Dynamics of Protein-RNA Interfaces Using All-Atom Molecular Dynamics Simulations.

Facing the current challenges posed by human health diseases requires the understanding of cell machinery at a molecular level. The interplay between proteins and RNA is key for any physiological phenomenon, as well protein-RNA interactions. To understand these interactions, many experimental techniques have been developed, spanning a very wide range of spatial and temporal resolutions. In particular, the knowledge of tridimensional structures of protein-RNA complexes provides structural, mechanical, and dynamical pieces of information essential to understand their functions. To get insights into the dynamics of protein-RNA complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on nine different protein-RNA complexes with different functions and interface size by taking into account the bound and unbound forms. First, we characterized structural changes upon binding and, for the RNA part, the change in the puckering. Second, we extensively analyzed the interfaces, their dynamics and structural properties, and the structural waters involved in the binding, as well as the contacts mediated by them. Based on our analysis, the interfaces rearranged during the simulation time showing alternative and stable residue-residue contacts with respect to the experimental structure.

Revealing the Molecular Interactions between Human ACE2 and the Receptor Binding Domain of the SARS-CoV-2 Wild-Type, Alpha and Delta Variants.

After a sudden and first spread of the pandemic caused by the novel SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) wild-type strain, mutants have emerged which have been associated with increased infectivity, inducing surges in the contagions. The first of the so-called variants of concerns, was firstly isolated in the United Kingdom and later renamed Alpha variant. Afterwards, in the middle of 2021, a new variant appeared called Delta. The latter is characterized by the presence of point mutations in the Spike protein of SARS-CoV-2, especially in the Receptor Binding Domain (RBD). When in its active conformation, the RBD can interact with the human receptor Angiotensin-Converting Enzyme 2 (ACE2) to allow the entry of the virions into cells. In this contribution, by using extended all-atom molecular dynamic simulations, complemented with machine learning post-processing, we analyze the changes in the molecular interaction network induced by these different strains in comparison with the wild-type. On one hand, although relevant variations are evidenced, only limited changes in the global stability indicators and in the flexibility profiles have been observed. On the other hand, key differences were obtained by tracking hydrophilic and hydrophobic molecular interactions, concerning both positioning at the ACE2/RBD interface and formation/disruption dynamic behavior.

Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction.

Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.5 Šand further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.

How Fragile We Are: Influence of Stimulator of Interferon Genes (STING) Variants on Pathogen Recognition and Immune Response Efficiency.

The stimulator of interferon genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP in the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines. In the human population, several STING variants exist and exhibit dramatic differences in their activity, impacting the efficiency of the host defense against infections. Understanding the molecular mechanisms of these variants opens perspectives for personalized medicine treatments against diseases such as viral infections, cancers, or autoinflammatory diseases. Through microsecond-scale molecular modeling simulations, contact analyses, and machine learning techniques, we reveal the dynamic behavior of four STING variants (wild type, G230A, R293Q, and G230A/R293Q) and rationalize the variability of efficiency observed experimentally. Our results show that the decrease in STING activity is linked to a stiffening of key structural elements of the binding cavity together with changes in the interaction patterns within the protein.

Atomistic-Level Description of the Covalent Inhibition of SARS-CoV-2 Papain-like Protease.

Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys111), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys111 deprotonation of the thiol group by a Brønsted-Lowry base; (iii) Cys111-S- addition to the ligand; and (iv) proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys111, that is, prior to the Cys111 deprotonation. For the second step, MD simulations revealed that His272 would deprotonate Cys111 after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys111 deprotonation, whose equilibrium is largely shifted toward the neutral Cys111 form. Although thermodynamically disfavored, if Cys111 is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys111-S- negative charge redistribution over the bound ligand, proton transfer from the initially protonated His272 is favored, finally leading to an irreversibly modified Cys111 and a restored His272. These results elucidate the selectivity of Cys111 to enable formation of a covalent bond, even if a weak proton acceptor is available, as His272.

Never Cared for What They Do: High Structural Stability of Guanine-Quadruplexes in the Presence of Strand-Break Damage.

DNA integrity is an important factor that assures genome stability and, more generally, the viability of cells and organisms. In the presence of DNA damage, the normal cell cycle is perturbed when cells activate their repair processes. Although efficient, the repair system is not always able to ensure complete restoration of gene integrity. In these cases, mutations not only may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold that induces apoptosis and programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiation are the most toxic due to the inherent difficultly of repair, which may lead to genomic instability. In this article we show, by using classical molecular simulation techniques, that compared to canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show remarkable structural stability, even in the presence of two strand breaks. Since G4-DNA is recognized for its regulatory roles in cell senescence and gene expression, including oncogenes, this stability may be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.

Structural and morphological changes of breast cancer cells induced by iron(II) complexes.

