Molecular mechanisms behind BRACO19 and human telomeric G-quadruplex interaction.

Human telomeres (HTs) can form DNA G-quadruplex (G4), an attractive target for anticancer and antiviral drugs. HT-G4s exhibit inherent structural polymorphism, posing challenges for understanding their specific recognition by ligands. Here, we aim to explore the impact of different topologies within a small segment of the HT (Tel22) on its interaction with BRACO19, a rationally designed G4 ligand with high quadruplex affinity, already employed in in-vivo treatments. Our multi-technique approach is based on the combined use of a set of contactless spectroscopic tools. Circular dichroism and UV resonance Raman spectroscopy probe ligand-induced conformational changes in the G4 sequence, while UV-visible absorption, coupled with steady-state fluorescence spectroscopy, provides further insights into the electronic features of the complex, exploiting the photoresponsive properties of BRACO19. Overall, we find that modifying the topology of the unbound Tel22 through cations (K+ or Na+), serves as a critical determinant for ligand interactions and binding modes, thus influencing the HT-G4's assembly capabilities. Furthermore, we show how fluorescence serves as a valuable probe for recognizing cation-driven multimeric structures, which may be present in living organisms, giving rise to pathological forms.

Conformational transitions in redissolved silk fibroin films and application for printable self-powered multistate resistive memory biomaterials.

3D printing of water stable proteins with elastic properties offers a broad range of applications including self-powered biomedical devices driven by piezoelectric biomaterials. Here, we present a study on water-soluble silk fibroin (SF) films. These films were prepared by mixing degummed silk fibers and calcium chloride (CaCl2) in formic acid, resulting in a silk I-like conformation, which was then converted into silk II by redissolving in phosphate buffer (PBS). Circular dichroism, Raman and infrared (IR) spectroscopies were used to investigate the transitions of secondary structure in silk I and silk II as the pH of the solvent and the sonication time were changed. We showed that a solvent with low pH (e.g. 4) maintains the silk I ß-turn structure; in contrast solvent with higher pH (e.g. 7.4) promotes ß-sheet features of silk II. Ultrasonic treatment facilitates the transition to water stable silk II only for the SF redissolved in PBS. SF from pH 7.4 solution has been printed using extrusion-based 3D printing. A self-powered memristor was realized, comprising an SF-based electric generator and an SF 3D-printed memristive unit connected in series. By exploiting the piezoelectric properties of silk II with higher ß-sheet content and Ca2+ ion transport phenomena, the application of an input voltage driven by a SF generator to SF 3D printed holey structures induces a variation from an initial low resistance state (LRS) to a high resistance state (HRS) that recovers in a few minutes, mimicking the transient memory, also known as short-term memory. Thanks to this holistic approach, these findings can contribute to the development of self-powered neuromorphic networks based on biomaterials with memory capabilities.

Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

Correction to "Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications".

[This corrects the article DOI: 10.1021/acsomega.3c04563.].

Decoding the Role of the Global Proteome Dynamics for Cellular Thermal Stability.

Molecular mechanisms underlying the thermal response of cells remain elusive. On the basis of the recent result that the short-time diffusive dynamics of the Escherichia coli proteome is an excellent indicator of temperature-dependent bacterial metabolism and death, we used neutron scattering (NS) spectroscopy and molecular dynamics (MD) simulations to investigate the sub-nanosecond proteome mobility in psychro-, meso-, and hyperthermophilic bacteria over a wide temperature range. The magnitude of thermal fluctuations, measured by atomic mean square displacements, is similar among all studied bacteria at their respective thermal cell death. Global roto-translational motions turn out to be the main factor distinguishing the bacterial dynamical properties. We ascribe this behavior to the difference in the average proteome net charge, which becomes less negative for increasing bacterial thermal stability. We propose that the chemical-physical properties of the cytoplasm and the global dynamics of the resulting proteome are fine-tuned by evolution to uphold optimal thermal stability conditions.

Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications.

In this study, we dissolved Bombyx mori degummed silk [i.e., silk fibroin (SF)] and salmon sperm deoxyribonucleic acid (DNA) in water and used a bioinspired spinning process to obtain an electrospun nanofibrous SF-based patch (ESF). We investigated the bidirectional macroscale actuation behavior of ESF in response to water vapor and its UV-blocking properties as well as those of ESF/DNA films. Fourier transform infrared (FTIR) results suggest that the formation of ß-sheet-rich structures promotes the actuation effect. ESF/DNA film with high-ordered and ß-sheet-rich structures exhibits higher electrical conductivity and is water-insoluble. Given the intrinsic ability of both SF and DNA to absorb UV radiation, we performed biological experiments on the viability of keratinocyte HaCaT cells after exposure to solar spectrum components. Our findings indicate that the ESF/DNA patch is photoprotective and can increase the cellular viability of keratinocytes after UV exposure. Furthermore, we demonstrated that ESF/DNA patches treated with water vapor can serve as suitable scaffolds for tissue engineering and can improve tissue regeneration when cellularized with HaCaT cells. The 3D shape morphing capability of these patches, along with their potential as UV filters, could offer significant practical advantages in tissue engineering.

