Interaction of the Immune System TIM-3 Protein with a Model Cellular Membrane Containing Phosphatidyl-Serine Lipids.

T cell transmembrane, Immunoglobulin, and Mucin (TIM) are important immune system proteins which are especially present in T-cells and regulated the immune system by sensing cell engulfment and apoptotic processes. Their role is exerted by the capacity to detect the presence of phosphatidyl-serine lipid polar head in the outer leaflet of cellular membranes (correlated with apoptosis). In this contribution by using equilibrium and enhanced sampling molecular dynamics simulation we unravel the molecular bases and the thermodynamics of TIM, and in particular TIM-3, interaction with phosphatidyl serine in a lipid bilayer. Since TIM-3 deregulation is an important factor of pro-oncogenic tumor micro-environment understanding its functioning at a molecular level may pave the way to the development of original immunotherapeutic approaches.

Modelling the effects of E/Z photoisomerization of a cyclocurcumin analogue on the properties of cellular lipid membranes.

The use of photosensitive molecules capable of isomerizing under light stimuli, and thus induce perturbation in biological systems, is becoming increasingly popular for potential light-activated chemotherapeutic purposes. We recently show that a cyclocurcumin derivative (CCBu), may be suitable for light-activated chemotherapy and may constitute a valuable alternative to traditional photodynamic therapy, due to its oxygen-independent mechanism of action, which allows the treatment of hypoxic solid tumors. In particular, we have shown that the E/Z photoisomerization of CCBu correlates with strong perturbations of model lipid bilayers. In this work, we perform all-atom classical molecular dynamics for a more complex bilayer, whose composition is, thus, much closer to eukaryotic outer cell membranes. We have evidenced important differences in the interaction pathway between CCBu and the complex lipid bilayer as compared to previous models, concerning both the membrane penetration capacity and the isomerization-induced perturbations. While we confirm that structural perturbations of the lipid membrane are induced by isomerization, we also show how the use of a simplified membrane model can result in an oversimplification of the system and hinder key physical and biological phenomena. Although, CCBu may be considered as a suitable candidate for light-activated chemotherapy, we also underline how the inclusion of bulkier substituents, inducing larger perturbations upon photoisomerization, may enhance its efficiency.

Mechanism of the Covalent Inhibition of Human Transmembrane Protease Serine 2 as an Original Antiviral Strategy.

The Transmembrane Protease Serine 2 (TMPRSS2) is a human enzyme which is involved in the maturation and post-translation of different proteins. In addition to being overexpressed in cancer cells, TMPRSS2 plays a further fundamental role in favoring viral infections by allowing the fusion of the virus envelope with the cellular membrane, notably in SARS-CoV-2. In this contribution, we resort to multiscale molecular modeling to unravel the structural and dynamical features of TMPRSS2 and its interaction with a model lipid bilayer. Furthermore, we shed light on the mechanism of action of a potential inhibitor (nafamostat), determining the free-energy profile associated with the inhibition reaction and showing the facile poisoning of the enzyme. Our study, while providing the first atomistically resolved mechanism of TMPRSS2 inhibition, is also fundamental in furnishing a solid framework for further rational design targeting transmembrane proteases in a host-directed antiviral strategy.

Predicting the Three-Dimensional Structure of the c-KIT Proto-Oncogene Promoter and the Dynamics of Its Strongly Coupled Guanine Quadruplexes.

Guanine quadruplexes (G4s) play essential protective and regulatory roles within cells, influencing gene expression. In several gene-promoter regions, multiple G4-forming sequences are in close proximity and may form three-dimensional arrangements. We analyze the interplay among the three neighboring G4s in the c-KIT proto-oncogene promoter (WK1, WSP, and WK2). We highlight that the three G4s are structurally linked and their cross-talk favors the formation of a parallel structure for WSP. Relying on all-atom molecular dynamic simulations exceeding the µs time scale and using enhanced sampling methods, we provide the first computationally resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that can ultimately be used as a starting point for drug discovery.

Molecular Basis of the pH-Controlled Maturation of the Tick-Borne Encephalitis Flavivirus.

Flaviviruses are enveloped viruses causing high public concerns. Their maturation spans several cellular compartments having different pH. Thus, complex control mechanisms are in place to avoid premature maturation. Here we report the dynamical behavior at neutral and acidic pH of the precursor of the membrane fusion protein E of tick-borne encephalitis, showing the different stabilizations of the E dimer and the role played by the small fusion-assisting protomer (pr). The comprehension, at atomic resolution, of the fine regulation of viral maturation will be fundamental to the development of efficient strategies against emerging viral threats.

