AMALPHI: A Machine Learning Platform for Predicting Drug-Induced PhospholIpidosis.

Drug-induced phospholipidosis (PLD) involves the accumulation of phospholipids in cells of multiple tissues, particularly within lysosomes, and it is associated with prolonged exposure to druglike compounds, predominantly cationic amphiphilic drugs (CADs). PLD affects a significant portion of drugs currently in development and has recently been proven to be responsible for confounding antiviral data during drug repurposing for SARS-CoV-2. In these scenarios, it has become crucial to identify potential safe drug candidates in advance and distinguish them from those that may lead to false in vitro antiviral activity. In this work, we developed a series of machine learning classifiers with the aim of predicting the PLD-inducing potential of drug candidates. The models were built on a high-quality chemical collection comprising 545 curated small molecules extracted from ChEMBL v30. The most effective model, obtained using the balanced random forest algorithm, achieved high performance, including an AUC value computed in validation as high as 0.90. The model was made freely available through a user-friendly web platform named AMALPHI (https://www.ba.ic.cnr.it/softwareic/amalphiportal/), which can represent a valuable tool for medicinal chemists interested in conducting an early evaluation of PLD inducer potential.

Study of metalation of thioredoxin by gold(I) therapeutic compounds using combined liquid chromatography/capillary electrophoresis with inductively coupled plasma/electrospray MS/MS detection.

The reactivity of thioredoxin (Trx1) with the Au(I) drug auranofin (AF) and two therapeutic N-heterocyclic carbene (NHC)2-Au(I) complexes (bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) and [1,3-diethyl-4,5-bis(4methoxyphenyl)imidazol-2-ylidene]gold(I) (Au4BC)) was investigated. Direct infusion (DI) electrospray ionization (ESI) mass spectrometry (MS) allowed information on the structure, stoichiometry, and kinetics of formation of Trx-Au adducts. The fragmentation of the formed adducts in the gas phase gave insights into the exact Au binding site within the protein, demonstrating the preference for Trx1 Cys32 or Cys35 of AF or the (NHC)2-Au(I) complex Au3BC, respectively. Reversed-phase HPLC suffered from the difficulty of elution of gold compounds, did not preserve the formed metal-protein adducts, and favored the loss of ligands (phosphine or NHC) from Au(I). These limitations were eliminated by capillary electrophoresis (CE) which enabled the separation of the gold compounds, Trx1, and the formed adducts. The ICP-MS/MS detection allowed the simultaneous quantitative monitoring of the gold and sulfur isotopes and the determination of the metallation extent of the protein. The hyphenation of the mentioned techniques was used for the analysis of Trx1-Au adducts for the first time.

Biophysical and Structural Characterization of the Interaction between Human Galectin-3 and the Lipopolysaccharide from Pseudomonas aeruginosa.

Given the significant involvement of galectins in the development of numerous diseases, the aim of the following work is to further study the interaction between galectin-3 (Gal3) and the LPS from Pseudomonas aeruginosa. This manuscript focused on the study of the interaction of the carbohydrate recognition domain of Gal3 with the LPS from Pseudomonas aeruginosa by means of different complementary methodologies, such as circular dichroism; spectrofluorimetry; dynamic and static light scattering and evaluation of the impact of Gal3 on the redox potential membranes of Escherichia coli and P. aeruginosa cells, as well as ITC and NMR studies. This thorough investigation reinforces the hypothesis of an interaction between Gal3 and LPS, unraveling the structural details and providing valuable insights into the formation of these intricate molecular complexes. Taken together, these achievements could potentially prompt the design of therapeutic drugs useful for the development of agonists and/or antagonists for LPS receptors such as galectins as adjunctive therapy for P. aeruginosa.

Disclosing the Preferential Mercury Chelation by SeCys Containing Peptides over Their Cys Analogues.

Methylmercury, mercury (II), and mercury (I) chlorides were found to react with vasopressin, a nonapeptide hormone cyclized by two cysteine residues, and its mono- and diselenium analogues to form several mercury-peptide adducts. The replacement of Cys by SeCys in vasopressin increased the reactivity toward methylmercury, with the predominant formation of -Se/S-Hg-Se-bridged structures and the consequent demethylation of methylmercury. In competitive experiments, CH3HgCl reacted preferentially with the diselenium analogue rather than with vasopressin. The diselenium peptide also showed the capability to displace the CH3Hg moiety bound to S in vasopressin. These results open a promising perspective for the use of selenopeptides for methylmercury chelation and detoxification strategies.

