Fighting Antimicrobial Resistance: Insights on How the Staphylococcus aureus NorA Efflux Pump Recognizes 2-Phenylquinoline Inhibitors by Supervised Molecular Dynamics (SuMD) and Molecular Docking Simulations.

The superbug Staphylococcus aureus (S. aureus) exhibits several resistance mechanisms, including efflux pumps, that strongly contribute to antimicrobial resistance. In particular, the NorA efflux pump activity is associated with S. aureus resistance to fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from cells. Thus, since efflux pump inhibitors (EPIs) are able to increase antibiotic concentrations in bacteria as well as restore their susceptibility to these agents, they represent a promising strategy to counteract bacterial resistance. Additionally, the very recent release of two NorA efflux pump cryo-electron microscopy (cryo-EM) structures in complex with synthetic antigen-binding fragments (Fabs) represents a real breakthrough in the study of S. aureus antibiotic resistance. In this scenario, supervised molecular dynamics (SuMD) and molecular docking experiments were combined to investigate for the first time the molecular mechanisms driving the interaction between NorA and efflux pump inhibitors (EPIs), with the ultimate goal of elucidating how the NorA efflux pump recognizes its inhibitors. The findings provide insights into the dynamic NorA-EPI intermolecular interactions and lay the groundwork for future drug discovery efforts aimed at the identification of novel molecules to fight antimicrobial resistance.

Identification of Anti-Influenza A Compounds Inhibiting the Viral Non-Structural Protein 1 (NS1) Using a Type I Interferon-Driven Screening Strategy.

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-ß expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.

Q-raKtion: A Semiautomated KNIME Workflow for Bioactivity Data Points Curation.

The recent increase of bioactivity data freely available to the scientific community and stored as activity data points in chemogenomic repositories provides a huge amount of ready-to-use information to support the development of predictive models. However, the benefits provided by the availability of such a vast amount of accessible information are strongly counteracted by the lack of uniformity and consistency of data from multiple sources, requiring a process of integration and harmonization. While different automated pipelines for processing and assessing chemical data have emerged in the last years, the curation of bioactivity data points is a less investigated topic, with useful concepts provided but no tangible tools available. In this context, the present work represents a first step toward the filling of this gap, by providing a tool to meet the needs of end-user in building proprietary high-quality data sets for further studies. Specifically, we herein describe Q-raKtion, a systematic, semiautomated, flexible, and, above all, customizable KNIME workflow that effectively aggregates information on biological activities of compounds retrieved by two of the most comprehensive and widely used repositories, PubChem and ChEMBL.

Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus NorA inhibitors.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1.

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

1,2,4-Triazolo[1,5-a]pyrimidines as a Novel Class of Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Activity.

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors.

Deciphering the Molecular Recognition Mechanism of Multidrug Resistance Staphylococcus aureus NorA Efflux Pump Using a Supervised Molecular Dynamics Approach.

The use and misuse of antibiotics has resulted in critical conditions for drug-resistant bacteria emergency, accelerating the development of antimicrobial resistance (AMR). In this context, the co-administration of an antibiotic with a compound able to restore sufficient antibacterial activity may be a successful strategy. In particular, the identification of efflux pump inhibitors (EPIs) holds promise for new antibiotic resistance breakers (ARBs). Indeed, bacterial efflux pumps have a key role in AMR development; for instance, NorA efflux pump contributes to Staphylococcus aureus (S. aureus) resistance against fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from the cells. Even though NorA efflux pump is known to be a potential target for EPIs development, the absence of structural information about this protein and the little knowledge available on its mechanism of action have strongly hampered rational drug discovery efforts in this area. In the present work, we investigated at the molecular level the substrate recognition pathway of NorA through a Supervised Molecular Dynamics (SuMD) approach, using a NorA homology model. Specific amino acids were identified as playing a key role in the efflux pump-mediated extrusion of its substrate, paving the way for a deeper understanding of both the mechanisms of action and the inhibition of such efflux pumps.

p38α MAPK and Type I Inhibitors: Binding Site Analysis and Use of Target Ensembles in Virtual Screening.

Mitogen-activated protein kinase p38α plays an essential role in the regulation of pro-inflammatory signaling, and selective blockade of this kinase could be efficacious in many pathological processes. Despite considerable research efforts focused on the discovery and development of p38α MAPK inhibitors, no drug targeting this protein has been approved for clinical use so far. We herein analyze the available crystal structures of p38α MAPK in complex with ATP competitive type I inhibitors, getting insights into ATP binding site conformation and its influence on automated molecular docking results. The use of target ensembles, rather than single conformations, resulted in a performance improvement in both the ability to reproduce experimental bound conformations and the capability of mining active molecules from compound libraries. The information gathered from this study can be exploited in structure-based drug discovery programs having as the ultimate aim the identification of novel p38α MAPK type I inhibitors.