Intra-protein interacting collective modes in the terahertz frequency region.

Understanding how proteins work requires a thorough understanding of their internal dynamics. Proteins support a wide range of motions, from the femtoseconds to seconds time scale, relevant to crucial biological functions. In this context, the term "protein collective dynamics" refers to the complex patterns of coordinated motions of numerous atoms throughout the protein in the sub-picosecond time scale (terahertz frequency region). It is hypothesized that these dynamics have a substantial impact on the regulation of functional dynamical mechanisms, including ligand binding and allosteric signalling, charge transport direction, and the regulation of thermodynamic and thermal transport properties. Using the theoretical framework of hydrodynamics, the collective dynamics of proteins had previously been described in a manner akin to that of simple liquids, i.e. in terms of a single acoustic-like excitation, related to intra-protein vibrational motions. Here, we employ an interacting-mode model to analyse the results from molecular dynamics simulations and we unveil that the vibrational landscape of proteins is populated by multiple acoustic-like and low-frequency optic-like modes, with mixed symmetry and interfering with each other. We propose an interpretation at the molecular level of the observed scenario that we relate to the side-chains and the hydrogen-bonded networks dynamics. The present insights provide a perspective for understanding the molecular mechanisms underlying the energy redistribution processes in the interior of proteins.

The hope and hype of ellagic acid and urolithins as ligands of SARS-CoV-2 Nsp5 and inhibitors of viral replication.

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A-D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.

Stacking Interactions and Flexibility of Human Telomeric Multimers.

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.

Development of Salmon Sperm DNA/Regenerated Silk Bio-Based Films for Biomedical Studies on Human Keratinocyte HaCaT Cells under Solar Spectrum.

In this study, we fabricated adhesive patches from silkworm-regenerated silk and DNA to safeguard human skin from the sun's rays. The patches are realized by exploiting the dissolution of silk fibers (e.g., silk fibroin (SF)) and salmon sperm DNA in formic acid and CaCl2 solutions. Infrared spectroscopy is used to investigate the conformational transition of SF when combined with DNA; the results indicated that the addition of DNA provides an increase in the SF crystallinity. UV-Visible absorption and circular dichroism spectroscopy showed strong absorption in the UV region and the presence of B-form of DNA once dispersed in the SF matrix, respectively. Water absorption measurements as well as thermal dependence of water sorption and thermal analysis, suggested the stability of the fabricated patches. Biological results on cellular viability (MTT assay) of keratinocyte HaCaT cells after exposures to the solar spectrum showed that both SF and SF/DNA patches are photo-protective by increasing the cellular viability of keratinocytes after UV component exposure. Overall, these SF/DNA patches promise applications in wound dressing for practical biomedical purposes.

Stability of Human Telomeric G-Quadruplexes Complexed with Photosensitive Ligands and Irradiated with Visible Light.

Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.

Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

Polymorphism and Ligand Binding Modulate Fast Dynamics of Human Telomeric G-Quadruplexes.

Telomeric G-quadruplexes (G4s) are promising targets in the design and development of anticancer drugs. Their actual topology depends on several factors, resulting in structural polymorphism. In this study, we investigate how the fast dynamics of the telomeric sequence AG3(TTAG3)3 (Tel22) depends on the conformation. By using Fourier transform Infrared spectroscopy, we show that, in the hydrated powder state, Tel22 adopts parallel and mixed antiparallel/parallel topologies in the presence of K+ and Na+ ions, respectively. These conformational differences are reflected in the reduced mobility of Tel22 in Na+ environment in the sub-nanosecond timescale, as probed by elastic incoherent neutron scattering. These findings are consistent with the G4 antiparallel conformation being more stable than the parallel one, possibly due to the presence of ordered hydration water networks. In addition, we study the effect of Tel22 complexation with BRACO19 ligand. Despite the quite similar conformation in the complexed and uncomplexed state, the fast dynamics of Tel22-BRACO19 is enhanced compared to that of Tel22 alone, independently of the ions. We ascribe this effect to the preferential binding of water molecules to Tel22 against the ligand. The present results suggest that the effect of polymorphism and complexation on the G4 fast dynamics is mediated by hydration water.

Diffusive Dynamics of Bacterial Proteome as a Proxy of Cell Death.