Understanding the Interactions of Guanine Quadruplexes with Peptides as Novel Strategies for Diagnosis or Tuning Biological Functions.

Guanine quadruplexes (G4s) are nucleic acid structures exhibiting a complex structural behavior and exerting crucial biological functions in both cells and viruses. The specific interactions of peptides with G4s, as well as an understanding of the factors driving the specific recognition are important for the rational design of both therapeutic and diagnostic agents. In this review, we examine the most important studies dealing with the interactions between G4s and peptides, highlighting the strengths and limitations of current analytic approaches. We also show how the combined use of high-level molecular simulation techniques and experimental spectroscopy is the best avenue to design specifically tuned and selective peptides, thus leading to the control of important biological functions.

Perturbation of Lipid Bilayers by Biomimetic Photoswitches Based on Cyclocurcumin.

The use of photoswitches which may be activated by suitable electromagnetic radiation is an attractive alternative to conventional photodynamic therapy. Here, we report all-atom molecular dynamics simulation of a biomimetic photoswitch derived from cyclocurcumin and experiencing E/Z photoisomerization. In particular, we show that the two isomers interact persistently with a lipid bilayer modeling a cellular membrane. Furthermore, the interaction with the membrane is strongly dependent on the concentration, and a transition between ordered and disordered arrangements of the photoswitches is observed. We also confirm that the structural parameters of the bilayer are differently affected by the two isomers and hence can be modulated through photoswitching, offering interesting perspectives for future applications.

G-Quadruplex Recognition by DARPIns through Epitope/Paratope Analogy.

We investigated the mechanisms leading to the specific recognition of Guanine Guadruplex (G4) by DARPins peptides, which can lead to the design of G4 s specific sensors. To this end we carried out all-atom molecular dynamic simulations to unravel the interactions between specific nucleic acids, including human-telomeric (h-telo), Bcl-2, and c-Myc, with different peptides, forming a DARPin/G4 complex. By comparing the sequences of DARPin with that of a peptide known for its high affinity for c-Myc, we show that the recognition cannot be ascribed to sequence similarity but, instead, depends on the complementarity between the three-dimensional arrangement of the molecular fragments involved: the α-helix/loops domain of DARPin and the G4 backbone. Our results reveal that DARPins tertiary structure presents a charged hollow region in which G4 can be hosted, thus the more complementary the structural shapes, the more stable the interaction.

Spatial and Temporal Resolution of the Oxygen-Independent Photoinduced DNA Interstrand Cross-Linking by a Nitroimidazole Derivative.

DNA damage is ubiquitous in nature and is at the basis of emergent treatments such as photodynamic therapy, which is based on the activation of highly oxidative reactive oxygen species by photosensitizing O2. However, hypoxia observed in solid tumors imposes the necessity to devise oxygen-independent modes of action able to induce DNA damage under a low oxygen concentration. The complexity of these DNA damage mechanisms in realistic environments grows exponentially when taking into account light absorption and subsequent excited-state population, photochemical and (photo)-redox reactions, the multiple species involved in different electronic states, noncovalent interactions, multiple reaction steps, and the large number of DNA reactive sites. This work tackles all the intricate reactivity of a photosensitizer based on a nitroimidazole derivative reacting toward DNA in solution under UV light exposition. This is performed through a combination of ground- and excited-state quantum chemistry, classical molecular dynamics, and hybrid QM/MM simulations to rationalize in detail the formation of DNA interstrand cross-links (ICLs) exerted by the noncanonical noncovalent photosensitizer. Unprecedented spatial and temporal resolution of these phenomena is achieved, revealing that the ICL is sequence-specific and that the fastest reactions take place at AT, GC, and GT steps involving either the opposite nucleobases or adjacent Watson-Crick base pairs. The N7 and O6 positions of guanine, the N7 and N3 sites of adenine, the N4 position of cytosine, and the O2 atom of thymine are deemed as the most nucleophile sites and are positively identified to participate in the ICL productions. This work provides a multiscale computational protocol to study DNA reactivity with noncovalent photosensitizers, and contributes to the understanding of therapies based on photoinduced DNA damage at molecular and electronic levels. In addition, we believe the depth understanding of these processes should assist the design of new photosensitizers considering their molecular size, electronic properties, and the observed regioselectivity toward nucleic acids.

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.

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.

