Streamlining Large Chemical Library Docking with Artificial Intelligence: the PyRMD2Dock Approach.

The present contribution introduces a novel computational protocol called PyRMD2Dock, which combines the Ligand-Based Virtual Screening (LBVS) tool PyRMD with the popular docking software AutoDock-GPU (AD4-GPU) to enhance the throughput of virtual screening campaigns for drug discovery. By implementing PyRMD2Dock, we demonstrate that it is possible to rapidly screen massive chemical databases and identify those with the highest predicted binding affinity to a target protein. Our benchmarking and screening experiments illustrate the predictive power and speed of PyRMD2Dock and highlight its potential to accelerate the discovery of novel drug candidates. Overall, this study showcases the value of combining AI-powered LBVS tools with docking software to enable effective and high-throughput virtual screening of ultralarge molecular databases in drug discovery. PyRMD and the PyRMD2Dock protocol are freely available on GitHub (https://github.com/cosconatilab/PyRMD) as an open-source tool.

Discovering Dually Active Anti-cancer Compounds with a Hybrid AI-structure-based Approach.

Cancer's persistent growth often relies on its ability to maintain telomere length and tolerate the accumulation of DNA damage. This study explores a computational approach to identify compounds that can simultaneously target both G-quadruplex (G4) structures and poly(ADP-ribose) polymerase (PARP)1 enzyme, offering a potential multipronged attack on cancer cells. We employed a hybrid virtual screening (VS) protocol, combining the power of machine learning with traditional structure-based methods. PyRMD, our AI-powered tool, was first used to analyze vast chemical libraries and to identify potential PARP1 inhibitors based on known bioactivity data. Subsequently, a structure-based VS approach selected compounds from these identified inhibitors for their G4 stabilization potential. This two-step process yielded 50 promising candidates, which were then experimentally validated for their ability to inhibit PARP1 and stabilize G4 structures. Ultimately, four lead compounds emerged as promising candidates with the desired dual activity and demonstrated antiproliferative effects against specific cancer cell lines. This study highlights the potential of combining Artificial Intelligence and structure-based methods for the discovery of multitarget anticancer compounds, offering a valuable approach for future drug development efforts.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

A novel smaller ß-defensin-derived peptide is active against multidrug-resistant bacterial strains.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human ß-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the ß-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human ß-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10-SPR19) as potential anti-HAT agents. The new molecules show Ki values in the low-micro/sub-micromolar range against rhodesain, coupled with k2nd values between 1314 and 6950 M-1 min-1. With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 µM towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agents.

PyRMD: A New Fully Automated AI-Powered Ligand-Based Virtual Screening Tool.

Artificial intelligence (AI) algorithms are dramatically redefining the current drug discovery landscape by boosting the efficiency of its various steps. Still, their implementation often requires a certain level of expertise in AI paradigms and coding. This often prevents the use of these powerful methodologies by non-expert users involved in the design of new biologically active compounds. Here, the random matrix discriminant (RMD) algorithm, a high-performance AI method specifically tailored for the identification of new ligands, was implemented in a new fully automated tool, PyRMD. This ligand-based virtual screening tool can be trained using target bioactivity data directly downloaded from the ChEMBL repository without manual intervention. The software automatically splits the available training compounds into active and inactive sets and learns the distinctive chemical features responsible for the compounds' activity/inactivity. PyRMD was designed to easily screen millions of compounds in hours through an automated workflow and intuitive input files, allowing fine tuning of each parameter of the calculation. Additionally, PyRMD features a wealth of benchmark metrics, to accurately probe the model performance, which were used here to gauge its predictive potential and limitations. PyRMD is freely available on GitHub (https://github.com/cosconatilab/PyRMD) as an open-source tool.

FEPrepare: A Web-Based Tool for Automating the Setup of Relative Binding Free Energy Calculations.

Relative binding free energy calculations in drug design are becoming a useful tool in facilitating lead binding affinity optimization in a cost- and time-efficient manner. However, they have been limited by technical challenges such as the manual creation of large numbers of input files to set up, run, and analyze free energy simulations. In this Application Note, we describe FEPrepare, a novel web-based tool, which automates the setup procedure for relative binding FEP calculations for the dual-topology scheme of NAMD, one of the major MD engines, using OPLS-AA force field topology and parameter files. FEPrepare provides the user with all necessary files needed to run a FEP/MD simulation with NAMD. FEPrepare can be accessed and used at https://feprepare.vi-seem.eu/.

Lead Discovery of SARS-CoV-2 Main Protease Inhibitors through Covalent Docking-Based Virtual Screening.