Metal-based complexes are well-established cancer chemotherapeutic drug candidates. Although our knowledge regarding their exact activity vs. toxicity profile is incomplete, changes in cell membrane biophysical properties and cytoskeletal structures have been implicated as part of the mechanism of action. Thus, in this work, we characterised the effects of iron(II)-based complexes on the structural and morphological properties of epithelial non-tumorigenic (MCF 10A) and tumorigenic (MDA-MB-231) breast cell lines using atomic force microscopy (AFM), flow cytometry and immunofluorescence microscopy. At 24 h of exposure, both the MCF 10A and MDA-MB-231 cells experienced a cell softening, and an increase in size followed by a re-stiffening at 96 h. In addition, the triple negative breast cancer cell line, MDA-MB-231, sustained a notable cytoskeletal and mitochondrial reorganization with increased actin stress fibers and cell-to-cell communication structures. An extensive all-atom molecular dynamic simulation suggests a possible direct and unassisted internalization of the metallodrug candidate, and confirmed that the cellular effects could not be ascribed to the simple physical interaction of the iron-based complexes with the biological membrane. These observations provide an insight into a link between the mechanisms of action of such iron-based complexes as anti-cancer treatment and cytoskeletal architecture.

In silico drug discovery of IKK-ß inhibitors from 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl) pyridine derivatives based on QSAR, docking, molecular dynamics and drug-likeness evaluation studies.

The Inhibitor of IKK-ß (nuclear factor kappa B kinase subunit beta), a specific modulator of NF-κB (nuclear factor-κB), is considered a valid target to discover new active compounds for various cancers and rheumatoid arthritis treatment. In this study a series of thirty 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl) pyridine derivatives was involved for a quantitative structure activity relationship model (QSAR) elaboration which allows the prediction of the pIC50 values of new designed compounds. The model can be used to predict the activity of new compounds within its applicability domain. Then a molecular docking study was carried out to identify the interactions between the compounds and the amino acids of the active site. After that, golden triangle, Veber's rule, and Lipinski's rule properties were calculated to identify the drug-likeness properties of the investigated compounds. Finally, in-silico-toxicity studies were performed to predict the toxicity of the new designed compounds. The analysis of the results of QSAR model and molecular docking succeeded to screen 21 interesting compounds with better inhibitory concentration having a good affinity to IKK-ß. All compounds were within the range set by Veber's rule and Lipinski's rule. the analysis of golden triangle showed that the thirty 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl) pyridine derivatives would not have clearance and cell membrane permeability problems except comp6 comp12,comp20, comp21, and comp26.As for the new designed compounds, their properties may have these problems, except two compounds which are: A8m, A8p. The A1m, A1p, A3p and A11m compounds were predicted to be nontoxic. These findings indicate that the novel potent candidate drugs have promising potential to IKK-ß enzyme inhibition and should motivate future experimental investigations.Communicated by Ramaswamy H. Sarma.

Structure and Dynamics of RNA Guanine Quadruplexes in SARS-CoV-2 Genome. Original Strategies against Emerging Viruses.

Guanine quadruplex (G4) structures in the viral genome have a key role in modulating viruses' biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modeling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of the RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.

Microscopic interactions between ivermectin and key human and viral proteins involved in SARS-CoV-2 infection.

The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CLpro and PLpro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.

The Iron Maiden. Cytosolic Aconitase/IRP1 Conformational Transition in the Regulation of Ferritin Translation and Iron Hemostasis.

Maintaining iron homeostasis is fundamental for almost all living beings, and its deregulation correlates with severe and debilitating pathologies. The process is made more complicated by the omnipresence of iron and by its role as a fundamental component of a number of crucial metallo proteins. The response to modifications in the amount of the free-iron pool is performed via the inhibition of ferritin translation by sequestering consensus messenger RNA (mRNA) sequences. In turn, this is regulated by the iron-sensitive conformational equilibrium between cytosolic aconitase and IRP1, mediated by the presence of an iron-sulfur cluster. In this contribution, we analyze by full-atom molecular dynamics simulation, the factors leading to both the interaction with mRNA and the conformational transition. Furthermore, the role of the iron-sulfur cluster in driving the conformational transition is assessed by obtaining the related free energy profile via enhanced sampling molecular dynamics simulations.

Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand.

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

Forever Young: Structural Stability of Telomeric Guanine Quadruplexes in the Presence of Oxidative DNA Lesions*.

Human telomeric DNA, in G-quadruplex (G4) conformation, is characterized by a remarkable structural stability that confers it the capacity to resist to oxidative stress producing one or even clustered 8-oxoguanine (8oxoG) lesions. We present a combined experimental/computational investigation, by using circular dichroism in aqueous solutions, cellular immunofluorescence assays and molecular dynamics simulations, that identifies the crucial role of the stability of G4s to oxidative lesions, related also to their biological role as inhibitors of telomerase, an enzyme overexpressed in most cancers associated to oxidative stress.