Don't help them to bury the light. The interplay between intersystem crossing and hydrogen transfer in photoexcited curcumin revealed by surface-hopping dynamics.

Curcumin is a natural compound extracted from turmeric (Curcuma longa), which has shown remarkable anti-inflammatory, antibacterial, and possibly anticancer properties. The intense absorption in the visible domain and the possibility of intersystem crossing make curcumin attractive also for photodynamic therapy purposes. In the present contribution, we unravel, thanks to non-adiabatic surface hopping dynamics, the interplay between intersystem crossing and hydrogen transfer in the enol form, i.e. the most stable tautomer of curcumin. Most notably, we show that while hydrogen transfer is ultrafast and happens in the sub-ps regime, intersystem crossing is still present, as shown by the non-negligible population of the triplet state manifold after 2 ps. Hence, while the hydrogen transfer channel can act as a deactivating channel, curcumin, also in the red-shifted absorption enol form, can still be regarded as potentially favorable for photodynamic therapy applications.

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.

Towards Iron(II) Complexes with Octahedral Geometry: Synthesis, Structure and Photophysical Properties.

The control of ligand-field splitting in iron (II) complexes is critical to slow down the metal-to-ligand charge transfer (MLCT)-excited states deactivation pathways. The gap between the metal-centered states is maximal when the coordination sphere of the complex approaches an ideal octahedral geometry. Two new iron(II) complexes (C1 and C2), prepared from pyridylNHC and pyridylquinoline type ligands, respectively, have a near-perfect octahedral coordination of the metal. The photophysics of the complexes have been further investigated by means of ultrafast spectroscopy and TD-DFT modeling. For C1, it is shown that-despite the geometrical improvement-the excited state deactivation is faster than for the parent pseudo-octahedral C0 complex. This unexpected result is due to the increased ligand flexibility in C1 that lowers the energetic barrier for the relaxation of 3MLCT into the 3MC state. For C2, the effect of the increased ligand field is not strong enough to close the prominent deactivation channel into the metal-centered quintet state, as for other Fe-polypyridine complexes.

Experimental and theoretical studies on thymine photodimerization mediated by oxidatively generated DNA lesions and epigenetic intermediates.

Interaction of nucleic acids with light is a scientific question of paramount relevance not only in the understanding of life functioning and evolution, but also in the insurgence of diseases such as malignant skin cancer and in the development of biomarkers and novel light-assisted therapeutic tools. This work shows that the UVA portion of sunlight, not absorbed by canonical DNA nucleobases, can be absorbed by 5-formyluracil (ForU) and 5-formylcytosine (ForC), two ubiquitous oxidatively generated lesions and epigenetic intermediates present in living beings in natural conditions. We measure the strong propensity of these molecules to populate triplet excited states able to transfer the excitation energy to thymine-thymine dyads, inducing the formation of cyclobutane pyrimidine dimers (CPDs). By using steady-state and transient absorption spectroscopy, NMR, HPLC, and theoretical calculations, we quantify the differences in the triplet-triplet energy transfer mediated by ForU and ForC, revealing that the former is much more efficient in delivering the excitation energy and producing the CPD photoproduct. Although significantly slower than ForU, ForC is also able to harm DNA nucleobases and therefore this process has to be taken into account as a viable photosensitization mechanism. The present findings evidence a rich photochemistry crucial to understand DNA damage photobehavior.

Impact of the Nucleosome Histone Core on the Structure and Dynamics of DNA-Containing Pyrimidine-Pyrimidone (6-4) Photoproduct.

The pyrimidine-pyrimidone (6-4) photoproduct (64-PP) is an important photoinduced DNA lesion constituting a mutational signature for melanoma. The structural impact of 64-PP on DNA complexed with histones affects the lesion mutagenicity and repair but remains poorly understood. Here we investigate the conformational dynamics of DNA-containing 64-PP within the nucleosome core particle by atomic-resolution molecular dynamics simulations and multiscale data analysis. We demonstrate that the histone core exerts important mechanical restraints that largely decrease global DNA structural fluctuations. However, the local DNA flexibility at the damaged site is enhanced due to imperfect structural adaptation to restraints imposed by the histone core. If 64-PP faces the histone core and is therefore not directly accessible by the repair protein, the complementary strand facing the solvent is deformed and exhibits higher flexibility than the corresponding strand in a naked, undamaged DNA. This may serve as an initial recognition signal for repair. Our simulations also pinpoint the structural role of proximal residues from the truncated histone tails.