Enhanced stability and photothermal efficiency of Indocyanine Green J-aggregates by nanoformulation with Calix[4]arene for photothermal therapy of cancers.

Photothermal therapy (PTT) is a method of growing attention, owing to its controllable process, high efficiency and minimal side effect. Indocyanine Green (ICG) is as Food and Drug Administration (FDA) approved agent that stands on the frontline of further developments of PTT toward clinics. However, the applicability of ICG-mediated PTT is limited by the rapid in vivo clearance and photo-degradation of ICG. To improve those parameters, nanosized ICG-loaded nanoparticles (ICG-J/CX) were fabricated in this study by co-assembly of anionic ICG J-aggregates (ICG-J) with cationic tetraguanidinium calix[4]arene (CX). This very simple approach produces ICG-J/CX with a well-defined nanometer range size and a close to neutral charge. The nanoparticles demonstrate high photothermal conversion efficiency (PCE) and dramatically improved photostability, as compared with ICG. The in vitro cellular uptake and cytotoxicity studies further demonstrated that the ICG-J/CX nanoparticles enhance uptake and photothermal efficiency in comparison with ICG or non-formulated ICG-J, overall demonstrating that ICG-J/CX mediated photothermal therapy have significant potential for attaining cancer treatment.

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.

How SARS-CoV-2 Alters the Regulation of Gene Expression in Infected Cells.

Nonstructural accessory proteins in viruses play a key role in hijacking the basic cellular mechanisms, which is essential to promote the virus survival and evasion of the immune system. The immonuglobulin-like open reading frame 8 (ORF8) protein expressed by SARS-CoV-2 accumulates in the nucleus and may influence the regulation of the gene expression in infected cells. In this contribution, by using microsecond time-scale all-atom molecular dynamics simulations, we unravel the structural bases behind the epigenetic action of ORF8. In particular, we highlight how the protein is able to form stable aggregates with DNA through a histone tail-like motif, and how this interaction is influenced by post-translational modifications, such as acetylation and methylation, which are known epigenetic markers in histones. Our work not only clarifies the molecular mechanisms behind the perturbation of the epigenetic regulation caused by the viral infection but also offers an unusual perspective which may foster the development of original antivirals.

Functionalized liposomes for targeted breast cancer drug delivery.

Despite the exceptional progress in breast cancer pathogenesis, prognosis, diagnosis, and treatment strategies, it remains a prominent cause of female mortality worldwide. Additionally, although chemotherapies are effective, they are associated with critical limitations, most notably their lack of specificity resulting in systemic toxicity and the eventual development of multi-drug resistance (MDR) cancer cells. Liposomes have proven to be an invaluable drug delivery system but of the multitudes of liposomal systems developed every year only a few have been approved for clinical use, none of which employ active targeting. In this review, we summarize the most recent strategies in development for actively targeted liposomal drug delivery systems for surface, transmembrane and internal cell receptors, enzymes, direct cell targeting and dual-targeting of breast cancer and breast cancer-associated cells, e.g., cancer stem cells, cells associated with the tumor microenvironment, etc.

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.

DDB2 represses epithelial-to-mesenchymal transition and sensitizes pancreatic ductal adenocarcinoma cells to chemotherapy.

Introduction: Damage specific DNA binding protein 2 (DDB2) is an UV-indiced DNA damage recognition factor and regulator of cancer development and progression. DDB2 has dual roles in several cancers, either as an oncogene or as a tumor suppressor gene, depending on cancer localization. Here, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC). Methods: The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 DDB2-low) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 DDB2-high). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays. Results: DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 overexpression increased expression of E-cadherin epithelial marker, and decreased levels of N-cadherin mesenchymal marker. Conversely, we observed opposite effects in DDB2 repression and enhanced transcription of SNAIL, ZEB1, and TWIST EMT transcription factors (EMT-TFs). Study of migration and invasion revealed that these properties were negatively correlated with DDB2 expression in both cell models. DDB2 overexpression sensitized cells to 5-fluorouracil, oxaliplatin and gemcitabine. Conclusion: Our study highlights the potential tumor suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target or biomarker for defining prognosis and predicting chemotherapy response in patients with PDAC.

