Molecular Basis of SARS-CoV-2 Infection and Rational Design of Potential Antiviral Agents: Modeling and Simulation Approaches

The emergence in late 2019 of the coronavirus SARS-CoV-2 has resulted in the breakthrough of the COVID-19 pandemic that is presently affecting a growing number of countries. The development of the pandemic has also prompted an unprecedented effort of the scientific community to understand the molecular bases of the virus infection and to propose rational drug design strategies able to alleviate the serious COVID-19 morbidity. In this context, a strong synergy between the structural biophysics and molecular modeling and simulation communities has emerged, resolving at the atomistic level the crucial protein apparatus of the virus and revealing the dynamic aspects of key viral processes. In this Review, we focus on how in silico studies have contributed to the understanding of the SARS-CoV-2 infection mechanism and the proposal of novel and original agents to inhibit the viral key functioning. This Review deals with the SARS-CoV-2 spike protein, including the mode of action that this structural protein uses to entry human cells, as well as with nonstructural viral proteins, focusing the attention on the most studied proteases and also proposing alternative mechanisms involving some of its domains, such as the SARS unique domain. We demonstrate that molecular modeling and simulation represent an effective approach to gather information on key biological processes and thus guide rational molecular design strategies

Flexibility within the Heads of Muscle Myosin-2 Molecules

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN·nm/rad2 is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor–lever junction

Investigating the Structure of the Papain-Inhibitor Complex using SPR and NMR

Cysteine proteases (CPs) are enzymes with a nucleophilic thiol in their active sites. Inhibitors of cysteine proteases (ICPs) occur naturally in bacterial pathogens and some protozoa. In parasites, ICPs are often virulence factors, contributing to the formation and survival of amastigotes within host cells. These amastigotes have higher CP activity, therefore making both ICPs and CPs potential drug targets. Despite great genetic variability, ICPs contain highly conserved structural features, including a series of defined loops that play a significant role in binding CPs. Papain, a CP from Carica papaya, complexes with ICP from Leishmania mexicana. Although the individual 3-D structures of ICP and papain have been determined, as of this work, the structure of the papain-ICP complex has only been predicted, not solved. This research details the development of a technique for determining quaternary structure of the papain-ICP complex using paramagnetic relaxation enhancement NMR (PRE-NMR). A paramagnetic tag (MTSL) was added to various cysteine-mutants of ICP to measure distances to reductively 13C-methylated papain. The modification of ICP with MTSL was quantified using EPR, and the effects of labeling on the binding kinetics of papain and ICP were determined using SPR. 13C-methyl peak perturbations due to PRE were observed when papain was bound to spin-labeled E102C-ICP and K27C-ICP. Intermolecular distances were predicted using modeling software and a working model of the complex was created. Data from additional mutants will help to further determine complex structure and perfect the model.The penultimate chapter of this dissertation includes work towards the development of a method for studying protein-protein interactions using atomic force microscopy. Papain-ICP was used as a model system, with the intention to apply this method to the study of another system: filamentous actin (f-actin) and the actin-binding domain of abelson tyrosine-protein kinase (ABL2-FABD). The creation of nanopores on an AFM sensor chip surface was successful. ICP monomers bound selectively into the pores. Attempts to form the papain-ICP complex on the chip surface were unsuccessful, and future work is needed to perfect this method. The final chapter of this dissertation is a literature review outlining previous work in this area

Experimental study and computational modelling of cruzain cysteine protease inhibition by dipeptidyl nitriles

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases

Microrheology to explore protein and cell dynamics

In this thesis, I explore the applications of optical tweezers and passive video particle tracking microrheology for bioanalytical applications. Microrheology is a branch of rheology that has the same principles as conventional bulk rheology, but which works on micron length scales. Microrheological techniques relate the free or the driven motion of micron-sized tracer particles suspended in the fluid under investigation to the ‘elastic’ and ‘viscous’ components of the material. These components can be related to the dynamics of the molecules that make up the fluid, and thus microrheology has the potential to reveal new information about the microscopic properties of complex materials. Optical tweezers are sensitive instruments that have been used to apply forces on the order of pN and to measure the displacements down to nm of objects ranging in size from 10 nm to over 100μm, making them an essential tool for microrheology. Here, we have developed a new set of analytical methods for microrheological measurements of biological and bio-analytical systems. In particular, we have developed two new self-consistent procedures for measuring the linear viscoelastic properties of materials across the widest frequency range achievable with optical tweezers (Phys.Review E. (2010) 81:2, and J. Optics (2011) 13:4). Furthermore, we present a straightforward procedure for measuring the in vivo linear viscoelastic properties of single cells via passive video particle tracking microrheology of single beads attached to the cells’ exterior. Notably, the procedure presented here represents an alternative methodology that can be extended to many experimental formats and provides a simple addition to existing cellular physiology studies. In addition, we introduce new methodologies for deriving the concentration scaling laws of polymer and biopolymer solutions from microrheological measurements carried out with optical tweezers. These methods have been adopted to investigate the concentration scaling laws of in vitro reconstituted actin solutions and actin/myosin solution

From Byte to Bench to Bedside: Molecular Dynamics Simulations and Drug Discovery

Molecular dynamics (MD) simulations and computer-aided drug design (CADD) have advanced substantially over the past two decades, thanks to continuous computer hardware and software improvements. Given these advancements, MD simulations are poised to become even more powerful tools for investigating the dynamic interactions between potential small-molecule drugs and their target proteins, with significant implications for pharmacological research.Comment: 15 pages including references, 0 figure