Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Reaction of 3-Cl/OMe-Substituted 5-Nitrobenzisothiazoles with Hydrazine: Structural and Computational Evidence for Rearrangement Pathways Implicating Intramolecular Formation of Pivotal Meisenheimer Complexes.

In projected structure-activity relationship studies of the novel diheteroarylamide-based anti-HIV agent 2 (1C8), one objective was to evaluate the influence of incorporating the central amide motif in 2 into a five-membered pyrazolone ring, as found in 3. It was envisaged that compound 3 could be prepared through reaction of 3-hydrazino-5-nitrobenzisothiazole 5 with the methyl ester of 4-chloropyridine-3-carboxylic acid, followed by N-methylation of the pyridine nitrogen. However, the reaction of 3-methoxyl-5-nitrobenzisothiazole with hydrazine resulted in formation of ring-opened hydrazonate product 18. In the corresponding reaction with 3-chloro-5-nitrobenzisothiazole, a different rearrangement product 19 was formed, in which two 2,1-benzisothiazole units are joined by a sulfur bridge. Meisenheimer complex formation, favored by the presence of the 5-nitro substituent on the benzisothiazole ring, was postulated to be a key feature in the formation of these deep-seated rearrangement products. Support for the proposed formation of the pivotal Meisenheimer complexes and their subsequent evolution to the observed products in which the benzisothiazole sulfur atom is either expelled or maintained in the isomeric 2,1-benzisothiazole system was obtained by density function theory calculations.

Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication.

We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2ß, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2ß, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents.

Simulated adhesion between realistic hydrocarbon materials: effects of composition, roughness, and contact point.

The work of adhesion is an interfacial materials property that is often extracted from atomic force microscope (AFM) measurements of the pull-off force for tips in contact with flat substrates. Such measurements rely on the use of continuum contact mechanics models, which ignore the atomic structure and contain other assumptions that can be challenging to justify from experiments alone. In this work, molecular dynamics is used to examine work of adhesion values obtained from simulations that mimic such AFM experiments and to examine variables that influence the calculated work of adhesion. Ultrastrong carbon-based materials, which are relevant to high-performance AFM and nano- and micromanufacturing applications, are considered. The three tips used in the simulations were composed of amorphous carbon terminated with hydrogen (a-C-H), and ultrananocrystalline diamond with and without hydrogen (UNCD-H and UNCD, respectively). The model substrate materials used were amorphous carbon with hydrogen termination (a-C-H) and without hydrogen (a-C); ultrananocrystalline diamond with (UNCD-H) and without hydrogen (UNCD); and the (111) face of single crystal diamond with (C(111)-H) and without a monolayer of hydrogen (C(111)). The a-C-H tip was found to have the lowest work of adhesion on all substrates examined, followed by the UNCD-H and then the UNCD tips. This trend is attributable to a combination of roughness on both the tip and sample, the degree of alignment of tip and substrate atoms, and the surface termination. Continuum estimates of the pull-off forces were approximately 2-5 times larger than the MD value for all but one tip-sample pair.

The anticancer potential of the CLK kinases inhibitors 1C8 and GPS167 revealed by their impact on the epithelial-mesenchymal transition and the antiviral immune response.

The diheteroarylamide-based compound 1C8 and the aminothiazole carboxamide-related compound GPS167 inhibit the CLK kinases, and affect the proliferation of a broad range of cancer cell lines. A chemogenomic screen previously performed with GPS167 revealed that the depletion of components associated with mitotic spindle assembly altered sensitivity to GPS167. Here, a similar screen performed with 1C8 also established the impact of components involved in mitotic spindle assembly. Accordingly, transcriptome analyses of cells treated with 1C8 and GPS167 indicated that the expression and RNA splicing of transcripts encoding mitotic spindle assembly components were affected. The functional relevance of the microtubule connection was confirmed by showing that subtoxic concentrations of drugs affecting mitotic spindle assembly increased sensitivity to GPS167. 1C8 and GPS167 impacted the expression and splicing of transcripts in pathways relevant to tumor progression, including MYC targets and the epithelial mesenchymal transition (EMT). Finally, 1C8 and GPS167 altered the expression and alternative splicing of transcripts involved in the antiviral immune response. Consistent with this observation, depleting the double-stranded RNA sensor DHX33 suppressed GPS167-mediated cytotoxicity on HCT116 cells. Our study uncovered molecular mechanisms through which 1C8 and GPS167 affect cancer cell proliferation as well as processes critical for metastasis.