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.

Autophagy and evasion of the immune system by SARS-CoV-2. Structural features of the non-structural protein 6 from wild type and Omicron viral strains interacting with a model lipid bilayer.

The viral cycle of SARS-CoV-2 is based on a complex interplay with the cellular machinery, which is mediated by specific proteins eluding or hijacking the cellular defense mechanisms. Among the complex pathways induced by the viral infection, autophagy is particularly crucial and is strongly influenced by the action of the non-structural protein 6 (Nsp6) interacting with the endoplasmic reticulum membrane. Importantly, differently from other non-structural proteins, Nsp6 is mutated in the recently emerged Omicron variant, suggesting a possible different role of autophagy. In this contribution we explore, for the first time, the structural properties of Nsp6 thanks to long-timescale molecular dynamics simulations and machine learning analysis, identifying the interaction patterns with the lipid membrane. We also show how the mutation brought by the Omicron variant may indeed modify some of the specific interactions, and more particularly help anchor the viral protein to the lipid bilayer interface.

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.

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.

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.

Flavagline synthetic derivative induces senescence in glioblastoma cancer cells without being toxic to healthy astrocytes.

Glioblastoma (GBM) is one of the most aggressive types of cancer, which begins within the brain. It is the most invasive type of glioma developed from astrocytes. Until today, Temozolomide (TMZ) is the only standard chemotherapy for patients with GBM. Even though chemotherapy extends the survival of patients, there are many undesirable side effects, and most cases show resistance to TMZ. FL3 is a synthetic flavagline which displays potent anticancer activities, and is known to inhibit cell proliferation, by provoking cell cycle arrest, and leads to apoptosis in a lot of cancer cell lines. However, the effect of FL3 in glioblastoma cancer cells has not yet been examined. Hypoxia is a major problem for patients with GBM, resulting in tumor resistance and aggressiveness. In this study, we explore the effect of FL3 in glioblastoma cells under normoxia and hypoxia conditions. Our results clearly indicate that this synthetic flavagline inhibits cell proliferation and induced senescence in glioblastoma cells cultured under both conditions. In addition, FL3 treatment had no effect on human brain astrocytes. These findings support the notion that the FL3 molecule could be used in combination with other chemotherapeutic agents or other therapies in glioblastoma treatments.

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.

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.

Effects of Bioactive Marine-Derived Liposomes on Two Human Breast Cancer Cell Lines.

Breast cancer is the leading cause of death from cancer among women. Higher consumption of dietary marine n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) is associated with a lower risk of breast cancer. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two n-3 LC-PUFAs found in fish and exert anticancer effects. In this study, natural marine- derived lecithin that is rich in various polyunsaturated fatty acids (PUFAs) was extracted from salmon heads and transformed into nanoliposomes. These nanoliposomes were characterized and cultured with two breast cancer lines (MCF-7 and MDA-MB- 231). The nanoliposomes decreased the proliferation and the stiffness of both cancer cell types. These results suggest that marine-derived lecithin possesses anticancer properties, which may have an impact on developing new liposomal delivery strategies for breast cancer treatment.

New desulfured troglitazone derivatives: Improved synthesis and biological evaluation.

Breast cancer is a major medical threat which cannot be sufficiently addressed by current therapies because of spontaneous or acquired treatment resistance. Besides, triple-negative breast cancer (TNBC) tumors do not respond to targeted therapies, thus new therapeutic strategies are needed. In this context, we designed and prepared new desulfured troglitazone (TGZ)-derived molecules and evaluated them in vitro for their anti-proliferative activity, with a special focus on triple-negative breast cancer cell lines. Optimization of the synthetic strategies and deracemization of the lead compound were performed to give highly active compound 10 with low-micromolar potency. Further studies revealed that this compound triggers apoptosis rather than cell cycle arrest as observed with TGZ.