Probing the Allosteric Inhibition Mechanism of a Spike Protein Using Molecular Dynamics Simulations and Active Compound Identifications.

The receptor recognition of the novel coronavirus SARS-CoV-2 relies on the "down-to-up" conformational change in the receptor-binding domain (RBD) of the spike (S) protein. Therefore, understanding the process of this change at the molecular level facilitates the design of therapeutic agents. With the help of coarse-grained molecular dynamic simulations, we provide evidence showing that the conformational dynamics of the S protein are globally cooperative. Importantly, an allosteric path was discovered that correlates the motion of the RBD with the motion of the junction between the subdomain 1 (SD1) and the subdomain 2 (SD2) of the S protein. Building on this finding, we designed non-RBD binding modulators to inhibit SARS-CoV-2 by prohibiting the conformational change of the S protein. Their inhibition effect and function stages at inhibiting SARS-CoV-2 were evaluated experimentally. In summary, our studies establish a molecular basis for future therapeutic agent design through allosteric effects.

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.

In quest of small-molecules as potent non-competitive inhibitors against influenza.

A series of scaffolds namely aurones, 3-indolinones, 4-quinolones and cinnamic acid-piperazine hybrids, was designed, synthesized and investigated in vitro against influenza A/H1N1pdm09 virus. Designed molecules adopted different binding mode i.e., in 430-cavity of neuraminidase, unlike sialic acid and oseltamivir in molecular docking studies. All molecules reduced the viral titer and exhibited non-cytotoxicity along with cryo-protective property towards MDCK cells. Molecules (Z)-2-(3'-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2f), (Z)-2-(4'-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2g) and 2-(2'-Methoxy-phenyl)-1H-quinolin-4-one (3a) were the most interesting molecules identified in this research, endowed with robust potencies showing low-nanomolar EC50 values of 4.0 nM, 6.7 nM and 4.9 nM, respectively, compared to reference competitive and non-competitive inhibitors: oseltamivir (EC50 = 12.7 nM) and quercetin (EC50 = 0.56 µM), respectively. Besides, 2f, 2g and 3a exhibited good neuraminidase inhibitory activity in sub-micromolar range (IC50 = 0.52 µM, 3.5 µM, 1.3 µM respectively). Moreover, these molecules were determined as non-competitive inhibitors similar to reference non-competitive inhibitor quercetin unlike reference competitive inhibitor oseltamivir in kinetics studies.

Flexible Synthetic Carbohydrate Receptors as Inhibitors of Viral Attachment.

Carbohydrate-receptor interactions are often involved in the docking of viruses to host cells, and this docking is a necessary step in the virus life cycle that precedes infection and, ultimately, replication. Despite the conserved structures of the glycans involved in docking, they are still considered "undruggable", meaning these glycans are beyond the scope of conventional pharmacological strategies. Recent advances in the development of synthetic carbohydrate receptors (SCRs), small molecules that bind carbohydrates, could bring carbohydrate-receptor interactions within the purview of druggable targets. Here we discuss the role of carbohydrate-receptor interactions in viral infection, the evolution of SCRs, and recent results demonstrating their ability to prevent viral infections in vitro. Common SCR design strategies based on boronic ester formation, metal chelation, and noncovalent interactions are discussed. The benefits of incorporating the idiosyncrasies of natural glycan-binding proteins-including flexibility, cooperativity, and multivalency-into SCR design to achieve nonglucosidic specificity are shown. These studies into SCR design and binding could lead to new strategies for mitigating the grave threat to human health posed by enveloped viruses, which are heavily glycosylated viroids that are the cause of some of the most pressing and untreatable diseases, including HIV, Dengue, Zika, influenza, and SARS-CoV-2.

Site mapping and small molecule blind docking reveal a possible target site on the SARS-CoV-2 main protease dimer interface.

The SARS-CoV-2 virus is causing COVID-19 resulting in an ongoing pandemic with serious health, social, and economic implications. Much research is focused in repurposing or identifying new small molecules which may interact with viral or host-cell molecular targets. An important SARS-CoV-2 target is the main protease (Mpro), and the peptidomimetic α-ketoamides represent prototypical experimental inhibitors. The protease is characterised by the dimerization of two monomers each which contains the catalytic dyad defined by Cys145 and His41 residues (active site). Dimerization yields the functional homodimer. Here, our aim was to investigate small molecules, including lopinavir and ritonavir, α-ketoamide 13b, and ebselen, for their ability to interact with the Mpro. The sirtuin 1 agonist SRT1720 was also used in our analyses. Blind docking to each monomer individually indicated preferential binding of the ligands in the active site. Site-mapping of the dimeric protease indicated a highly reactive pocket in the dimerization region at the domain III apex. Blind docking consistently indicated a strong preference of ligand binding in domain III, away from the active site. Molecular dynamics simulations indicated that ligands docked both to the active site and in the dimerization region at the apex, formed relatively stable interactions. Overall, our findings do not obviate the superior potency with respect to inhibition of protease activity of covalently-linked inhibitors such as α-ketoamide 13b in the Mpro active site. Nevertheless, along with those from others, our findings highlight the importance of further characterisation of the Mpro active site and any potential allosteric sites.