Small Molecule Drugs Targeting Viral Polymerases.

Small molecules that specifically target viral polymerases-crucial enzymes governing viral genome transcription and replication-play a pivotal role in combating viral infections. Presently, approved polymerase inhibitors cover nine human viruses, spanning both DNA and RNA viruses. This review provides a comprehensive analysis of these licensed drugs, encompassing nucleoside/nucleotide inhibitors (NIs), non-nucleoside inhibitors (NNIs), and mutagenic agents. For each compound, we describe the specific targeted virus and related polymerase enzyme, the mechanism of action, and the relevant bioactivity data. This wealth of information serves as a valuable resource for researchers actively engaged in antiviral drug discovery efforts, offering a complete overview of established strategies as well as insights for shaping the development of next-generation antiviral therapeutics.

Equisetum arvense standardized dried extract hinders age-related osteosarcopenia.

Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ's anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.

Computer-Aided Identification of Kinase-Targeted Small Molecules for Cancer: A Review on AKT Protein.

AKT (also known as PKB) is a serine/threonine kinase that plays a pivotal regulatory role in the PI3K/AKT/mTOR signaling pathway. Dysregulation of AKT activity, especially its hyperactivation, is closely associated with the development of various human cancers and resistance to chemotherapy. Over the years, a wide array of AKT inhibitors has been discovered through experimental and computational approaches. In this regard, herein we present a comprehensive overview of AKT inhibitors identified using computer-assisted drug design methodologies (including docking-based and pharmacophore-based virtual screening, machine learning, and quantitative structure-activity relationships) and successfully validated small molecules endowed with anticancer activity. Thus, this review provides valuable insights to support scientists focused on AKT inhibition for cancer treatment and suggests untapped directions for future computer-aided drug discovery efforts.

Small Molecules Targeting DNA Polymerase Theta (POLθ) as Promising Synthetic Lethal Agents for Precision Cancer Therapy.

Synthetic lethality (SL) is an innovative strategy in targeted anticancer therapy that exploits tumor genetic vulnerabilities. This topic has come to the forefront in recent years, as witnessed by the increased number of publications since 2007. The first proof of concept for the effectiveness of SL was provided by the approval of poly(ADP-ribose)polymerase inhibitors, which exploit a SL interaction in BRCA-deficient cells, although their use is limited by resistance. Searching for additional SL interactions involving BRCA mutations, the DNA polymerase theta (POLθ) emerged as an exciting target. This review summarizes, for the first time, the POLθ polymerase and helicase inhibitors reported to date. Compounds are described focusing on chemical structure and biological activity. With the aim to enable further drug discovery efforts in interrogating POLθ as a target, we propose a plausible pharmacophore model for POLθ-pol inhibitors and provide a structural analysis of the known POLθ ligand binding sites.

New C-6 functionalized quinoline NorA inhibitors strongly synergize with ciprofloxacin against planktonic and biofilm growing resistant Staphylococcus aureus strains.

Antimicrobial resistance (AMR) represents a global health issue threatening our social lifestyle and the world economy. Efflux pumps are widely involved in AMR by playing a primary role in the development of specific mechanisms of resistance. In addition, they seem to be involved in the process of biofilm formation and maintenance, contributing to enhance the risk of creating superbugs difficult to treat. Accordingly, the identification of non-antibiotic molecules able to block efflux pumps, namely efflux pump inhibitors (EPIs), could be a promising strategy to counteract AMR and restore the antimicrobial activity of ineffective antibiotics. Herein, we enlarge the knowledge about the structure-activity relationship of 2-phenylquinoline Staphylococcus aureus NorA EPIs by reporting a new series of very potent C-6 functionalized derivatives. Best compounds significantly inhibited ethidium bromide efflux in a NorA-overexpressing S. aureus strain (SA-1199B) and strongly synergized at very low concentrations (0.20-0.78 µg/mL) with ciprofloxacin (CPX) against CPX-resistant S. aureus strains (SA-1199B and SA-K2378), as proved by checkerboard and time-kill experiments. In addition, some of these EPIs (9b and 10a) produced a post-antibiotic effect of 1.2 h and strongly enhanced antibiofilm activity of CPX against SA-1199B strain. Interestingly, at the concentrations used to reach synergy with CPX against resistant S. aureus strains, most of the EPI compounds did not show any human cell toxicity. Finally, by exploiting the recent released crystal structure of NorA, we observed that best EPI 9b highlighted a favourable docking pose, establishing some interesting interactions with key residues.

