Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization.

The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.

Inhibition of ACE2-Spike Interaction by an ACE2 Binder Suppresses SARS-CoV-2 Entry.

The emergence of SARS-CoV-2 variants is a significant concern in developing effective therapeutics and vaccines in the middle of the ongoing COVID-19 pandemic. Here, we have identified a novel small molecule that inhibited the interactions between SARS-CoV-2 spike RBDs and ACE2 by modulating ACE2 without impairing its enzymatic activity necessary for normal physiological functions. Furthermore, the identified compounds suppressed viral infection in cultured cells by inhibiting the entry of ancestral and variant SARS-CoV-2. Our study suggests that targeting ACE2 could be a novel therapeutic strategy to inhibit SARS-CoV-2 entry into host cells and prevent the development of COVID-19.

Investigation of Molecular Details of Keap1-Nrf2 Inhibitors Using Molecular Dynamics and Umbrella Sampling Techniques.

In this study, we investigate the atomistic details of Keap1-Nrf2 inhibitors by in-depth modeling techniques, including molecular dynamics (MD) simulations, and the path-based free energy method of umbrella sampling (US). The protein-protein interaction (PPI) of Keap1-Nrf2 is implicated in several neurodegenerative diseases like cancer, diabetes, and cardiomyopathy. A better understanding of the five sub-pocket binding sites for Nrf2 (ETGE and DLG motifs) inside the Kelch domain would expedite the inhibitor design process. We selected four protein-ligand complexes with distinct co-crystal ligands and binding occupancies inside the Nrf2 binding site. We performed 100 ns of MD simulation for each complex and analyzed the trajectories. From the results, it is evident that one ligand (1VV) has flipped inside the binding pocket, whereas the remaining three were stable. We found that Coulombic (Arg483, Arg415, Ser363, Ser508, and Ser602) and Lennard-Jones (Tyr525, Tyr334, and Tyr572) interactions played a significant role in complex stability. The obtained binding free energy values from US simulations were consistent with the potencies of simulated ligands. US simulation highlight the importance of basic and aromatic residues in the binding pocket. A detailed description of the dissociation process brings valuable insight into the interaction of the four selected protein-ligand complexes, which could help in the future to design more potent PPI inhibitors.

Theoretical Model of EphA2-Ephrin A1 Inhibition.

This work aims at the theoretical description of EphA2-ephrin A1 inhibition by small molecules. Recently proposed ab initio-based scoring models, comprising long-range components of interaction energy, is tested on lithocholic acid class inhibitors of this protein⁻protein interaction (PPI) against common empirical descriptors. We show that, although limited to compounds with similar solvation energy, the ab initio model is able to rank the set of selected inhibitors more effectively than empirical scoring functions, aiding the design of novel compounds.

Structure-based design, synthesis and evaluation of a novel family of PEX5-PEX14 interaction inhibitors against Trypanosoma.

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

Homo- and Heterodimerization of Proteins in Cell Signaling: Inhibition and Drug Design.

Protein dimerization controls many physiological processes in the body. Proteins form homo-, hetero-, or oligomerization in the cellular environment to regulate the cellular processes. Any deregulation of these processes may result in a disease state. Protein-protein interactions (PPIs) can be inhibited by antibodies, small molecules, or peptides, and inhibition of PPI has therapeutic value. PPI drug discovery research has steadily increased in the last decade, and a few PPI inhibitors have already reached the pharmaceutical market. Several PPI inhibitors are in clinical trials. With advancements in structural and molecular biology methods, several methods are now available to study protein homo- and heterodimerization and their inhibition by drug-like molecules. Recently developed methods to study PPI such as proximity ligation assay and enzyme-fragment complementation assay that detect the PPI in the cellular environment are described with examples. At present, the methods used to design PPI inhibitors can be classified into three major groups: (1) structure-based drug design, (2) high-throughput screening, and (3) fragment-based drug design. In this chapter, we have described some of the experimental methods to study PPIs and their inhibition. Examples of homo- and heterodimers of proteins, their structural and functional aspects, and some of the inhibitors that have clinical importance are discussed. The design of PPI inhibitors of epidermal growth factor receptor heterodimers and CD2-CD58 is discussed in detail.

Direct and Propagated Effects of Small Molecules on Protein-Protein Interaction Networks.

Networks of protein-protein interactions (PPIs) link all aspects of cellular biology. Dysfunction in the assembly or dynamics of PPI networks is a hallmark of human disease, and as such, there is growing interest in the discovery of small molecules that either promote or inhibit PPIs. PPIs were once considered undruggable because of their relatively large buried surface areas and difficult topologies. Despite these challenges, recent advances in chemical screening methodologies, combined with improvements in structural and computational biology have made some of these targets more tractable. In this review, we highlight developments that have opened the door to potent chemical modulators. We focus on how allostery is being used to produce surprisingly robust changes in PPIs, even for the most challenging targets. We also discuss how interfering with one PPI can propagate changes through the broader web of interactions. Through this analysis, it is becoming clear that a combination of direct and propagated effects on PPI networks is ultimately how small molecules re-shape biology.