Phylogenomics of Neogastropoda: the backbone hidden in the bush.

The molluscan order Neogastropoda encompasses over 15,000 almost exclusively marine species playing important roles in benthic communities and in the economies of coastal countries. Neogastropoda underwent intensive cladogenesis in early stages of diversification, generating a 'bush' at the base of their evolutionary tree, that has been hard to resolve even with high throughput molecular data. In the present study to resolve the bush, we use a variety of phylogenetic inference methods and a comprehensive exon capture dataset of 1,817 loci (79.6% data occupancy) comprising 112 taxa of 48 out of 60 Neogastropoda families. Our results show consistent topologies and high support in all analyses at (super)family level, supporting monophyly of Muricoidea, Mitroidea, Conoidea, and, with some reservations, Olivoidea and Buccinoidea. Volutoidea and Turbinelloidea as currently circumscribed are clearly paraphyletic. Despite our analyses consistently resolving most backbone nodes, three prove problematic: First, uncertain placement of Cancellariidae, as the sister group to either a Ficoidea-Tonnoidea clade, or to the rest of Neogastropoda, leaves monophyly of Neogastropoda unresolved. Second, relationships are contradictory at the base of the major 'core Neogastropoda' grouping. Third, coalescence-based analyses reject monophyly of the Buccinoidea in relation to Vasidae. We analysed phylogenetic signal of targeted loci in relation to potential biases, and we propose most probable resolutions in the latter two recalcitrant nodes. The uncertain placement of Cancellariidae may be explained by orthology violations due to differential paralog loss shortly after the whole genome duplication, which should be resolved with a curated set of longer loci.

DeTox: a pipeline for the detection of toxins in venomous organisms.

Venomous organisms have independently evolved the ability to produce toxins 101 times during their evolutionary history, resulting in over 200 000 venomous species. Collectively, these species produce millions of toxins, making them a valuable resource for bioprospecting and understanding the evolutionary mechanisms underlying genetic diversification. RNA-seq is the preferred method for characterizing toxin repertoires, but the analysis of the resulting data remains challenging. While early approaches relied on similarity-based mapping to known toxin databases, recent studies have highlighted the importance of structural features for toxin detection. The few existing pipelines lack an integration between these complementary approaches, and tend to be difficult to run for non-experienced users. To address these issues, we developed DeTox, a comprehensive and user-friendly tool for toxin research. It combines fast execution, parallelization and customization of parameters. DeTox was tested on published transcriptomes from gastropod mollusks, cnidarians and snakes, retrieving most putative toxins from the original articles and identifying additional peptides as potential toxins to be confirmed through manual annotation and eventually proteomic analysis. By integrating a structure-based search with similarity-based approaches, DeTox allows the comprehensive characterization of toxin repertoire in poorly-known taxa. The effect of the taxonomic bias in existing databases is minimized in DeTox, as mirrored in the detection of unique and divergent toxins that would have been overlooked by similarity-based methods. DeTox streamlines toxin annotation, providing a valuable tool for efficient identification of venom components that will enhance venom research in neglected taxa.

Whole Genome Duplication and Gene Evolution in the Hyperdiverse Venomous Gastropods.

The diversity of venomous organisms and the toxins they produce have been increasingly investigated, but taxonomic bias remains important. Neogastropods, a group of marine predators representing almost 22% of the known gastropod diversity, evolved a wide range of feeding strategies, including the production of toxins to subdue their preys. However, whether the diversity of these compounds is at the origin of the hyperdiversification of the group and how genome evolution may correlate with both the compounds and species diversities remain understudied. Among the available gastropods genomes, only eight, with uneven quality assemblies, belong to neogastropods. Here, we generated chromosome-level assemblies of two species belonging to the Tonnoidea and Muricoidea superfamilies (Monoplex corrugatus and Stramonita haemastoma). The two obtained high-quality genomes had 3 and 2.2 Gb, respectively, and 92-89% of the total assembly conformed 35 pseudochromosomes in each species. Through the analysis of syntenic blocks, Hox gene cluster duplication, and synonymous substitutions distribution pattern, we inferred the occurrence of a whole genome duplication event in both genomes. As these species are known to release venom, toxins were annotated in both genomes, but few of them were found in homologous chromosomes. A comparison of the expression of ohnolog genes (using transcriptomes from osphradium and salivary glands in S. haemastoma), where both copies were differentially expressed, showed that most of them had similar expression profiles. The high quality of these genomes makes them valuable reference in their respective taxa, facilitating the identification of genome-level processes at the origin of their evolutionary success.

