The Structural Dispersity of Oligoethylene Glycol-Containing Polymer Brushes Determines Their Interfacial Properties.

Ought to their bioinert properties and facile synthesis, poly[(oligoethylene glycol)methacrylate]s (POEGMAs) have been raised as attractive alternatives to poly(ethylene glycols) (PEGs) in an array of (bio)material applications, especially when they are applied as polymer brush coatings. However, commercially available OEG-methacrylate (macro)monomers feature a broad distribution of OEG lengths, thus generating structurally polydisperse POEGMAs when polymerized through reversible deactivation radical polymerization. Here, we demonstrate that the interfacial physicochemical properties of POEGMA brushes are significantly affected by their structural dispersity, i.e., the degree of heterogeneity in the length of side OEG segments. POEGMA brushes synthesized from discrete (macro)monomers obtained through chromatographic purification of commercial mixtures show increased hydration and reduced adhesion when compared to their structurally polydisperse analogues. The observed alteration of interfacial properties is directly linked to the presence of monodisperse OEG side chains, which hamper intramolecular and intermolecular hydrophobic interactions while simultaneously promoting the association of water molecules. These phenomena provide structurally homogeneous POEGMA brushes with a more lubricious and protein repellent character with respect to their heterogeneous counterparts. More generally, in contrast to what has been assumed until now, the properties of POEGMA brushes cannot be anticipated while ruling out the effect of dispersity by (macro)monomer feeds. Simultaneously, side chain dispersity of POEGMAs emerges as a critical parameter for determining the interfacial characteristics of brushes.

Targeting the Major Groove of the Palindromic d(GGCGCC)2 Sequence by Oligopeptide Derivatives of Anthraquinone Intercalators.

GC-rich sequences are recurring motifs in oncogenes and retroviruses and could be targeted by noncovalent major-groove therapeutic ligands. We considered the palindromic sequence d(G1G2C3G4C5C6)2, and designed several oligopeptide derivatives of the anticancer intercalator mitoxantrone. The stability of their complexes with an 18-mer oligonucleotide encompassing this sequence in its center was validated using polarizable molecular dynamics. We report the most salient structural features of two novel compounds, having a dialkylammonium group as a side chain on both arms. The anthraquinone ring is intercalated in the central d(CpG)2 sequence with its long axis perpendicular to that of the two base pairs. On each strand, this enables each ammonium group to bind in-register to O6/N7 of the two facing G bases upstream. We subsequently designed tris-intercalating derivatives, each dialkylammonium substituted with a connector to an N9-aminoacridine intercalator extending our target range from a six- to a ten-base-pair palindromic sequence, d(C1G2G3G4C5G6C7C8C9G10)2. The structural features of the complex of the most promising derivative are reported. The present design strategy paves the way for designing intercalator-oligopeptide derivatives with even higher selectivity, targeting an increased number of DNA bases, going beyond ten.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Insight into the LFA-1/SARS-CoV-2 Orf7a Complex by Protein-Protein Docking, Molecular Dynamics, and MM-GBSA Calculations.

In the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome, open reading frames (ORFs) encode for viral accessory proteins. Among these, Orf7a structurally resembles the members of the immunoglobulin (Ig) superfamily and intracellular adhesion molecules (ICAMs), in particular. ICAMs are involved in integrin binding through lymphocyte function-associated antigen 1 (LFA-1). Based on such considerations and on previous findings on SARS-CoV, it has been postulated that the formation of the LFA-1/Orf7a complex could contribute to SARS-CoV-2 infectivity and pathogenicity. With the current work, we aim at providing insight into this macromolecular assembly, taking advantage of the recently reported SARS-CoV-2 Orf7a structure. Protein-protein docking, molecular dynamics (MD) simulations, and a Molecular Mechanical-Generalized Born Surface Area (MM-GBSA)-based stage were enrolled to provide refined models.

Selenoxide Elimination Triggers Enamine Hydrolysis to Primary and Secondary Amines: A Combined Experimental and Theoretical Investigation.

We discuss a novel selenium-based reaction mechanism consisting in a selenoxide elimination-triggered enamine hydrolysis. This one-pot model reaction was studied for a set of substrates. Under oxidative conditions, we observed and characterized the formation of primary and secondary amines as elimination products of such compounds, paving the way for a novel strategy to selectively release bioactive molecules. The underlying mechanism was investigated using NMR, mass spectrometry and density functional theory (DFT).