Inhibition of nonsense-mediated mRNA decay (NMD) by a new chemical molecule reveals the dynamic of NMD factors in P-bodies.

In mammals, nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that degrades mRNA harboring a premature termination codon to prevent the synthesis of truncated proteins. To gain insight into the NMD mechanism, we identified NMD inhibitor 1 (NMDI 1) as a small molecule inhibitor of the NMD pathway. We characterized the mode of action of this compound and demonstrated that it acts upstream of hUPF1. NMDI 1 induced the loss of interactions between hSMG5 and hUPF1 and the stabilization of hyperphosphorylated isoforms of hUPF1. Incubation of cells with NMDI 1 allowed us to demonstrate that NMD factors and mRNAs subject to NMD transit through processing bodies (P-bodies), as is the case in yeast. The results suggest a model in which mRNA and NMD factors are sequentially recruited to P-bodies.

The aberrant upregulation of exon 10-inclusive SREK1 through SRSF10 acts as an oncogenic driver in human hepatocellular carcinoma.

Deregulation of alternative splicing is implicated as a relevant source of molecular heterogeneity in cancer. However, the targets and intrinsic mechanisms of splicing in hepatocarcinogenesis are largely unknown. Here, we report a functional impact of a Splicing Regulatory Glutamine/Lysine-Rich Protein 1 (SREK1) variant and its regulator, Serine/arginine-rich splicing factor 10 (SRSF10). HCC patients with poor prognosis express higher levels of exon 10-inclusive SREK1 (SREK1L). SREK1L can sustain BLOC1S5-TXNDC5 (B-T) expression, a targeted gene of nonsense-mediated mRNA decay through inhibiting exon-exon junction complex binding with B-T to exert its oncogenic role. B-T plays its competing endogenous RNA role by inhibiting miR-30c-5p and miR-30e-5p, and further promoting the expression of downstream oncogenic targets SRSF10 and TXNDC5. Interestingly, SRSF10 can act as a splicing regulator for SREK1L to promote hepatocarcinogenesis via the formation of a SRSF10-associated complex. In summary, we demonstrate a SRSF10/SREK1L/B-T signalling loop to accelerate the hepatocarcinogenesis.

New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights.

Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.

2-Trifluoromethylthiazole-5-carboxamides: Analogues of a Stilbene-Based Anti-HIV Agent that Impact HIV mRNA Processing.

The observation that stilbene 3 (5350150) blocks HIV replication through its impact on HIV mRNA processing prompted a program to develop non-cytotoxic analogues that maintain its mechanism of action. This initially involved replacement of the central double bond in 3 by an amide function and the quinoline motif by a 2-aminobenzothiazole subunit, as in 12jj (R' = Cl), 12pp (R = NO2), and 12vv (R = CF3). On the basis of the possible CF3 ↔ NO2 bioisostere relationship in 12vv and 12pp, compound 23 was prepared and also found to be active. In the final step, the thiazole compounds 28 (GPS488) (EC50 = 1.66 µM) and 29 (GPS491) (EC50 = 0.47 µM) were prepared and evaluated. Similar activity and cell viability values (therapeutic index (TI = CC50/EC50) values of 50-100) were observed in primary peripheral blood mononuclear cells. Furthermore, they remained active against a panel of HIV mutant strains displaying resistance to individual drugs used in antiretroviral therapy. It was determined that compound 29 suppressed expression of the HIV-1 structural protein Gag and altered HIV-1 RNA accumulation, decreasing the abundance of RNAs encoding the structural proteins while increasing levels of viral RNAs encoding the regulatory proteins, a pattern similar to that seen for compound 3.