GENERA: A Combined Genetic/Deep-Learning Algorithm for Multiobjective Target-Oriented De Novo Design.

This study introduces a new de novo design algorithm called GENERA that combines the capabilities of a deep-learning algorithm for automated drug-like analogue design, called DeLA-Drug, with a genetic algorithm for generating molecules with desired target-oriented properties. Specifically, GENERA was applied to the angiotensin-converting enzyme 2 (ACE2) target, which is implicated in many pathological conditions, including COVID-19. The ability of GENERA to de novo design promising candidates for a specific target was assessed using two docking programs, PLANTS and GLIDE. A fitness function based on the Pareto dominance resulting from computed PLANTS and GLIDE scores was applied to demonstrate the algorithm's ability to perform multiobjective optimizations effectively. GENERA can quickly generate focused libraries that produce better scores compared to a starting set of known ACE-2 binders. This study is the first to utilize a DL-based algorithm designed for analogue generation as a mutational operator within a GA framework, representing an innovative approach to target-oriented de novo design.

Peptoids: Smart and Emerging Candidates for the Diagnosis of Cancer, Neurological and Autoimmune Disorders.

Early detection of fatal and disabling diseases such as cancer, neurological and autoimmune dysfunctions is still desirable yet challenging to improve quality of life and longevity. Peptoids (N-substituted glycine oligomers) are a relatively new class of peptidomimetics, being highly versatile and capable of mimicking the architectures and the activities of the peptides but with a marked resistance to proteases and a propensity to cross the cellular membranes over the peptides themselves. For these properties, they have gained an ever greater interest in applications in bioengineering and biomedical fields. In particular, the present manuscript is to our knowledge the only review focused on peptoids for diagnostic applications and covers the last decade's literature regarding peptoids as tools for early diagnosis of pathologies with a great impact on human health and social behavior. The review indeed provides insights into the peptoid employment in targeted cancer imaging and blood-based screening of neurological and autoimmune diseases, and it aims to attract the scientific community's attention to continuing and sustaining the investigation of these peptidomimetics in the diagnosis field considering their promising peculiarities.

Advances in Nucleic Acid Research: Exploring the Potential of Oligonucleotides for Therapeutic Applications and Biological Studies.

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.

The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

DeLA-Drug: A Deep Learning Algorithm for Automated Design of Druglike Analogues.

In this paper, we present a deep learning algorithm for automated design of druglike analogues (DeLA-Drug), a recurrent neural network (RNN) model composed of two long short-term memory (LSTM) layers and conceived for data-driven generation of similar-to-bioactive compounds. DeLA-Drug captures the syntax of SMILES strings of more than 1 million compounds belonging to the ChEMBL28 database and, by employing a new strategy called sampling with substitutions (SWS), generates molecules starting from a single user-defined query compound. Remarkably, the algorithm preserves druglikeness and synthetic accessibility of the known bioactive compounds present in the ChEMBL28 repository. The absence of any time-demanding fine-tuning procedure enables DeLA-Drug to perform a fast generation of focused libraries for further high-throughput screening and makes it a suitable tool for performing de novo design even in low-data regimes. To provide a concrete idea of its applicability, DeLA-Drug was applied to the cannabinoid receptor subtype 2 (CB2R), a known target involved in different pathological conditions such as cancer and neurodegeneration. DeLA-Drug, available as a free web platform (http://www.ba.ic.cnr.it/softwareic/deladrugportal/), can help medicinal chemists interested in generating analogues of compounds already available in their laboratories and, for this reason, good candidates for an easy and low-cost synthesis.

A Comparative Molecular Dynamics Study of Selected Point Mutations in the Shwachman-Bodian-Diamond Syndrome Protein SBDS.

The Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease whose majority of patients display mutations in a ribosome assembly protein named Shwachman-Bodian-Diamond Syndrome protein (SBDS). A specific therapy for treating this rare disease is missing, due to the lack of knowledge of the molecular mechanisms responsible for its pathogenesis. Starting from the observation that SBDS single-point mutations, localized in different domains of the proteins, are responsible for an SDS phenotype, we carried out the first comparative Molecular Dynamics simulations on three SBDS mutants, namely R19Q, R126T and I212T. The obtained 450-ns long trajectories were compared with those returned by both the open and closed forms of wild type SBDS and strongly indicated that two distinct conformations (open and closed) are both necessary for the proper SBDS function, in full agreement with recent experimental observations. Our study supports the hypothesis that the SBDS function is governed by an allosteric mechanism involving domains I and III and provides new insights into SDS pathogenesis, thus offering a possible starting point for a specific therapeutic option.

Design, Synthesis, and Anticancer Activity of a Selenium-Containing Galectin-3 and Galectin-9N Inhibitor.

Galectins are soluble ß-D-galactoside-binding proteins whose implication in cancer progression and disease outcome makes them prominent targets for therapeutic intervention. In this frame, the development of small inhibitors that block selectively the activity of galectins represents an important strategy for cancer therapy which is, however, still relatively underdeveloped. To this end, we designed here a rationally and efficiently novel diglycosylated compound, characterized by a selenoglycoside bond and the presence of a lipophilic benzyl group at both saccharide residues. The relatively high binding affinity of the new compound to the carbohydrate recognition domain of two galectins, galectin 3 and galectin 9, its good antiproliferative and anti-migration activity towards melanoma cells, as well as its anti-angiogenesis properties, pave the way for its further development as an anticancer agent.

Exploring the Molecular Interactions of Symmetrical and Unsymmetrical Selenoglycosides with Human Galectin-1 and Galectin-3.

Galectins (Gals) are small cytosolic proteins that bind ß-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors.

High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations.

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvß3 without cross reacting with αvß5 and αIIbß3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvß3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions.

New Advances in Short Peptides: Looking Forward.

It is beyond doubt that short peptides hold significant promise in bio-medicine, as the most versatile molecules, both structurally and functionally [...].

Diphenylalanine Motif Drives Self-Assembling in Hybrid PNA-Peptide Conjugates.

Peptides and nucleic acids can self-assemble to give supramolecular structures that find application in different fields, ranging from the delivery of drugs to the obtainment of materials endowed with optical properties. Forces that stabilize the "suprastructures" typically are hydrogen bonds or aromatic interactions; in case of nucleic acids, Watson-Crick pairing drives self-assembly while, in case of peptides, backbone hydrogen bonds and interactions between aromatic side chains trigger the formation of structures, such as nanotubes or ribbons. Molecules containing both aromatic peptides and nucleic acids could in principle exploit different forces to self-assemble. In this work we meant to investigate the self-assembly of mixed systems, with the aim to understand which forces play a major role and determine formation/structure of aggregates. We therefore synthesized conjugates of the peptide FF to the peptide nucleic acid dimer "gc" and characterized their aggregates by different spectroscopic techniques, including NMR, CD and fluorescence.

Structure-Based Prediction of hERG-Related Cardiotoxicity: A Benchmark Study.

Drug-induced blockade of the human ether-à-go-go-related gene (hERG) channel is today considered the main cause of cardiotoxicity in postmarketing surveillance. Hence, several ligand-based approaches were developed in the last years and are currently employed in the early stages of a drug discovery process for in silico cardiac safety assessment of drug candidates. Herein, we present the first structure-based classifiers able to discern hERG binders from nonbinders. LASSO regularized support vector machines were applied to integrate docking scores and protein-ligand interaction fingerprints. A total of 396 models were trained and validated based on: (i) high-quality experimental bioactivity information returned by 8337 curated compounds extracted from ChEMBL (version 25) and (ii) structural predictor data. Molecular docking simulations were performed using GLIDE and GOLD software programs and four different hERG structural models, namely, the recently published structures obtained by cryoelectron microscopy (PDB codes: 5VA1 and 7CN1) and two published homology models selected for comparison. Interestingly, some classifiers return performances comparable to ligand-based models in terms of area under the ROC curve (AUCMAX = 0.86 ± 0.01) and negative predictive values (NPVMAX = 0.81 ± 0.01), thus putting forward the herein proposed computational workflow as a valuable tool for predicting hERG-related cardiotoxicity without the limitations of ligand-based models, typically affected by low interpretability and a limited applicability domain. From a methodological point of view, our study represents the first example of a successful integration of docking scores and protein-ligand interaction fingerprints (IFs) through a support vector machine (SVM) LASSO regularized strategy. Finally, the study highlights the importance of using hERG structural models accounting for ligand-induced fit effects and allowed us to select the best-performing protein conformation (made available in the Supporting Information, SI) to be employed for a reliable structure-based prediction of hERG-related cardiotoxicity.

