Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth.

Glioblastoma (GBM) is the most common malignant primary brain cancer that, despite recent advances in the understanding of its pathogenesis, remains incurable. GBM contains a subpopulation of cells with stem cell-like properties called cancer stem cells (CSCs). Several studies have demonstrated that CSCs are resistant to conventional chemotherapy and radiation thus representing important targets for novel anti-cancer therapies. Proton sensing receptors expressed by CSCs could represent important factors involved in the adaptation of tumours to the extracellular environment. Accordingly, the expression of acid-sensing ion channels (ASICs), proton-gated sodium channels mainly expressed in the neurons of peripheral (PNS) and central nervous system (CNS), has been demonstrated in several tumours and linked to an increase in cell migration and proliferation. In this paper we report that the ASIC3 isoform, usually absent in the CNS and present in the PNS, is enriched in human GBM CSCs while poorly expressed in the healthy human brain. We propose here a novel therapeutic strategy based on the pharmacological activation of ASIC3, which induces a significant GBM CSCs damage while being non-toxic for neurons. This approach might offer a promising and appealing new translational pathway for the treatment of glioblastoma.

4-(3-Phenylsulfonylindol-2-yl)-1-(pyridin-2-yl)piperazinyl-methanones as Potent Inhibitors of both SARS-CoV-2 and HCoV-OC43 Viruses.

SARS-CoV-2 and HCoV-OC43 belong to the same ß genus of the Coronaviridae family. SARS-CoV-2 was responsible for the recent COVID-19 pandemic, and HCoV-OC43 is the etiological agent of mild upper respiratory tract infections. SARS-COV-2 and HCoV-OC43 co-infections were found in children with respiratory symptoms during the COVID-19 pandemic. The two ß-coronaviruses share a high degree of homology between the 3CLpro active sites, so much so that the safer HCoV-OC43 has been suggested as a tool for the identification of new anti-SARS-COV-2 agents. Compounds 5 and 24 inhibited effectively both Wuhan and British SARS-CoV-2 patient isolates in Vero E6 cells and the HCoV-OC43 in MRC-5 cells at low micromolar concentrations. The inhibition was apparently exerted via targeting the 3CLpro active sites of both viruses. Compounds 5 and 24 at 100 µM inhibited the SARS-CoV-2 3CLpro activity of 61.78 and 67.30%, respectively. These findings highlight 5 and 24 as lead compounds of a novel class of antiviral agents with the potential to treat SARS-COV-2 and HCoV-OC43 infections.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

Stabilization of the retromer complex: Analysis of novel binding sites of bis-1,3-phenyl guanylhydrazone 2a to the VPS29/VPS35 interface.

The stabilization of the retromer protein complex can be effective in the treatment of different neurological disorders. Following the identification of bis-1,3-phenyl guanylhydrazone 2a as an effective new compound for the treatment of amyotrophic lateral sclerosis, in this work we analyze the possible binding sites of this molecule to the VPS35/VPS29 dimer of the retromer complex. Our results show that the affinity for different sites of the protein assembly depends on compound charge and therefore slight changes in the cell microenvironment could promote different binding states. Finally, we describe a novel binding site located in a deep cleft between VPS29 and VPS35 that should be further explored to select novel molecular chaperones for the stabilization of the retromer complex.

Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2.

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).

New Class of Benzodiazepinone Derivatives as Pro-Death Agents Targeting BIR Domains in Cancer Cells.

Inhibitor of Apoptosis Proteins (IAPs) are validated targets for cancer therapy, and the deregulation of their activities within the NF-κB pathway correlates with chemoresistance events, even after treatment with IAPs-antagonists in the clinic (Smac-mimetics). The molecule FC2 was identified as a NF-κB pathway modulator in MDA-MB-231 adenocarcinoma cancer cells after virtual screening of the Chembridge library against the Baculoviral IAP Repeat 1 (BIR1) domain of cIAP2 and XIAP. An improved cytotoxic effect is observed when FC2 is combined with Smac-mimetics or with the cytokine Tumor Necrosis Factor (TNF). Here, we propose a library of 22 derivatives of FC2, whose scaffold was rationally modified starting from the position identified as R1. The cytotoxic effect of FC2 derivatives was evaluated in MDA-MB-231 and binding to the cIAP2- and XIAP-BIR1 domains was assessed in fluorescence-based techniques and virtual docking. Among 22 derivatives, 4m and 4p display improved efficacy/potency in MDA-MB-231 cells and low micromolar binding affinity vs the target proteins. Two additional candidates (4b and 4u) display promising cytotoxic effects in combination with TNF, suggesting the connection between this class of molecules and the NF-κB pathway. These results provide the rationale for further FC2 modifications and the design of novel IAP-targeting candidates supporting known therapies.