A novel class of inhibitors that target SRSF10 and promote p53-mediated cytotoxicity on human colorectal cancer cells.

The elevated expression of the splicing regulator SRSF10 in metastatic colorectal cancer (CRC) stimulates the production of the pro-tumorigenic BCLAF1-L splice variant. We discovered a group of small molecules with an aminothiazole carboxamide core (GPS167, GPS192 and others) that decrease production of BCLAF1-L. While additional alternative splicing events regulated by SRSF10 are affected by GPS167/192 in HCT116 cells (e.g. in MDM4, WTAP, SLK1 and CLK1), other events are shifted in a SRSF10-independent manner (e.g. in MDM2, NAB2 and TRA2A). GPS167/192 increased the interaction of SRSF10 with the CLK1 and CLK4 kinases, leading us to show that GPS167/192 can inhibit CLK kinases preferentially impacting the activity of SRSF10. Notably, GPS167 impairs the growth of CRC cell lines and organoids, inhibits anchorage-independent colony formation, cell migration, and promotes cytoxicity in a manner that requires SRSF10 and p53. In contrast, GPS167 only minimally affects normal colonocytes and normal colorectal organoids. Thus, GPS167 reprograms the tumorigenic activity of SRSF10 in CRC cells to elicit p53-dependent apoptosis.

The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation.

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

The effectiveness of, and predictors of response to, inpatient treatment of anorexia nervosa.

OBJECTIVE: The inpatient treatment of anorexia nervosa lacks a clear evidence base. We sought to determine the effectiveness of, and predictors of response to, a specialist inpatient programme for adults with anorexia nervosa, and to survey satisfaction with the same. METHOD: Demographic and clinical data were collected, at three time points, for 90 consecutive admission episodes over a three-year period. RESULTS: Both a completers and an intention-to-treat analysis indicated the effectiveness of the programme. A longer length of hospital stay was associated with a greater degree of change in BMI, but no other predictors of treatment outcome were detected. Participants reported a high degree of satisfaction with the programme. CONCLUSION: Adults suffering from anorexia nervosa improved significantly with a specialist programme delivered in an inpatient setting. Future research should investigate the potential role of factors other than obvious demographic and clinical history variables in determining treatment outcome.

The relationship between parental bonding, social problem solving and eating pathology in an anorexic inpatient sample.

OBJECTIVE: Parental relationships and maladaptive problem solving have been associated with anorexic symptomatology. This study investigates the relationship between perceived parental bonding, social problem solving and eating psychopathology. METHODS: Forty three female inpatients with anorexia nervosa and 76 student controls were assessed using the Parental Bonding Instrument, the Social Problem Solving Inventory and the Eating Disorders Examination or the Eating Disorders Examination-Questionnaire. RESULTS: The anorexic group reported significantly lower levels of parental care than the student control group and used more negative and avoidance style coping. In the anorexic group, disordered eating was significantly correlated with low maternal care and high control. Maternal bonding was found to mediate the relationship between avoidance style coping and eating pathology. CONCLUSIONS: Findings suggest a relationship between maternal bonding, the use of maladaptive problem solving techniques and eating disorder pathology in inpatients with anorexia nervosa.

General psychopathology in anorexia nervosa: the role of psychosocial factors.

The aim of the present study was to investigate psychosocial correlates of comorbid psychopathology. Data were collected from a total of 90 female inpatients with anorexia nervosa (AN). Higher levels of general psychopathology were detected in depression, interpersonal sensitivity, obsessive-compulsive and anxiety subscales of the Symptom Checklist (SCL)-90. Regression analysis also revealed that higher levels of psychopathology across SCL-90 subscales in AN patients are significantly associated with an earlier age of onset of the condition, higher levels of anorectic psychopathology as measured by Eating Disorders Examination, lower self-esteem as measured by Multidimensional Self-Esteem Inventory and social support levels as measured by Quality of Social Network and Social Support Questionnaire. Considering the high levels of general psychopathology in people with AN, routine clinical practice should aim for a comprehensive assessment of such. Given the strong association between psychosocial factors such as self-esteem, social support and general psychopathology, psychological therapies could play an important role in facilitating emotional recovery.