Discovery of SI 1/20 and SI 1/22 as Mutual Prodrugs of 5-Fluorouracil and Imidazole-Based Heme Oxygenase 1 Inhibitor with Improved Cytotoxicity in DU145 Prostate Cancer Cells.

In this work, we extend the concept of 5-fluorouracil/heme oxygenase 1 (5-FU/HO-1) inhibitor hybrid as an effective strategy for enhancing 5-FU-based anticancer therapies. For this purpose, we designed and synthesized new mutual prodrugs, named SI 1/20 and SI 1/22, in which the two active parent drugs (i. e., 5-FU and an imidazole-based HO-1 inhibitor) were connected through an easily cleavable succinic linker. Experimental hydrolysis rate, and in silico ADMET predictions were indicative of good drug-likeness and pharmacokinetic properties. Novel hybrids significantly reduced the viability of prostate DU145 cancer cells compared to the parent compounds 5-FU and HO-1 inhibitor administered alone or in combination. Interestingly, both compounds showed statistically significant lower toxicity, than 5-FU at the same dose, against non-tumorigenic human benign prostatic hyperplasia (BPH-1) cell line. Moreover, the newly synthesized mutual prodrugs inhibited the HO-1 activity both in a cell-free model and in vitro, as well as downregulated the HO-1 expression and increased the reactive oxygen species (ROS) levels.

Structure-activity relationships of mixed σ1R/σ2R ligands with antiproliferative and anticancer effects.

The overexpression of σ receptors (σRs) in various types of tumors has prompted a deep investigation of their role in cancer pathophysiology. Consequently, σR ligands have been widely studied in vitro and in vivo for their antiproliferative effects as a novel potential class of chemotherapeutic agents, both alone and in combination with other anticancer drugs. A growing body of evidence highlights that σR ligands can inhibit cancer cells' survival, migration, and proliferation, thanks to the modulation of a wide panel of tumorigenic pathways. In addition to their antitumor activity, σR ligands are gaining momentum as radiotracers for PET and SPECT imaging applications. The purpose of this review is to report on recent advances in the development of σR ligands. In particular, herein, we describe the structure-activity relationships of structurally diverse mixed σ1R/σ2R ligands that showed promising antitumor profiles towards a variety of cancer cell lines.

Expansion and Neofunctionalization of Actinoporin-like Genes in Mediterranean Mussel (Mytilus galloprovincialis).

Pore-forming toxins are an important component of the venom of many animals. Actinoporins are potent cytolysins that were first detected in the venom of sea anemones; however, they are occasionally found in animals other than cnidarians and are expanded in a few predatory gastropods. Here, we report the presence of 27 unique actinoporin-like genes with monophyletic origin in Mytilus galloprovincialis, which we have termed mytiporins. These mytiporins exhibited a remarkable level of molecular diversity and gene presence-absence variation, which warranted further studies aimed at elucidating their functional role. We structurally and functionally characterized mytiporin-1 and found significant differences from the archetypal actinoporin fragaceatoxin C. Mytiporin-1 showed weaker permeabilization activity, no specificity towards sphingomyelin, and weak activity in model lipid systems with negatively charged lipids. In contrast to fragaceatoxin C, which forms octameric pores, functional mytiporin-1 pores on negatively charged lipid membranes were hexameric. Similar hexameric pores were observed for coluporin-26 from Cumia reticulata and a conoporin from Conus andremenezi. This indicates that also other molluscan actinoporin-like proteins differ from fragaceatoxin C. Although the functional role of mytiporins in the context of molluscan physiology remains to be elucidated, the lineage-specific gene family expansion event that characterizes mytiporins indicates that strong selective forces acted on their molecular diversification. Given the tissue distribution of mytiporins, this process may have broadened the taxonomic breadth of their biological targets, which would have important implications for digestive processes or mucosal immunity.

Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research.

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

Examination of the Novel Sigma-1 Receptor Antagonist, SI 1/28, for Antinociceptive and Anti-allodynic Efficacy against Multiple Types of Nociception with Fewer Liabilities of Use.