A new sensitive and subunit-selective molecular tool for investigating protein kinase A in the brain.

Despite cellular complexity, a limited number of small molecules act as intracellular second messengers. Protein kinase A (PKA) is the main transducer of the information carried by cyclic adenosine monophosphate (cAMP). Recently, cellular imaging has achieved major technical advancements, although the search for more specific and sensitive low-molecular-weight probes to explore subcellular events involving second messengers is still in progress. The convergent synthesis of a novel, fluorescent small molecule comprising the cAMP structure and a rhodamine-based fluorescent residue, connected through a flexible linker, is described here. The interaction motif of this compound with PKA was investigated in silico using a blind docking approach, comparing its theoretical binding energy with the one calculated for cAMP. Moreover, the predicted pharmacokinetic properties were also computed and discussed. The new probe was tested on three areas of the mouse central nervous system (parietal cerebral cortex, hippocampus, and cerebellar cortex) with different fixation methods demonstrating remarkable selectivity towards the PKA RIα subunit. The probe showed overall better performances when compared to other commercially available fluorescent cAMP analogues, acting at lower concentrations, and providing stable labeling.

Investigation of the molecular reactivity of bioactive oxiranylmethyloxy anthraquinones.

The design of a multitarget and multifunctional small molecule containing two functional groups reacting through different mechanisms represents an attractive goal for the medicinal chemist. The preparation of two bifunctional oxiranylmethyloxy anthraquinones, previously investigated as anticancer agents, is described here. These compounds combine a planar, DNA-intercalating and pro-oxidant anthraquinone scaffold and the alkylating epoxide functions which can covalently react with the nucleic acid. Their multilevel molecular reactivity was studied through a combination of analytical techniques: The DNA-binding properties were investigated using a mass spectrometry-based binding assay and by nuclear magnetic resonance, highlighting the formation of a covalent adduct with a nucleobase. Moreover, the contribution of the pro-oxidant redox cycling was evaluated.

Sequence-Selective Recognition of the d(GGCGCC)2 DNA Palindrome by Oligopeptide Derivatives of Mitoxantrone. Enabling for Simultaneous Targeting of the Two Guanine Bases Upstream from the Central Intercalation Site in Both Grooves and along Both Strands.

The d(GGCGCC)2 palindrome is encountered in several oncogenic and retroviral sequences. In order to target it, we previously designed several oligopeptide derivatives of the mitoxantrone and ametantrone anticancer intercalators. These have two arms with a cationic side-chain in the major groove, each destined to bind along each strand O6/N7 of the two successive guanine bases (G1-G2/G1'-G2') upstream from the central anthraquinone intercalation site. We retained from a previous study (El Hage et al., 2022) a tris-intercalating molecule with two outer 9-aminoacridine (9-AA) intercalators, denoted as III. We sought enhancements in both affinity and selectivity by simultaneously targeting the minor groove of the extracyclic -NH2 groups of these bases and G4-G4' of the intercalation site. We considered derivatives of distamycin, having each pyrrole ring replaced by an imidazole to act as an in-register electron acceptor from the -NH2 group of a target guanine. We substituted the C6 and C7 carbons of anthraquinone, or the C8 and C9 ones of anthracycline, by an (imidazole-amide)3 chain. Four different derivatives of III were designed with different connectors to the anthraquinone/anthracycline and 9-AA. Polarizable molecular dynamics simulations of their complexes with a double-stranded DNA 18-mer with a central d(C GGGC GCCC G)2 palindrome sequence showed in-register minor groove binding to -NH2 of G1-G2/G1'-G2' to coexist with major groove recognition of O6/N7. Up to 12 H-bonds could be stabilized in the minor groove coexisting with four bidentate interactions of the alkyl diammonium moieties in the major groove. Since there is no mutual interference, the binding enthalpies, ΔH, contributed by each groove could add up and enable significant enhancements of the affinity constants. As was the case for their Lys precursor, these derivatives are amenable to chemical syntheses and in vitro and in vivo tests, for which the present results provide an incentive. The construction of derivatives III-A-III-D is modular. For in vitro experiments, this should enable unraveling the most important structural elements to further optimize both ΔH and TΔS and sequence selectivity and how this could translate to in vivo tests.