Salicylaldehyde derivatives as new protein kinase CK2 inhibitors.

Protein kinase CK2 is a Ser/Thr kinase, with a constitutive activity, that is considered as a promising target for cancer therapy. The currently available CK2 inhibitors lack the potency and the pharmacological properties necessary to be suitable and successful in clinical settings. We report the development of new potent CK2 inhibitors from salicylaldehyde derivatives identified by automated screening of a proprietary small-molecule library. Docking simulations and analysis of the structure-activity relationship for the hits allowed to determine their binding modes on CK2, and to carry out the optimization of their structures. This strategy led to the discovery of potent CK2 inhibitors with novel structures, one of which was able to inhibit CK2 activity in living cells and promote tumor cell death. The essential features required for potent CK2 inhibitory activity of this class of compounds are discussed.

Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

5-Substituted pyrido[2,3-d]pyrimidine, an inhibitor against three receptor tyrosine kinases.

NP506, the 3-{2,4-dimethyl-5-[2-oxo-5-(N'-phenylhydrazinocarbonyl)-1,2-dihydro-indol-3-ylidenemethyl]-1H-pyrrol-3-yl}-propionic acid, was designed as FGF receptor 1 inhibitor by computational study and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation than SU6668 with minimum effective dose of 10 microM. NP506 inhibited the tyrosine phosphorylation in FGF, VEGF, and PDGF receptors and the activation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal-kinase (JNK) and AKT after the rhFGF-2 stimulation. The introduction of the phenyl hydrazide motif to the position 5 of the pyrido[2,3-d]pyrimidine scaffold led to the inhibitory effect in two signaling pathways: inhibition of AKT activation in the phosphatidyl inositol 3'-kinase (PI13K)/AKT signaling pathway and the inhibition of ERK and JNK activation in MAPK pathway.

2-Aminothiazole-4-carboxamides Enhance Readthrough of Premature Termination Codons by Aminoglycosides.

Nonsense mutations introduce a premature termination codon (PTC) and are the underlying cause of multiple rare genetic diseases and cancers. Although certain aminoglycosides bind to eukaryotic ribosomes enabling incorporation of an amino acid at the PTC and formation of full-length protein, they are inefficient and toxic at therapeutic doses. Library screening in assays that measure readthrough at a PTC in the TP53 gene in human HDQ-P1 cells identified six novel 2-aminothiazole-4-carboxamide derivatives that potentiate the PTC readthrough (PTCR) efficiency of G418 when used in combination. The two most potent compounds incorporated a 4-indazole motif on the 2-aminothiazole nitrogen and a hydrophobic aryl substituent on the carboxamide nitrogen. These compounds are valuable tools to further investigate the therapeutic potential of aminoglycoside-induced PTCR.

BENA435, a new cell-permeant photoactivated green fluorescent DNA probe.

N'-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye.

Bilateral macular sub-retinal fluid and retinal pigment epithelial detachment associated with type 2 membrano-proliferative glomerulonephritis.

Choroidal neovascularisation (CNV) and idiopathic central serous chorioretinopathy (ICSC) are recognised ocular complications related to type 2 membrano-proliferative glomerulonephritis. We report a 38-year-old white male who presented with a 10-day history of blurring of vision, micropsia and metamorphopsia. He had been diagnosed recently to have type 2 membrano-proliferative glomerulonephritis. On examination, there was bilateral retinal pigment epithelial (RPE) detachment with overlying sub-retinal fluid without any drusen. Fundus fluorescein angiography (FFA) and optical coherence tomography (OCT) confirmed the diagnosis of atypical ICSC. Three months later, sub-retinal fluid and RPE detachment resolved and VA had recovered to 6/6. The case highlights the importance of ophthalmological assessment in these patients to recognise sight-threatening complications.