Neuropathic pain is a significant problem with few effective treatments lacking adverse effects. The sigma-1 receptor (S1R) is a potential therapeutic target for neuropathic pain, as antagonists for this receptor effectively ameliorate pain in both preclinical and clinical studies. The current research examines the antinociceptive and anti-allodynic efficacy of SI 1/28, a recently reported benzylpiperazine derivative and analog of the S1R antagonist SI 1/13, that was 423-fold more selective for S1R over the sigma-2 receptor (S2R). In addition, possible liabilities of respiration, sedation, and drug reinforcement caused by SI 1/28 have been evaluated. Inflammatory and chemical nociception, chronic nerve constriction injury (CCI) induced mechanical allodynia, and adverse effects of sedation in a rotarod assay, conditioned place preference (CPP), and changes in breath rate and locomotor activity were assessed after i.p. administration of SI 1/28. Pretreatment with SI 1/28 produced dose-dependent antinociception in the formalin test, with an ED50 (and 95% C.I.) value of 13.2 (7.42-28.3) mg/kg, i.p. Likewise, SI 1/28 produced dose-dependent antinociception against visceral nociception and anti-allodynia against CCI-induced neuropathic pain. SI 1/28 demonstrated no impairment of locomotor activity, conditioned place preference, or respiratory depression. In summary, SI 1/28 proved efficacious in the treatment of acute inflammatory pain and chronic neuropathy without liabilities at therapeutic doses, supporting the development of S1R antagonists as therapeutics for chronic pain.

Mutual Prodrugs of 5-Fluorouracil: From a Classic Chemotherapeutic Agent to Novel Potential Anticancer Drugs.

The development of potent antitumor agents with a low toxicological profile against healthy cells is still one of the greatest challenges facing medicinal chemistry. In this context, the "mutual prodrug" approach has emerged as a potential tool to overcome undesirable physicochemical features and mitigate the side effects of approved drugs. Among broad-spectrum chemotherapeutics available for clinical use today, 5-fluorouracil (5-FU) is one of the most representative, also included in the World Health Organization model list of essential medicines. Unfortunately, severe side effects and drug resistance phenomena are still the primary limits and drawbacks in its clinical use. This review describes the progress made over the last ten years in developing 5-FU-based mutual prodrugs to improve the therapeutic profile and achieve targeted delivery to cancer tissues.

Recent Advances in the Development of Sigma Receptor Ligands as Cytotoxic Agents: A Medicinal Chemistry Perspective.

Since their discovery as distinct receptor proteins, the specific physiopathological role of sigma receptors (σRs) has been deeply investigated. It has been reported that these proteins, classified into two subtypes indicated as σ1 and σ2, might play a pivotal role in cancer growth, cell proliferation, and tumor aggressiveness. As a result, the development of selective σR ligands with potential antitumor properties attracted significant attention as an emerging theme in cancer research. This perspective deals with the recent advances of σR ligands as novel cytotoxic agents, covering articles published between 2010 and 2020. An up-to-date description of the medicinal chemistry of selective σ1R and σ2R ligands with antiproliferative and cytotoxic activities has been provided, including major pharmacophore models and comprehensive structure-activity relationships for each main class of σR ligands.

Development of New Benzylpiperazine Derivatives as σ1 Receptor Ligands with in Vivo Antinociceptive and Anti-Allodynic Effects.

σ-1 receptors (σ1R) modulate nociceptive signaling, driving the search for selective antagonists to take advantage of this promising target to treat pain. In this study, a new series of benzylpiperazinyl derivatives has been designed, synthesized, and characterized for their affinities toward σ1R and selectivity over the σ-2 receptor (σ2R). Notably, 3-cyclohexyl-1-{4-[(4-methoxyphenyl)methyl]piperazin-1-yl}propan-1-one (15) showed the highest σ1R receptor affinity (Ki σ1 = 1.6 nM) among the series with a significant improvement of the σ1R selectivity (Ki σ2/Ki σ1= 886) compared to the lead compound 8 (Ki σ2/Ki σ1= 432). Compound 15 was further tested in a mouse formalin assay of inflammatory pain and chronic nerve constriction injury (CCI) of neuropathic pain, where it produced dose-dependent (3-60 mg/kg, i.p.) antinociception and anti-allodynic effects. Moreover, compound 15 demonstrated no significant effects in a rotarod assay, suggesting that this σ1R antagonist did not produce sedation or impair locomotor responses. Overall, these results encourage the further development of our benzylpiperazine-based σ1R antagonists as potential therapeutics for chronic pain.

The new COST Action European Venom Network (EUVEN)-synergy and future perspectives of modern venomics.