Temperature variations have a big impact on bacterial metabolism and death, yet an exhaustive molecular picture of these processes is still missing. For instance, whether thermal death is determined by the deterioration of the whole or a specific part of the proteome is hotly debated. Here, by monitoring the proteome dynamics of E. coli, we clearly show that only a minor fraction of the proteome unfolds at the cell death. First, we prove that the dynamical state of the E. coli proteome is an excellent proxy for temperature-dependent bacterial metabolism and death. The proteome diffusive dynamics peaks at about the bacterial optimal growth temperature, then a dramatic dynamical slowdown is observed that starts just below the cell's death temperature. Next, we show that this slowdown is caused by the unfolding of just a small fraction of proteins that establish an entangling interprotein network, dominated by hydrophobic interactions, across the cytoplasm. Finally, the deduced progress of the proteome unfolding and its diffusive dynamics are both key to correctly reproduce the E. coli growth rate.

Biocompatible and Printable Ionotronic Sensing Materials Based on Silk Fibroin and Soluble Plant-Derived Polyphenols.

The emergence of ionotronic materials has been recently exploited for interfacing electronics and biological tissues, improving sensing with the surrounding environment. In this paper, we investigated the synergistic effect of regenerated silk fibroin (RS) with a plant-derived polyphenol (i.e., chestnut tannin) on ionic conductivity and how water molecules play critical roles in regulating ion mobility in these materials. In particular, we observed that adding tannin to RS increases the ionic conductivity, and this phenomenon is accentuated by increasing the hydration. We also demonstrated how silk-based hybrids could be used as building materials for scaffolds where human fibroblast and neural progenitor cells can highly proliferate. Finally, after proving their biocompatibility, RS hybrids demonstrate excellent three-dimensional (3D) printability via extrusion-based 3D printing to fabricate a soft sensor that can detect charged objects by sensing the electric fields that originate from them. These findings pave the way for a viable option for cell culture and novel sensors, with the potential base for tissue engineering and health monitoring.

Role of fast dynamics in the complexation of G-quadruplexes with small molecules.

G-quadruplexes (G4s) formed by the human telomeric sequence AG3 (TTAG3)3 (Tel22) play a key role in cancer and aging. We combined elastic incoherent neutron scattering (EINS) and quasielastic incoherent neutron scattering (QENS) to characterize the internal dynamics of Tel22 G4s and to assess how it is affected by complexation with two standard ligands, Berberine and BRACO19. We show that the interaction with the two ligands induces an increase of the overall mobility of Tel22 as quantified by the mean squared displacements (MSD) of hydrogen atoms. At the same time, the complexes display a lower stiffness than G4 alone. Two different types of motion characterize the G4 nanosecond timescale dynamics. Upon complexation, an increasing fraction of G4 atomic groups participate in this fast dynamics, along with an increase in the relevant characteristic length scales. We suggest that the entropic contribution to the conformational free energy of these motions might be crucial for the complexation mechanisms.

The Combination of Molnupiravir with Nirmatrelvir or GC376 Has a Synergic Role in the Inhibition of SARS-CoV-2 Replication In Vitro.

Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic; however, the need for orally administered antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using five two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were performed in triplicate, each being repeated at least three times. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p < 0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p < 0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p = 0.01) and 13.08 (p < 0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro.

BCL2 G quadruplex-binding small molecules: Current status and prospects for the development of next-generation anticancer therapeutics.

B cell lymphoma 2 (BCL2) overexpression in a range of human tumors is often related to chemotherapy resistance and poor prognosis. GC-rich regions upstream of the P1 promoter in human BCL2 can form G-quadruplex (G4) structures through the stacking of four Hoogsteen-paired guanine bases. Stabilizing the G4 fold implies the inhibition of BCL2 expression and, thus, small molecules that selectively bind to the G4 are promising anticancer candidates. In this review, we discuss the structural aspects, binding affinity, selectivity, and biological activity of well-characterized BCL2 G4 binding ligands in vitro and in vivo. We also explore future directions in the research and development of G4-based anticancer therapeutics.

Solvent Vibrations as a Proxy of the Telomere G-Quadruplex Rearrangements across Thermal Unfolding.

G-quadruplexes (G4s) are noncanonical forms of DNA involved in many key genome functions. Here, we exploited UV Resonance Raman scattering to simultaneously explore the vibrational behavior of a human telomeric G4 (Tel22) and its aqueous solvent as the biomolecule underwent thermal melting. We found that the OH stretching band, related to the local hydrogen-bonded network of a water molecule, was in strict relation with the vibrational features of the G4 structure as a function of temperature. In particular, the modifications to the tetrahedral ordering of the water network were strongly coupled to the DNA rearrangements, showing changes in temperature that mirrored the multi-step melting process of Tel22. The comparison between circular dichroism and Raman results supported this view. The present findings provide novel insights into the impact of the molecular environment on G4 conformation. Improving current knowledge on the solvent structural properties will also contribute to a better understanding of the role played by water arrangement in the complexation of G4s with ligands.