The Bioactive Gamma-Oryzanol from Oryza sativa L. Promotes Neuronal Differentiation in Different In Vitro and In Vivo Models.

Gamma-oryzanol (ORY), found in rice (Oryza sativa L.), is a mixture of ferulic acid esters with triterpene alcohols, well-known for its antioxidant and anti-inflammatory properties. Our past research demonstrated its positive impact on cognitive function in adult mice, influencing synaptic plasticity and neuroprotection. In this study, we explored whether ORY can exert neuro-differentiating effects by using different experimental models. For this purpose, chemical characterization identified four components that are most abundant in ORY. In human neuroblastoma cells, we showed ORY's ability to stimulate neurite outgrowth, upregulating the expression of GAP43, BDNF, and TrkB genes. In addition, ORY was found to guide adult mouse hippocampal neural progenitor cells (NPCs) toward a neuronal commitment. Microinjection of ORY in zebrafish Tg (-3.1 neurog1:GFP) amplified neurog1-GFP signal, islet1, and bdnf mRNA levels. Zebrafish nrf2a and nrf2b morphants (MOs) were utilized to assess ORY effects in the presence or absence of Nrf2. Notably, ORY's ability to activate bdnf was nullified in nrf2a-MO and nrf2b-MO. Furthermore, computational analysis suggested ORY's single components have different affinities for the Keap1-Kelch domain. In conclusion, although more in-depth studies are needed, our findings position ORY as a potential source of bioactive molecules with neuro-differentiating potential involving the Nrf2 pathway.

Synthesis and Evaluation of Bithiazole Derivatives As Potential α-Sarcoglycan Correctors.

4'-Methyl-4,5'-bithiazoles were previously identified as cystic fibrosis transmembrane regulator (CFTR) correctors, thus being able to correct folding defective mutants of the channel regulating chloride transport through the membrane. Additionally, bithiazole derivative C17 was reported to recover α-sarcoglycan in vitro and in vivo. We report here the synthesis of two new derivatives of C17, in which the two sides of the bithiazole scaffold were modified. The synthesized compounds and the corresponding precursors were tested in myogenic cells to evaluate the expression of α-sarcoglycan. The results highlighted that both substitutions of the bithiazole scaffold are important to achieve the maximum recovery of the α-sarcoglycan mutant. Nonetheless, partial preservation of the activity was observed. Accordingly, this paves the way to further derivatizations/optimization and target fishing studies, which were preliminarily performed in this study as a proof of concept, allowing the investigation of the molecular mechanisms leading to the α-sarcoglycan correction.

Combining computational and experimental evidence on the activity of antimalarial drugs on papain-like protease of SARS-CoV-2: A repurposing study.

The development of inhibitors that target the papain-like protease (PLpro) has the potential to counteract the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent causing coronavirus disease 2019 (COVID-19). Based on a consideration of its several downstream effects, interfering with PLpro would both revert immune suppression exerted by the virus and inhibit viral replication. By following a repurposing strategy, the current study evaluates the potential of antimalarial drugs as PLpro inhibitors, and thereby the possibility of their use for treatment of SARS-CoV-2 infection. Computational tools were employed for structural analysis, molecular docking, and molecular dynamics simulations to screen antimalarial drugs against PLpro, and in silico data were validated by in vitro experiments. Virtual screening highlighted amodiaquine and methylene blue as the best candidates, and these findings were complemented by the in vitro results that indicated amodiaquine as a µM PLpro deubiquitinase inhibitor. The results of this study demonstrate that the computational workflow adopted here can correctly identify active compounds. Thus, the highlighted antimalarial drugs represent a starting point for the development of new PLpro inhibitors through structural optimization.

Synthesis of 3-Acyloxyindolenines by TiCl3-Mediated Reductive Cyclization of 2-(ortho-Nitroaryl)-Substituted Enol Esters.