Venom research is a highly multidisciplinary field that involves multiple subfields of biology, informatics, pharmacology, medicine, and other areas. These different research facets are often technologically challenging and pursued by different teams lacking connection with each other. This lack of coordination hampers the full development of venom investigation and applications. The COST Action CA19144-European Venom Network was recently launched to promote synergistic interactions among different stakeholders and foster venom research at the European level.

Becoming a limpet: An 'intermittent limpetization' process driven by host features in the kleptoparasitic gastropod family Capulidae.

A coiled shell is the most evident feature of the typical Bauplan of a gastropod mollusc. However, at least 54 families independently evolved an apparently simplified shell morphology: the limpet. Species with this largely uncoiled, depressed shell morphology occur in almost every aquatic habitat and are associated to a number of different lifestyles and diets. The marine gastropod family Capulidae includes 18 recognised genera, the large majority of which are coiled, but with a number of limpet-like species. Capulid shell plasticity is also associated to a broad range of feeding ecologies, from obligate suspension feeders to kleptoparasites. To investigate the evolution of the limpet-like shell in the family Capulidae we performed an ancestral state reconstruction analysis on a time-calibrated phylogenetic tree (COI, 16S, and ITS2) including 16 species representing a good deal of its morphological diversity. Our results identified at least three capulid lineages that independently evolved limpet-like shells, suggesting that a recurrent limpetization process characterizes this family. One of the limpet-like genera was undescribed and was here named Cryocapulus n. gen. We suggest that capulids evolved from a coiled suspension feeder lineage and that the shift to kleptoparasitism, which occurred in the family ancestor, may have represented a strategy to save energy through the exploitation of the water current produced by the host. Probably the major drivers of shell evolution in capulids are related to their ecology, most of them being kleptoparasites, include the shape and the kind of host substrate, and lead to the repeated acquisition of a limpet-like shape.

In Vitro Antioxidant and Anti-Glycation Activity of Resveratrol and Its Novel Triester with Trolox.

Resveratrol (RSV) is well known for its many beneficial activities, but its unfavorable physicochemical properties impair its effectiveness after systemic and topical administration; thus, several strategies have been investigated to improve RSV efficacy. With this aim, in this work, we synthesized a novel RSV triester with trolox, an analogue of vitamin E with strong antioxidant activity. The new RSV derivative (RSVTR) was assayed in vitro to evaluate its antioxidant and anti-glycation activity compared to RSV. RSVTR chemical stability was assessed at pH 2.0, 6.8, and 7.2 and different storage temperatures (5 °C, 22 °C, and 37 °C). An influence of pH stronger than that of temperature on RSVTR half-life values was pointed out, and RSVTR greatest stability was observed at pH 7.2 and 5 °C. RSVTR showed a lower antioxidant ability compared to RSV (determined by the oxygen radical absorbance capacity assay) while its anti-glycation activity (evaluated using the Maillard reaction) was significantly greater than that of RSV. The improved ability to inhibit the glycation process was attributed to a better interaction of RSVTR with albumin owing to its increased topological polar surface area value and H-bond acceptor number compared to RSV. Therefore, RSVTR could be regarded as a promising anti-glycation agent worthy of further investigations.

Synthesis and Molecular Modelling Studies of New 1,3-Diaryl-5-Oxo-Proline Derivatives as Endothelin Receptor Ligands.

The synthesis of seventeen new 1,3-diaryl-5-oxo-proline derivatives as endothelin receptor (ETR) ligands is described. The structural configuration of the new molecules was determined by analyzing selected signals in proton NMR spectra. In vitro binding assays of the human ETA and ETB receptors allowed us to identify compound 31h as a selective ETAR ligand. The molecular docking of the selected compounds and the ETA antagonist atrasentan in the ETAR homology model provided insight into the structural elements required for the affinity and the selectivity of the ETAR subtype.

Macroevolutionary Analyses Suggest That Environmental Factors, Not Venom Apparatus, Play Key Role in Terebridae Marine Snail Diversification.