Virucidal Activity of the Pyridobenzothiazolone Derivative HeE1-17Y against Enveloped RNA Viruses.

Pyridobenzothiazolone derivatives are a promising class of broad-spectrum antivirals. However, the mode of action of these compounds remains poorly understood. The HeE1-17Y derivative has already been shown to be a potent compound against a variety of flaviviruses of global relevance. In this work, the mode of action of HeE1-17Y has been studied for West Nile virus taking advantage of reporter replication particles (RRPs). Viral infectivity was drastically reduced by incubating the compound with the virus before infection, thus suggesting a direct interaction with the viral particles. Indeed, RRPs incubated with the inhibitor appeared to be severely compromised in electron microscopy analysis. HeE1-17Y is active against other enveloped viruses, including SARS-CoV-2, but not against two non-enveloped viruses, suggesting a virucidal mechanism that involves the alteration of the viral membrane.

Structure-based identification of a new IAP-targeting compound that induces cancer cell death inducing NF-κB pathway.

Inhibitors of apoptosis proteins (IAPs) are validated onco-targets, as their overexpression correlates with cancer onset, progression, diffusion and chemoresistance. IAPs regulate cell death survival pathways, inflammation, and immunity. Targeting IAPs, by impairing their protein-protein interaction surfaces, can affect events occurring at different stages of cancer development. To this purpose, we employed a rational virtual screening approach to identify compounds predicted to interfere with the assembly of pro-survival macromolecular complexes. One of the candidates, FC2, was shown to bind in vitro the BIR1 domains of both XIAP and cIAP2. Moreover, we demonstrated that FC2 can induce cancer cell death as a single agent and, more potently, in combination with the Smac-mimetic SM83 or with the cytokine TNF. FC2 determined a prolonged activation of the NF-κB pathway, accompanied to a stabilization of XIAP-TAB1 complex. This candidate molecule represents a valuable lead compound for the development of a new class of IAP-antagonists for cancer treatment.

A novel hotspot of gelsolin instability triggers an alternative mechanism of amyloid aggregation.

Gelsolin comprises six homologous domains, named G1 to G6. Single point substitutions in this protein are responsible for AGel amyloidosis, a hereditary disease causing progressive corneal lattice dystrophy, cutis laxa, and polyneuropathy. Although several different amyloidogenic variants of gelsolin have been identified, only the most common mutants present in the G2 domain have been thoroughly characterized, leading to clarification of the functional mechanism. The molecular events underlying the pathological aggregation of 3 recently identified mutations, namely A551P, E553K and M517R, all localized at the interface between G4 and G5, are here explored for the first time. Structural studies point to destabilization of the interface between G4 and G5 due to three structural determinants: ß-strand breaking, steric hindrance and/or charge repulsion, all implying impairment of interdomain contacts. Such rearrangements decrease the temperature and pressure stability of gelsolin but do not alter its susceptibility to furin cleavage, the first event in the canonical aggregation pathway. These variants also have a greater tendency to aggregate in the unproteolysed forms and exhibit higher proteotoxicity in a C. elegans-based assay. Our data suggest that aggregation of G4G5 variants follows an alternative, likely proteolysis-independent, pathway.

