Biophysical and computational fragment-based approaches to targeting protein–protein interactions: applications in structure-guided drug discovery

Abstract

Drug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein-protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids - so-called hotspots - may contribute the majority of interaction-free energy. The challenges posed by protein-protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein-protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods - nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry - to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein-protein interactions and have therapeutic value.

Developing Antagonists for the Met-HGF/SF Protein–Protein Interaction Using a Fragment-Based Approach

“…The large number of promiscuous binders found in our initial fragment screen reflects the challenges faced when no known inhibitor/small molecule binder or control protein containing a disabled binding site can be utilized (50). Therefore, screening campaigns for protein interfaces are likely to require additional selection steps to ensure identification of unique fragment hits (21). The progression from fragment hit to lead compound is challenging even in projects when structural information is available.…”

“…Protein-protein interactions (PPI) are generally large and lack the large cavities that characterize many enzyme active sites, such as protein kinases and proteases, and receptors such as G-protein-coupled receptors. However, the observation that protein surfaces often display small deep pockets (19,20), sometimes closely clustered, offers a basis for alternative design approaches (21). Examples of such inhibitors can be found in the literature, although the approaches by which they were discovered are diverse (21) ranging from virtual screening (22) to fragmentscreening campaigns (23,24) and mimicking the interaction partner by rational design (25).…”

“…However, the observation that protein surfaces often display small deep pockets (19,20), sometimes closely clustered, offers a basis for alternative design approaches (21). Examples of such inhibitors can be found in the literature, although the approaches by which they were discovered are diverse (21) ranging from virtual screening (22) to fragmentscreening campaigns (23,24) and mimicking the interaction partner by rational design (25). A further and major advantage of targeting the extracellular domain of Met is that the compounds would not have to cross the cell membrane, therefore allowing for more flexibility in drug design.…”

Drug-Based Disruption of Protein Complexes with Unknown Structure: Towards a Novel Platform for Drug Discovery

“…Such approaches are usually based on alanine scanning, assessing the impact of individual residues on the binding free energy. In this way, an effective epitope significantly smaller than the PPA region is determined [4,7] and fragment-based lead discovery may be subsequently exploited to generate candidate ligands for competitive binding [4,8,9].…”

“…Such approaches are usually based on alanine scanning, assessing the impact of individual residues on the binding free energy. In this way, an effective epitope significantly smaller than the PPA region is determined [4,7] and fragment-based lead discovery may be subsequently exploited to generate candidate ligands for competitive binding [4,8,9].The problem of therapeutic disruption of a PPA becomes especially difficult when the structure of the targeted binding partner is unknown. As argued in this editorial piece, even in such cases, it is possible to implement a drug discovery platform based solely on sequence-based predictors of binding epitopes.…”