Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier

Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo–glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial

Polyphenolic C-glucosidic ellagitannins present in oak-aged wine inhibit HIV-1 nucleocapsid protein

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone implicated in several steps of the virus replication cycle and an attractive new target for drug development. In reverse transcription, NC destabilizes nucleic acid secondary structures and catalyzes the annealing of HIV-1 TAR RNA to its DNA copy (cTAR) to form the heteroduplex TAR/cTAR. A screening program led to the identification of the plant polyphenols acutissimins A and B as potent inhibitors of NC in different assays. These two flavano-ellagitannins, which are found in wine aged in oak barrels, exhibited different mechanisms of protein inhibition and higher potency relatively to their epimers, epiacutissimins A and B, and to simpler structures notably representing hydrolytic fragments and metabolites therefrom

Sequence-specific interactions of drugs interfering with the topoisomerase–DNA cleavage complex

AbstractDNA-processing enzymes, such as the topoisomerases (tops), represent major targets for potent anticancer (and antibacterial) agents. The drugs kill cells by poisoning the enzymes' catalytic cycle. Understanding the molecular details of top poisoning is a fundamental requisite for the rational development of novel, more effective antineoplastic drugs. In this connection, sequence-specific recognition of the top–DNA complex is a key step to preferentially direct the action of the drugs onto selected genomic sequences. In fact, the (reversible) interference of drugs with the top–DNA complex exhibits well-defined preferences for DNA bases in the proximity of the cleavage site, each drug showing peculiarities connected to its structural features. A second level of selectivity can be observed when chemically reactive groups are present in the structure of the top-directed drug. In this case, the enzyme recognizes or generates a unique site for covalent drug–DNA binding. This will further subtly modulate the drug's efficiency in stimulating DNA damage at selected sites. Finally, drugs can discriminate not only among different types of tops, but also among different isoenzymes, providing an additional level of specific selection. Once the molecular basis for DNA sequence-dependent recognition has been established, the above-mentioned modes to generate selectivity in drug poisoning can be rationally exploited, alone or in combination, to develop tailor-made drugs targeted at defined loci in cancer cells

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidylanthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved Nterminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA

Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage

Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the −1 position, i.e. immediately 5′ of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria

AAV-mediated transcription factor EB (TFEB) gene delivery ameliorates muscle pathology and function in the murine model of Pompe Disease

Pompe disease (PD) is a metabolic myopathy due to acid alpha-glucosidase deficiency and characterized by extensive glycogen storage and impaired autophagy. We previously showed that modulation of autophagy and lysosomal exocytosis by overexpression of the transcription factor EB (TFEB) gene was effective in improving muscle pathology in PD mice injected intramuscularly with an AAV-TFEB vector. Here we have evaluated the effects of TFEB systemic delivery on muscle pathology and on functional performance, a primary measure of efficacy in a disorder like PD. We treated 1-month-old PD mice with an AAV2.9-MCK-TFEB vector. An animal cohort was analyzed at 3 months for muscle and heart pathology. A second cohort was followed at different timepoints for functional analysis. In muscles from TFEB-treated mice we observed reduced PAS staining and improved ultrastructure, with reduced number and increased translucency of lysosomes, while total glycogen content remained unchanged. We also observed statistically significant improvements in rotarod performance in treated animals compared to AAV2.9-MCK-eGFP-treated mice at 5 and 8 months. Cardiac echography showed significant reduction in left-ventricular diameters. These results show that TFEB overexpression and modulation of autophagy result in improvements of muscle pathology and of functional performance in the PD murine model, with delayed disease progression

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of antiretrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidylanthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TARbinding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins

Mapping Drug Interactions at the Covalent Topoisomerase II-DNA Complex by Bisantrene/Amsacrine Congeners *

To identify structural determinants for the sequence-specific recognition of covalent topoisomerase II-DNA complexes by anti-cancer drugs, we investigated a number of bisantrene congeners, including a 10-azabioisoster, bearing one or two 4, 5-dihydro-1H-imidazol-2-yl hydrazone side chains at positions 1, 4, or 9 of the anthracene ring system. The studied bisantrene/amsacrine (m-AMSA) hybrid and bisantrene isomers were able to poison DNA topoisomerase II with an intermediate activity between those of bisantrene and m-AMSA. Moving the side chain from the central to a lateral ring (from C-9 to C-1/C-4) only slightly modified the drug DNA affinity, whereas it dramatically affected local base preferences of poison-stimulated DNA cleavage. In contrast, switching the planar aromatic systems of bisantrene and m-AMSA did not substantially alter the sequence specificity of drug action. A computer-assisted steric and electrostatic alignment analysis of the test compounds was in agreement with the experimental data, since a common pharmacophore was shared by bisantrene, m-AMSA, and 9-substituted analogs, whereas the 1-substituted isomer showed a radically changed pharmacophoric structure. Thus, the relative space occupancy and electron distribution of putative DNA binding (aromatic rings) and enzyme binding (side chains) moieties are fundamental in directing the specific action of topoisomerase II poisons and in determining the poison pharmacophore

Behind the Mirror: Chirality Tunes the Reactivity and Cytotoxicity of Chloropiperidines as Potential Anticancer Agents

The pressing demand for sustainable antitumor drugs prompted us to investigate 3-chloropiperidines as potential mustard-based anticancer agents. In this study, an explorative set of variously decorated monofunctional 3-chloropiperidines (M-CePs) was efficiently synthesized through a fast and affordable route providing high yields of pure racemates and enantiomers. Consistently with their reactivity, M-CePs were demonstrated to alkylate DNA in vitro. On a panel of carcinoma cell lines, M-CePs exhibited low nanomolar cytotoxicity indexes, which showed their remarkable activity against pancreatic cancer cells and in all cases performed strikingly better than the chlorambucil control. Very interestingly, stereochemistry modulated the activity of M-CePs in unexpected ways, pointing to additional molecular mechanisms of action beyond the direct damage of genomic DNA. This encouraging combination of efficacy and sustainability suggests they are valid candidates for anticancer agent development