serve as an entry point for interested readers.
Protein-Protein Interaction Hotspots
Protein-protein interfaces (PPI) are have historically been considered poorly druggable, largely due to their large and mostly flat surface areas. At the same time, disrupting or stabilising them can have great therapeutic potential, so PPIs are of great interest as drug discovery targets. During the last couple of decades, an increasing number of drugs targeting PPIs have been approved, including Venetoclax, a fragment-based drug design success story. Part of this change in attitude (and success rate) came from an increased understanding of the energetics of PPIs. Mutagenesis studies showed that a few, crucial “hotspot” residues confer a disproportionate amount of the binding energy at these interfaces, and that disrupting (or stabilising) those interactions could be a viable strategy for drug design. The below papers go into the details of what these hotspots are, what are their properties, and how these can and have been used in drug design campaigns.
Ran, X. & Gestwicki, J. E. Inhibitors of protein–protein interactions (PPIs): an analysis of scaffold choices and buried surface area. Curr. Opin. Chem. Biol. 44, 75–86 (2018). Link
Zerbe, B. S., Hall, D. R., Vajda, S., Whitty, A. & Kozakov, D. Relationship between Hot Spot Residues and Ligand Binding Hot Spots in Protein–Protein Interfaces. J. Chem. Inf. Model. 52, 2236–2244 (2012). Link
Atomic Hotspots
Atomic hotspots can be thought of as the propensity for a given “probe” atom to be found on the surface of the protein. Two well-known examples of methods for detecting such hotspots are GRID and SuperStar. Both are computational methods that use atomic probes to scan the surfaces of target proteins for areas that favour noncovalent interactions involved in ligand-binding. While such methods have contributed to drug design campaigns, one known limitation (suggested by their definition) is that they do not take into account the molecular context of these interactions, which would be present in a ligand.
Goodford, P. J. A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules. J. Med. Chem. 28, 849–857 (1985).
Verdonk, M. L., Cole, J. C. & Taylor, R. SuperStar: A knowledge-based approach for identifying interaction sites in proteins. J. Mol. Biol. 289, 1093–1108 (1999).
Fragment and Small Molecule Hotspots
This type of hotspots are areas of the target binding site that make a disproportionately large contribution to binding affinity in the context of small molecule ligands. Both computational and experimental methods have been used to find such hotspots. Some of these look at the consensus sites identified by the crystallographic presence of bound small molecules, or by their residence times in cosolvent molecular dynamics simulations. In addition, “warm spots” have also been proposed. These are areas in the vicinity of hotspots, into which fragments can be elaborated. Below are some methods for finding hotspots experimentally and computationally. This list is not meant to provide an exhaustive overview, but to lightly scratch the surface of this vast and exciting field, and hopefully provide a starting point for further exploration.
Mattos, C. et al. Multiple solvent crystal structures: Probing binding sites, plasticity and hydration. J. Mol. Biol. 357, 1471–1482 (2006).
Brenke, R. et al. Fragment-based identification of druggable ‘hot spots’ of proteins using Fourier domain correlation techniques. Bioinformatics25, 621–627 (2009). Link
Alvarez-Garcia, D. & Barril, X. Molecular simulations with solvent competition quantify water displaceability and provide accurate interaction maps of protein binding sites. J. Med. Chem. 57, 8530–8539 (2014). Link
Faller, C. E., Raman, E. P., MacKerell Jr, A. D. & Guvench, O. Site Identification by Ligand Competitive Saturation (SILCS) simulations for fragment-based drug design. Methods Mol. Biol. 1289, 75–87 (2015). Link
Radoux, C. J., Olsson, T. S. G., Pitt, W. R., Groom, C. R. & Blundell, T. L. Identifying Interactions that Determine Fragment Binding at Protein Hotspots. J. Med. Chem. 59, 4314–4325 (2016). Link
Rathi, P. C. et al. Predicting ‘Hot’ and ‘Warm’ Spots for Fragment Binding. J. Med. Chem. 60, 4036–4046 (2017). Link
O’Reilly, M. et al. Crystallographic screening using ultra-low-molecular-weight ligands to guide
drug design. Drug Discov. Today24, 1081–1086 (2019). Link