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.

Thermodynamics of the Interaction between the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus-2 and the Receptor of Human Angiotensin-Converting Enzyme 2. Effects of Possible Ligands.

Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 1000000 deaths all over the world and still lacks a medical treatment despite the attention of the whole scientific community. Human angiotensin-converting enzyme 2 (ACE2) was recently recognized as the transmembrane protein that serves as the point of entry of SARS-CoV-2 into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the protein complex. Moreover, the free energy of binding between ACE2 and the active receptor binding domain of the SARS-CoV-2 spike protein is evaluated quantitatively, providing for the first time the thermodynamics of virus-receptor recognition. Furthermore, the action of different ACE2 ligands is also examined in particular in their capacity to disrupt SARS-CoV-2 recognition, also providing via a free energy profile the quantification of the ligand-induced decreased affinity. These results improve our knowledge on molecular grounds of the SARS-CoV-2 infection and allow us to suggest rationales that could be useful for the subsequent wise molecular design for the treatment of COVID-19 cases.

Recent advances in iron-complexes as drug candidates for cancer therapy: reactivity, mechanism of action and metabolites.

In this perspective, we discuss iron-complexes as drug candidates that are promising alternatives to conventional platinum-based chemotherapies owing to their broad range of reactivities and to the targeting of different biological systems. Breakthroughs in the comprehension of iron complexes' structure-activity relationship contributed to the clarification of their metabolization pathways, sub-cellular localization and influence on iron homeostasis, while enlightening the primary molecular targets of theses likely multi-target metallodrugs. Both the antiproliferative activity and elevated safety index observed among the family of iron complexes showed encouraging results as per their therapeutic potential and selectivity also with the aim of reducing chemotherapy side-effects, and facilitated more pre-clinical investigations. The purpose of this perspective is to summarize the recent advances that contributed in unveiling the intricate relationships between the structural modifications on iron-complexes and their reactivity, cellular trafficking and global mechanisms of action to broaden their use as anticancer drugs and advance to clinical evaluation.

Photoinduced DNA Lesions in Dormant Bacteria: The Peculiar Route Leading to Spore Photoproducts Characterized by Multiscale Molecular Dynamics*.

Some bacterial species enter a dormant state in the form of spores to resist to unfavorable external conditions. Spores are resistant to a wide series of stress agents, including UV radiation, and can last for tens to hundreds of years. Due to the suspension of biological functions, such as DNA repair, they accumulate DNA damage upon exposure to UV radiation. Differently from active organisms, the most common DNA photoproducts in spores are not cyclobutane pyrimidine dimers, but rather the so-called spore photoproducts. This noncanonical photochemistry results from the dry state of DNA and its binding to small, acid-soluble proteins that drastically modify the structure and photoreactivity of the nucleic acid. Herein, multiscale molecular dynamics simulations, including extended classical molecular dynamics and quantum mechanics/molecular mechanics based dynamics, are used to elucidate the coupling of electronic and structural factors that lead to this photochemical outcome. In particular, the well-described impact of the peculiar DNA environment found in spores on the favored formation of the spore photoproduct, given the small free energy barrier found for this path, is rationalized. Meanwhile, the specific organization of spore DNA precludes the photochemical path that leads to cyclobutane pyrimidine dimer formation.

Role of RNA Guanine Quadruplexes in Favoring the Dimerization of SARS Unique Domain in Coronaviruses.

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.

Molecular Bases of DNA Packaging in Bacteria Revealed by All-Atom Molecular Dynamics Simulations: The Case of Histone-Like Proteins in Borrelia burgdorferi.

DNA compaction is essential to ensure the packaging of the genetic material in living cells and also plays a key role in the epigenetic regulation of gene expression. In both humans and bacteria, DNA packaging is achieved by specific well-conserved proteins. Here, by means of all-atom molecular dynamics simulations, including the determination of relevant free-energy profiles, we rationalize the molecular bases for this remarkable process in bacteria, illustrating the crucial role played by positively charged amino acids of a small histone-like protein. We also present compelling evidence that this histone-like protein alone can induce strong bending of a DNA duplex around its core domain, a process that requires overcoming a major free-energy barrier.

Cooperative Effects of Cytosine Methylation on DNA Structure and Dynamics.

The behavior of the structural parameters of DNA considering different levels of methylation in CpG islands is studied by means of full-atom molecular dynamics simulations and electronic circular dichroism, both in an artificial model system and in a gene promoter sequence. It is demonstrated that methylation although intrinsically brings quite local perturbations may, if its level is high enough, induce cooperative effects that strongly modify the DNA backbone torsional parameters altering the helicity as compared to the nonmethylated case. Because methylation of the CpG island is correlated with the regulation of gene expression, understanding the structural modifications induced in DNA is crucial to characterize all the fine equilibria into play in epigenetics phenomena.