From Serendipity to Rational Identification of the 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one Core as a New Chemotype of AKT1 Inhibitors for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy whose prognosis is globally poor. In more than 60% of AML patients, the PI3K/AKTs/mTOR signaling pathway is aberrantly activated because of oncogenic driver alterations and further enhanced by chemotherapy as a mechanism of drug resistance. Against this backdrop, very recently we have started a multidisciplinary research project focused on AKT1 as a pharmacological target to identify novel anti-AML agents. Indeed, the serendipitous finding of the in-house compound T187 as an AKT1 inhibitor has paved the way to the rational identification of new active small molecules, among which T126 has emerged as the most interesting compound with IC50 = 1.99 ± 0.11 µM, ligand efficiency of 0.35, and a clear effect at low micromolar concentrations on growth inhibition and induction of apoptosis in AML cells. The collected results together with preliminary SAR data strongly indicate that the 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one derivative T126 is worthy of future biological experiments and medicinal chemistry efforts aimed at developing a novel chemical class of AKT1 inhibitors as anti-AML agents.

From Quinoline to Quinazoline-Based S. aureus NorA Efflux Pump Inhibitors by Coupling a Focused Scaffold Hopping Approach and a Pharmacophore Search.

Antibiotic resistance breakers, such as efflux pump inhibitors (EPIs), represent a powerful alternative to the development of new antimicrobials. Recently, by using previously described EPIs, we developed pharmacophore models able to identify inhibitors of NorA, the most studied efflux pump of Staphylococcus aureus. Herein we report the pharmacophore-based virtual screening of a library of new potential NorA EPIs generated by an in-silico scaffold hopping approach of the quinoline core. After chemical synthesis and biological evaluation of the best virtual hits, we found the quinazoline core as the best performing scaffold. Accordingly, we designed and synthesized a series of functionalized 2-arylquinazolines, which were further evaluated as NorA EPIs. Four of them exhibited a strong synergism with ciprofloxacin and a good inhibition of ethidium bromide efflux on resistant S. aureus strains coupled with low cytotoxicity against human cell lines, thus highlighting a promising safety profile.

Pharmacological inactivation of the prion protein by targeting a folding intermediate.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

The Compelling Demand for an Effective PrPC-Directed Therapy against Prion Diseases.

Decades of research efforts have conclusively provided overwhelming evidence that the cellular prion protein (PrPC) plays a central role in prion diseases, a set of fatal and incurable neurodegenerative disorders for which no therapy is yet available. In this Viewpoint, we provide an overview of the drug discovery strategies in the field, highlighting the current therapeutic hypotheses targeting, whether directly or indirectly, PrPC as well as the antiprion agents closest to clinical application.

New Insights on KCa3.1 Channel Modulation.

The human intermediate conductance calcium-activated potassium channel, KCa3.1, is involved in several pathophysiological conditions playing a critical role in cell secretory machinery and calcium signalling. The recent cryo-EM analysis provides new insights for understanding the modulation by both endogenous and pharmacological agents. A typical feature of this channel is the low open probability in saturating calcium concentrations and its modulation by potassium channel openers (KCOs), such as benzo imidazolone 1-EBIO, without changing calcium-dependent activation. In this paper, we proposed a model of KCOs action in the modulation of channel activity. The KCa3.1 channel has a very rich pharmacological profile with several classes of molecules that selectively interact with different binding sites of the channel. Among them, benzo imidazolones can be openers (positive modulators such as 1-EBIO, DC-EBIO) or blockers (negative modulators such as NS1619). Through computation modelling techniques, we identified the 1,4-benzothiazin-3-one as a promising scaffold to develop new KCa3.1 channel modulators. Further studies are needed to explore the potential use of 1-4 benzothiazine- 3-one in KCa3.1 modulation and its pharmacological application.

Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase.

Treatment of dengue virus (DENV) and other flavivirus infections is an unmet medical need. The highly conserved flaviviral NS5 RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target that interacts with NS3 and viral RNA within the replication complex assembly. Biochemical and cell-based evidence indicate that targeting cavity B may lead to dual RdRp and NS5-NS3 interaction inhibitors. By ligand-based design around 1H-pyrido[2,1-b][1,3]benzothiazol-1-one (PBTZ) 1, we identified new potent and selective DENV inhibitors that exert dual inhibition of NS5 RdRp and NS3-NS5 interaction, likely through binding cavity B. Resistance studies with compound 4 generated sequence variants in the 3'-untranslated region of RNA while further biochemical experiments demonstrated its ability to block also RNA-NS5 interaction, required for correct RNA synthesis in cells. These findings shed light on the potential mechanism of action for this class of compounds, underlying how PBTZs are very promising lead candidates for further evaluation.