During almost all 2020, coronavirus disease 2019 (COVID-19) pandemic has constituted the major risk for the worldwide health and economy, propelling unprecedented efforts to discover drugs for its prevention and cure. At the end of the year, these efforts have culminated with the approval of vaccines by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA) giving new hope for the future. On the other hand, clinical data underscore the urgent need for effective drugs to treat COVID-19 patients. In this work, we embarked on a virtual screening campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro chymotrypsin-like cysteine protease employing our in-house database of peptide and non-peptide ligands characterized by different types of warheads acting as Michael acceptors. To this end, we employed the AutoDock4 docking software customized to predict the formation of a covalent adduct with the target protein. In vitro verification of the inhibition properties of the most promising candidates allowed us to identify two new lead inhibitors that will deserve further optimization. From the computational point of view, this work demonstrates the predictive power of AutoDock4 and suggests its application for the in silico screening of large chemical libraries of potential covalent binders against the SARS-CoV-2 Mpro enzyme.

Tetrahydroquinazole-based secondary sulphonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation against isoforms I, II, IV, and IX, and computational studies.

A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquinazole scaffold was synthesised and biologically evaluated for their inhibitory activity against human carbonic anhydrase (hCA) I, II, IV, and IX. Of note, several compounds were identified showing submicromolar potency and excellent selectivity for the tumour-related hCA IX isoform. Structure-activity relationship data attained for various substitutions were rationalised by molecular modelling studies in terms of both inhibitory activity and selectivity.

Carbonic anhydrase activation profile of indole-based derivatives.

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.

Disulfide Bond Replacement with 1,4- and 1,5-Disubstituted [1,2,3]-Triazole on C-X-C Chemokine Receptor Type 4 (CXCR4) Peptide Ligands: Small Changes that Make Big Differences.

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.

Identification of novel indole derivatives acting as inhibitors of the Keap1-Nrf2 interaction.

Nine indole derivatives (9a-i) were tested as potential inhibitors of the Keap1-Nrf2 interaction. This class of compounds increases the intracellular levels of the transcription factor Nrf2 and the consequent expression of enzymes encoded by genes containing the antioxidant response element (ARE). In the ARE-luciferase reporter assay only 9e-g revealed to be remarkably more active than t-butylhydroxyquinone (t-BHQ), with 9g standing out as the best performing compound. While 9e and 9f are weak acids, 9g is an ampholyte prevailing as a zwitterion in neutral aqueous solutions. The ability of 9e-g to significantly increase levels of Nrf2, NADPH:quinone oxidoreductase 1, and transketolase (TKT) gave further support to the hypothesis that these compounds act as inhibitors of the Keap1-Nrf2 interaction. Docking simulations allowed us to elucidate the nature of the putative interactions between 9g and Keap1.

Novel 2-substituted-benzimidazole-6-sulfonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation against isoforms I, II, IX and XII and molecular docking studies.

Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors (CAIs) with a proper selectivity for certain isoforms, a series of derivatives featuring the 2-substituted-benzimidazole-6-sulfonamide scaffold, conceived as frozen analogs of Schiff bases and secondary amines previously reported in the literature as CAIs, were investigated. Enzyme inhibition assays on physiologically relevant human CA I, II, IX and XII isoforms revealed a number of potent CAIs, showing promising selectivity profiles towards the transmembrane tumor-associated CA IX and XII enzymes. Computational studies were attained to clarify the structural determinants behind the activities and selectivity profiles of the novel inhibitors.

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers.

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.

Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Tandem application of ligand-based virtual screening and G4-OAS assay to identify novel G-quadruplex-targeting chemotypes.

BACKGROUND: G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. METHODS: A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MTT assays. RESULTS: A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4-forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells. CONCLUSIONS: The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands. GENERAL SIGNIFICANCE: Expanding the repertoire of the available G4-targeting chemotypes with improved physico-chemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Discovery of the first dual G-triplex/G-quadruplex stabilizing compound: a new opportunity in the targeting of G-rich DNA structures?

BACKGROUND: Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. METHODS: Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. RESULTS: The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. CONCLUSIONS: A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. GENERAL SIGNIFICANCE: The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.

Development of novel 1,4-benzodiazepine-based Michael acceptors as antitrypanosomal agents.

Novel 1,4-benzodiazepines, endowed with a Michael acceptor moiety, were designed taking advantage of a computational prediction of their pharmacokinetic parameters. Among all the synthesized derivatives, we identified a new lead compound (i.e., 4a), bearing a vinyl ketone warhead and endowed with a promising antitrypanosomal activity against Trypanosoma brucei brucei (IC50=5.29µM), coupled with a lack of cytotoxicity towards mammalian cells (TC50 >100µM).