In the presence of TiCl3, the reductive cyclization of tetrasubstituted enol esters bearing a 2-(ortho-nitroaryl) substituent affords 3-acyloxy-2,3-disubstituted indolenines in good yields. A domino process involving the partial reduction of nitro to a nitroso group followed by 5-center-6π-electrocyclization, 1,2-acyloxy migration, and the further reduction of the resulting nitrone intermediate accounts for the reaction outcome. The so-obtained indolenines are converted smoothly to 2,2-disubstituted oxindoles via a sequence of saponification and semipinacol rearrangement.

Photoactivated semi-synthetic derivative of osajin selectively interacts with G-quadruplex DNA.

Natural and synthetic small molecules targeting G-quadruplex are currently being studied. These peculiar DNA arrangements arise in guanine-rich sequences located in telomeres, oncogene promoters and in several viruses. Two semi-synthetic derivatives of osajin, a natural isoflavone from Maclura pomifera, were prepared and screened for their binding affinity towards G-quadruplex and double stranded DNA (dsDNA) using electrospray mass spectrometry (ESI-MS). Although an overall decrease in affinity for DNA was observed, one of the derivatives showed improved selectivity for G-quadruplex over dsDNA, retaining structure stabilization properties. Binding motif was studied by collision-induced dissociation (CID) assays and molecular docking, that suggested stacking as the preferential interaction pattern. Moreover, the compound selectively induced DNA damage on the G-quadruplex sequence upon UV irradiation, due to the presence of the photoreactive aryl tosylate group.

Amino-Acid-Anthraquinone Click Chemistry Conjugates Selectively Target Human Telomeric G-Quadruplexes.

Guanine-rich sequences are known to fold into G-quadruplex (G4) arrangements, which are present in oncogenes and in the telomeric regions of chromosomes. In particular, G4s represent an obstacle to functioning of telomerase, an enzyme overexpressed in cancer cells causing their immortalization. Therefore, G4 stabilization using small molecules represents an appealing strategy for the medicinal chemist. Ligands based on an anthraquinone scaffold, to which peptidic side chains were attached by an amide bond, were previously reported. We envisioned improving this ligand concept leveraging the click chemistry approach, which, besides representing a flexible, high yielding synthetic strategy, allows an elongation of the side chains and an increase of π-π stacking and H-bond interactions with the nucleobases through the triazole ring. Compounds were tested for their ability to interact with G4 DNA with a multiple analytical approach, demonstrating an elevated aptitude to stabilize the G4 and high selectivity over double stranded DNA.

A computational approach to drug repurposing against SARS-CoV-2 RNA dependent RNA polymerase (RdRp).

The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused a worldwide outbreak of coronavirus disease 19 (COVID-19), which rapidly evolved as a global concern. The efforts of the scientific community are pointed towards the identification of promptly available therapeutic options. RNA-dependent RNA polymerase (RdRp) is a promising target for developing small molecules to contrast SARS-CoV-2 replication. Modern computational tools can boost identification and repurposing of known drugs targeting RdRp. We here report the results regarding the screening of a database containing more than 8800 molecules, including approved, experimental, nutraceutical, illicit, withdrawn and investigational compounds. The molecules were docked against the cryo-electron microscopy structure of SARS-CoV-2 RdRp, optimized by means of molecular dynamics (MD) simulations. The adopted three-stage ensemble docking study underline that compounds formerly developed as kinase inhibitors may interact with RdRp.Communicated by Ramaswamy H. Sarma.

Enhanced G-quadruplex selectivity of flavonoid glycoside rutin over quercetin.

In drug discovery, ligand-mediated stabilization of G-quadruplexes is pursued for regulating gene expression and key cellular processes. Electrospray ionization mass spectrometry (ESI-MS) has been optimized for screening putative DNA-binding small molecules of natural and synthetic origin. Several flavonoids were reported to interact with G-quadruplex, and quercetin is among them. In this contribution, the interaction with G-quadruplex DNA of rutin, a glycoside of quercetin extracted from flower buds of Styphnolobium japonicum (L.) Schott, was investigated by means of ESI-MS and molecular docking. While rutin and quercetin showed similar G-quadruplex binding affinity values, rutin was characterized by enhanced selectivity for G-quadruplex over double stranded DNA. Moreover, collision-induced dissociation (CID) assays demonstrated that rutin stabilizes the G-quadruplex arrangement more efficiently, and molecular docking predicted stacking as the preferential interaction pattern.