Discovery of a Novel Class of Norovirus Inhibitors with High Barrier of Resistance.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting in ~219,000 deaths annually and a societal cost of ~USD60 billion. There are no antivirals or vaccines available to treat and/or prevent HuNoV. In this study, we performed a large-scale phenotypical antiviral screening using the mouse norovirus (MNV), which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre (Sapienza University, Rome). Compound 3-((3,5-dimethylphenyl)sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide (compound 1) was identified as an inhibitor of MNV replication with an EC50 of 0.5 ± 0.1 µM. A series of 10 analogs were synthesized of which compound 6 showed an improved potency/selectivity (EC50 0.2 ± 0.1 µM) against MNV; good activity was also observed against the HuNoV GI replicon (EC50 1.2 ± 0.6 µM). Time-of-drug-addition studies revealed that analog 6 acts at a time point that coincides with the onset of viral RNA replication. After six months of selective pressure, two compound 6res variants were independently selected, both harboring one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype. In this study, we present a class of novel norovirus inhibitors with a high barrier to resistance and in vitro antiviral activity.

Synthesis and Characterization of Novel Mono- and Bis-Guanyl Hydrazones as Potent and Selective ASIC1 Inhibitors Able to Reduce Brain Ischemic Insult.

Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

Ligand Binding in Allosteric Flavoproteins: Part 2. Quantitative Analysis of the Redox-Dependent Interaction of the Apoptosis-Inducing Factor (AIF) with Its Protein Partner.

To perform their action usually flavoproteins interact transiently with a variety of molecular partners, whose binding is reciprocally affected and often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe an approach for the quantitative characterization of the redox-controlled interaction of the mammalian apoptosis inducing factor (AIF) with one of its known protein partners, namely, the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). In particular, we report a protocol for the titration of the flavoprotein in both in its oxidized and reduced states with CHCHD4, using an implementation of the MicroScale Thermophoresis (MST) technique.

Combined in silico and in vitro approaches identified the antipsychotic drug lurasidone and the antiviral drug elbasvir as SARS-CoV2 and HCoV-OC43 inhibitors.

The current emergency of the novel coronavirus SARS-CoV2 urged the need for broad-spectrum antiviral drugs as the first line of treatment. Coronaviruses are a large family of viruses that already challenged humanity in at least two other previous outbreaks and are likely to be a constant threat for the future. In this work we developed a pipeline based on in silico docking of known drugs on SARS-CoV1/2 RNA-dependent RNA polymerase combined with in vitro antiviral assays on both SARS-CoV2 and the common cold human coronavirus HCoV-OC43. Results showed that certain drugs displayed activity for both viruses at a similar inhibitory concentration, while others were specific. In particular, the antipsychotic drug lurasidone and the antiviral drug elbasvir showed promising activity in the low micromolar range against both viruses with good selectivity index.

Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+: Hints to Understand Protein Activity.

The guanylyl cyclase-activating protein 1, GCAP1, activates or inhibits retinal guanylyl cyclase (retGC) depending on cellular Ca2+ concentrations. Several point mutations of GCAP1 have been associated with impaired calcium sensitivity that eventually triggers progressive retinal degeneration. In this work, we demonstrate that the recombinant human protein presents a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and, more importantly, by protein binding to Ca2+ or Mg2+. Based on small-angle X-ray scattering data, protein-protein docking, and molecular dynamics simulations we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer. The different propensity of human GCAP1 to dimerize suggests structural differences induced by cation binding potentially involved in the regulation of retGC activity.

Discovery of Zika Virus NS2B/NS3 Inhibitors That Prevent Mice from Life-Threatening Infection and Brain Damage.

Zika virus (ZIKV) infection, which initially was endemic only in Africa and Asia, is rapidly spreading throughout Europe, Oceania, and the Americas. Although there have been enormous efforts, there is still no approved drug to treat ZIKV infection. Herein, we report the synthesis and biological evaluation of agents with noncompetitive mechanism of the ZIKV NS2B/NS3 protease inhibition through the binding to an allosteric site. Compounds 1 and 2 showed potent activity in both enzymatic and cellular assays. Derivative 1 efficiently reduced the ZIKV protein synthesis and the RNA replication and prevented the mice from life-threatening infection and the brain damage caused by ZIKV infection in a ZIKV mouse model.

Computational and Experimental Characterization of NF023, A Candidate Anticancer Compound Inhibiting cIAP2/TRAF2 Assembly.