More Is Always Better Than One: The N-Terminal Domain of the Spike Protein as Another Emerging Target for Hampering the SARS-CoV-2 Attachment to Host Cells.

Although the approved vaccines are proving to be of utmost importance in containing the Coronavirus disease 2019 (COVID-19) threat, they will hardly be resolutive as new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, a single-stranded RNA virus) variants might be insensitive to the immune response they induce. In this scenario, developing an effective therapy is still a dire need. Different targets for therapeutic antibodies and diagnostics have been identified, among which the SARS-CoV-2 spike (S) glycoprotein, particularly its receptor-binding domain, has been defined as crucial. In this context, we aim to focus attention also on the role played by the S N-terminal domain (S1-NTD) in the virus attachment, already recognized as a valuable target for neutralizing antibodies, in particular, building on a cavity mapping indicating the presence of two druggable pockets and on the recent literature hypothesizing the presence of a ganglioside-binding domain. In this perspective, we aim at proposing S1-NTD as a putative target for designing small molecules hopefully able to hamper the SARS-CoV-2 attachment to host cells.

Silver (I) N-Heterocyclic Carbene Complexes: A Winning and Broad Spectrum of Antimicrobial Properties.

The evolution of antibacterial resistance has arisen as the main downside in fighting bacterial infections pushing researchers to develop novel, more potent and multimodal alternative drugs.Silver and its complexes have long been used as antimicrobial agents in medicine due to the lack of silver resistance and the effectiveness at low concentration as well as to their low toxicities compared to the most commonly used antibiotics. N-Heterocyclic Carbenes (NHCs) have been extensively employed to coordinate transition metals mainly for catalytic chemistry. However, more recently, NHC ligands have been applied as carrier molecules for metals in anticancer applications. In the present study we selected from literature two NHC-carbene based on acridinescaffoldand detailed nonclassicalpyrazole derived mono NHC-Ag neutral and bis NHC-Ag cationic complexes. Their inhibitor effect on bacterial strains Gram-negative and positivewas evaluated. Imidazolium NHC silver complex containing the acridine chromophore showed effectiveness at extremely low MIC values. Although pyrazole NHC silver complexes are less active than the acridine NHC-silver, they represent the first example of this class of compounds with antimicrobial properties. Moreover all complexesare not toxic and they show not significant activity againstmammalian cells (Hek lines) after 4 and 24 h. Based on our experimental evidence, we are confident that this promising class of complexes could represent a valuable starting point for developing candidates for the treatment of bacterial infections, delivering great effectiveness and avoiding the development of resistance mechanisms.

PHA-680626 Is an Effective Inhibitor of the Interaction between Aurora-A and N-Myc.

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

Peptide-Protein Interactions: From Drug Design to Supramolecular Biomaterials.

The self-recognition and self-assembly of biomolecules are spontaneous processes that occur in Nature and allow the formation of ordered structures, at the nanoscale or even at the macroscale, under thermodynamic and kinetic equilibrium as a consequence of specific and local interactions. In particular, peptides and peptidomimetics play an elected role, as they may allow a rational approach to elucidate biological mechanisms to develop new drugs, biomaterials, catalysts, or semiconductors. The forces that rule self-recognition and self-assembly processes are weak interactions, such as hydrogen bonding, electrostatic attractions, and van der Waals forces, and they underlie the formation of the secondary structure (e.g., α-helix, ß-sheet, polyproline II helix), which plays a key role in all biological processes. Here, we present recent and significant examples whereby design was successfully applied to attain the desired structural motifs toward function. These studies are important to understand the main interactions ruling the biological processes and the onset of many pathologies. The types of secondary structure adopted by peptides during self-assembly have a fundamental importance not only on the type of nano- or macro-structure formed but also on the properties of biomaterials, such as the types of interaction, encapsulation, non-covalent interaction, or covalent interaction, which are ultimately useful for applications in drug delivery.