Combining Electrospray Mass Spectrometry (ESI-MS) and Computational Techniques in the Assessment of G-Quadruplex Ligands: A Hybrid Approach to Optimize Hit Discovery.

Guanine-rich sequences forming G-quadruplexes (GQs) are present in several genomes, ranging from viral to human. Given their peculiar localization, the induction of GQ formation or GQ stabilization with small molecules represents a strategy for interfering with crucial biological functions. Investigating the recognition event at the molecular level, with the aim of fully understanding the triggered pharmacological effects, is challenging. Native electrospray ionization mass spectrometry (ESI-MS) is being optimized to study these noncovalent assemblies. Quantitative parameters retrieved from ESI-MS studies, such as binding affinity, the equilibrium binding constant, and sequence selectivity, will be overviewed. Computational experiments supporting the ESI-MS investigation and boosting its efficiency in the search for GQ ligands will also be discussed with practical examples. The combination of ESI-MS and in silico techniques in a hybrid high-throughput-screening workflow represents a valuable tool for the medicinal chemist, providing data on the quantitative and structural aspects of ligand-GQ interactions.

Evidence on selective binding to G-quadruplex DNA of isoflavones from Maclura pomifera by mass spectrometry and molecular docking.

G-quadruplex DNA stabilization mediated by small molecules is an attractive approach to modulate the transcription of guanine-rich sequences and contrast unregulated cell proliferation. Natural alkaloids have been reported to target this macromolecular arrangement, and such mechanism may be among these underlying the antiproliferative effect of some flavonoids. The binding affinity for G-quadruplex and double stranded DNA of 4 isoflavones from Maclura pomifera, namely osajin, pomiferin, scandenone and auriculasin, was evaluated using electrospray mass spectrometry (ESI-MS). The experiments pointed out that osajin and scandenone preferentially bind G-quadruplex. Moreover, since G-quadruplex stabilization is a crucial aim for triggering biological effects such as gene expression, collision-induced dissociation (CID) assays were performed to study the relative gas-phase kinetic stability of the DNA/ligand complexes. Osajin was identified as the best G-quadruplex stabilizer of the set, and in silico docking studies indicate that stacking is the preferred interaction motif of this natural compound.

Natural phosphodiesterase 5 (PDE5) inhibitors: a computational approach.

In 1998, sildenafil was marketed as the first FDA-approved oral drug for the treatment of erectile dysfunction (ED). During the last two decades, the commercialization of other synthetic phosphodiesterase 5 (PDE5) inhibitors has been paralleled by the rise of remedies based on natural molecules from different chemical classes (flavonoids, polyphenols and alkaloids in general). In this work, a set of in silico tools were applied to study a panel of 30 natural compounds claimed to be effective against ED in the scientific literature or in folk medicine. First, pharmacokinetic properties were analysed to exclude the compounds lacking in specific drug-like features. Estimated binding energy for PDE5 and selectivity towards other PDE isoforms were then considered to highlight some promising molecules. Finally, a detailed structural investigation of the interaction pattern with PDE in comparison with sildenafil was conducted for the best performing compound of the set.

Critical Review on the Chemical Aspects of Cannabidiol (CBD) and Harmonization of Computational Bioactivity Data.

Cannabidiol (CBD) is a non-psychotropic phytocannabinoid which represents one of the constituents of the "phytocomplex" of Cannabis sativa. This natural compound is attracting growing interest since when CBD-based remedies and commercial products were marketed. This review aims to exhaustively address the extractive and analytical approaches that have been developed for the isolation and quantification of CBD. Recent updates on cutting-edge technologies were critically examined in terms of yield, sensitivity, flexibility and performances in general, and are reviewed alongside original representative results. As an add-on to currently available contributions in the literature, the evolution of the novel, efficient synthetic approaches for the preparation of CBD, a procedure which is appealing for the pharmaceutical industry, is also discussed. Moreover, with the increasing interest on the therapeutic potential of CBD and the limited understanding of the undergoing biochemical pathways, the reader will be updated about recent in silico studies on the molecular interactions of CBD towards several different targets attempting to fill this gap. Computational data retrieved from the literature have been integrated with novel in silico experiments, critically discussed to provide a comprehensive and updated overview on the undebatable potential of CBD and its therapeutic profile.