Protein-protein interactions are the basis of many important physiological processes and are currently promising, yet difficult, targets for drug discovery. In this context, inhibitor of apoptosis proteins (IAPs)-mediated interactions are pivotal for cancer cell survival; the interaction of the BIR1 domain of cIAP2 with TRAF2 was shown to lead the recruitment of cIAPs to the TNF receptor, promoting the activation of the NF-κB survival pathway. In this work, using a combined in silico-in vitro approach, we identified a drug-like molecule, NF023, able to disrupt cIAP2 interaction with TRAF2. We demonstrated in vitro its ability to interfere with the assembly of the cIAP2-BIR1/TRAF2 complex and performed a thorough characterization of the compound's mode of action through 248 parallel unbiased molecular dynamics simulations of 300 ns (totaling almost 75 µs of all-atom sampling), which identified multiple binding modes to the BIR1 domain of cIAP2 via clustering and ensemble docking. NF023 is, thus, a promising protein-protein interaction disruptor, representing a starting point to develop modulators of NF-κB-mediated cell survival in cancer. This study represents a model procedure that shows the use of large-scale molecular dynamics methods to typify promiscuous interactors.

Retromer stabilization results in neuroprotection in a model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.

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.

Comprehensive response to Usutu virus following first isolation in blood donors in the Friuli Venezia Giulia region of Italy: Development of recombinant NS1-based serology and sensitivity to antiviral drugs.

Surveillance of Usutu virus is crucial to prevent future outbreaks both in Europe and in other countries currently naïve to the infection, such as the Americas. This goal remains difficult to achieve, notably because of the lack of large-scale cohort studies and the absence of commercially available diagnostic reagents for USUV. This work started with the first identification of USUV in a blood donor in the Friuli Venezia Giulia (FVG) Region in Northern-Eastern Italy, which is endemic for West Nile virus. Considering that only one IgG ELISA is commercially available, but none for IgM, a novel NS1 antigen based IgG/M ELISA has been developed. This assay tested successfully for the detection of Usutu virus in blood donors with the identification of a second case of transmission and high levels of exposure. Furthermore, two pan-flavivirus antiviral drugs, that we previously characterized to be inhibitors of other flavivirus infectivity, were successfully tested for inhibition of Usutu virus with inhibitory concentrations in the low micromolar range. To conclude, this work identifies North-Eastern Italy as endemic for Usutu virus with implications for the screening of transfusion blood. A novel NS1-based ELISA test has been implemented for the detection of IgM/G that will be of importance as a tool for the diagnosis and surveillance of Usutu virus infection. Finally, Usutu virus is shown to be sensitive to a class of promising pan-flavivirus drugs.

Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro.

Sofosbuvir, a licensed nucleotide analog targeting hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), has been recently evaluated as a broad anti-Flavivirus lead candidate revealing activity against Zika and Dengue viruses both in vitro and in animal models. In this study, the in vitro antiviral activity of sofosbuvir against West Nile virus (WNV) was determined by plaque assay (PA) and Immunodetection Assay (IA) in human cell lines and by enzymatic RdRp assay. By PA, the sofosbuvir half-maximal inhibitory concentration (IC50) was 1.2 ± 0.3 µM in Huh-7, 5.3 ± 0.9 µM in U87, 7.8 ± 2.5 µM in LN-18 and 63.4 ± 14.1 µM in A549 cells. By IA, anti-WNV activity was confirmed in both hepatic (Huh-7, 1.7 ± 0.5 µM) and neuronal (U87, 7.3 ± 2.0 µM) cell types. Sofosbuvir was confirmed to inhibit the purified WNV RdRp (IC50 11.1 ± 4.6 µM). In vitro resistance selection experiments were performed by propagating WNV in the Huh-7 cell line with two-fold increasing concentrations of sofosbuvir. At 80 µM, a significantly longer time for viral breakthrough was observed compared with lower concentrations (18 vs. 7-9 days post infection; p = 0.029), along with the detection of the S604T mutation, corresponding to the well-known S282T substitution in the motif B of HCV NS5B, which confers resistance to sofosbuvir. Molecular docking experiments confirmed that the S604T mutation within the catalytic site of RdRp affected the binding mode of sofosbuvir. To our knowledge, this is the first report of the antiviral activity of sofosbuvir against WNV as well as of selection of mutants in vitro.