How species diversification occurs remains an unanswered question in predatory marine invertebrates, such as sea snails of the family Terebridae. However, the anatomical disparity found throughput the Terebridae provides a unique perspective for investigating diversification patterns in venomous predators. In this study, a new dated molecular phylogeny of the Terebridae is used as a framework for investigating diversification of the family through time, and for testing the putative role of intrinsic and extrinsic traits, such as shell size, larval ecology, bathymetric distribution, and anatomical features of the venom apparatus, as drivers of terebrid species diversification. Macroevolutionary analysis revealed that when diversification rates do not vary across Terebridae clades, the whole family has been increasing its global diversification rate since 25 Ma. We recovered evidence for a concurrent increase in diversification of depth ranges, while shell size appeared to have undergone a fast divergence early in terebrid evolutionary history. Our data also confirm that planktotrophy is the ancestral larval ecology in terebrids, and evolutionary modeling highlighted that shell size is linked to larval ecology of the Terebridae, with species with long-living pelagic larvae tending to be larger and have a broader size range than lecithotrophic species. Although we recovered patterns of size and depth trait diversification through time and across clades, the presence or absence of a venom gland (VG) did not appear to have impacted Terebridae diversification. Terebrids have lost their venom apparatus several times and we confirm that the loss of a VG happened in phylogenetically clustered terminal taxa and that reversal is extremely unlikely. Our findings suggest that environmental factors, and not venom, have had more influence on terebrid evolution.

Venom Diversity and Evolution in the Most Divergent Cone Snail Genus Profundiconus.

Profundiconus is the most divergent cone snail genus and its unique phylogenetic position, sister to the rest of the family Conidae, makes it a key taxon for examining venom evolution and diversity. Venom gland and foot transcriptomes of Profundiconus cf. vaubani and Profundiconusneocaledonicus were de novo assembled, annotated, and analyzed for differential expression. One hundred and thirty-seven venom components were identified from P. cf. vaubani and 82 from P. neocaledonicus, with only four shared by both species. The majority of the transcript diversity was composed of putative peptides, including conotoxins, profunditoxins, turripeptides, insulin, and prohormone-4. However, there were also a significant percentage of other putative venom components such as chymotrypsin and L-rhamnose-binding lectin. The large majority of conotoxins appeared to be from new gene superfamilies, three of which are highly different from previously reported venom peptide toxins. Their low conotoxin diversity and the type of insulin found suggested that these species, for which no ecological information are available, have a worm or molluscan diet associated with a narrow dietary breadth. Our results indicate that Profundiconus venom is highly distinct from that of other cone snails, and therefore important for examining venom evolution in the Conidae family.

[1]Benzothieno[3,2-d]pyrimidine derivatives as ligands for the serotonergic 5-HT7 receptor.

The present paper describes the synthesis and the binding properties for the serotonergic 5-HT7 and 5-HT1A receptors of three new series (A-C) of (benzo)thienopyrimidinone derivatives. All series exhibit a basic moiety at the 2-position of the heterocyclic scaffold such as N,N-dialkylaminoalkylthio chain in series A and phenylpiperazine, benzylpiperazine, or benzylpiperidine alkyl chain in series B and C. Compounds endowed with the best binding properties at 5-HT7R belong to the B and C types. In particular, derivatives B2 and C1 (RSC4) exhibit notable affinity for 5-HT7R (Ki = 9.08 and 0.85 nM, respectively) and selectivity over the 5-HT1AR (254- and 48-fold, respectively). The structure-affinity relationships for these three new classes of 5-HT7R ligands are discussed and, in order to rationalize and deeply investigate the observed results, molecular modeling studies were performed. In particular, the binding poses of the synthesized compounds were studied by docking them in the two receptor proteins suitably built by homology modeling. The calculated binding energies resulted in an excellent agreement with the experimental measured Ki, further validating the quality of the models.

Strategies to Improve Resveratrol Systemic and Topical Bioavailability: An Update.

In recent years, a great deal of attention has been paid to natural compounds due to their many biological effects. Polyphenols are a class of plant derivatives that have been widely investigated for preventing and treating many oxidative stress-related pathological conditions, such as neurodegenerative and cardiovascular diseases, cancer, diabetes mellitus and inflammation. Among these polyphenols, resveratrol (RSV) has attracted considerable interest owing to its high antioxidant and free radical scavenging activities. However, the poor water solubility and rapid metabolism of RSV lead to low bioavailability, thus limiting its clinical efficacy. After discussing the main biochemical mechanisms involved in RSV biological activities, this review will focus on the strategies attempted to improve RSV effectiveness, both for systemic and for topical administration. In particular, technological approaches involving RSV incorporation into different delivery systems such as liposomes, polymeric and lipid nanoparticles, microemulsions and cyclodextrins will be illustrated, highlighting their potential clinical applications. In addition, chemical modifications of this antioxidant aimed at improving its physicochemical properties will be described along with the results of in vitro and in vivo studies.