SignificanceA thorough characterization of the binding interaction between a drug and its molecular target is fundamental to successfully lead drug design. We demonstrate that this characterization is also possible using the recently developed method of funnel-metadynamics (FM), here applied to investigate the binding of berberine to DNA G-quadruplex. We computed a quantitatively well-characterized free-energy landscape that allows identifying two low-energy ligand binding modes and the presence of higher energy prebinding states. We validated the accuracy of our calculations by steady-state fluorescence experiments. The good agreement between the theoretical and experimental binding free-energy value demonstrates that FM is a most reliable method to study ligand/DNA interaction.

The synthesis, physico-chemical properties and biological effects of a new class of naphthalene diimides (NDIs) capable of reversibly binding telomeric DNA and alkylate it through an electrophilic quinone methide moiety (QM), are reported. FRET and circular dichroism assays showed a marked stabilization and selectivity towards telomeric G4 DNA folded in a hybrid topology. NDI-QMs' alkylating properties revealed a good reactivity on single nucleosides and selectivity towards telomeric G4. A selected NDI was able to significantly impair the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression. These findings points to our hybrid ligand-alkylating NDIs as possible tools for the development of novel targeted anticancer therapies.

Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety.

BackgroundBecause of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs).MethodsThe study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed.ResultsOverall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T).ConclusionsThe high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

Coronaviridae is a peculiar viral family, with a very large RNA genome and characteristic appearance, endowed with remarkable tendency to transfer from animals to humans. Since the beginning of the 21st century, three highly transmissible and pathogenic coronaviruses have crossed the species barrier and caused deadly pneumonia, inflicting severe outbreaks and causing human health emergencies of inconceivable magnitude. Indeed, in the past two decades, two human coronaviruses emerged causing serious respiratory illness: severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), causing more than 10,000 cumulative cases, with mortality rates of 10 % for SARS-CoV-1 and 34.4 % for MERS-CoV. More recently, the severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) has emerged in China and has been identified as the etiological agent of the recent COVID-19 pandemic outbreak. It has rapidly spread throughout the world, causing nearly 22 million cases and ∼ 770,000 deaths worldwide, with an estimated mortality rate of ∼3.6 %, hence posing serious challenges for adequate and effective prevention and treatment. Currently, with the exception of the nucleotide analogue prodrug remdesivir, and despite several efforts, there is no known specific, proven, pharmacological treatment capable of efficiently and rapidly inducing viral containment and clearance of SARS-CoV-2 infection as well as no broad-spectrum drug for other human pathogenic coronaviruses. Another confounding factor is the paucity of molecular information regarding the tendency of coronaviruses to acquire drug resistance, a gap that should be filled in order to optimize the efficacy of antiviral drugs. In this light, the present review provides a systematic update on the current knowledge of the marked global efforts towards the development of antiviral strategies aimed at coping with the infection sustained by SARS-CoV-2 and other human pathogenic coronaviruses, displaying drug resistance profiles. The attention has been focused on antiviral drugs mainly targeting viral protease, RNA polymerase and spike glycoprotein, that have been tested in vitro and/or in clinical trials as well as on promising compounds proven to be active against coronaviruses by an in silico drug repurposing approach. In this respect, novel insights on compounds, identified by structure-based virtual screening on the DrugBank database endowed by multi-targeting profile, are also reported. We specifically identified 14 promising compounds characterized by a good in silico binding affinity towards, at least, two of the four studied targets (viral and host proteins). Among which, ceftolozane and NADH showed the best multi-targeting profile, thus potentially reducing the emergence of resistant virus strains. We also focused on potentially novel pharmacological targets for the development of compounds with anti-pan coronavi...