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Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802—a highly potent DENV inhibitor that blocks the NS3–NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

PDF file, 55KB, ClARE responds to androgen-like substances.

PDF file, 37KB, MEL-3 has no agonistic properties on ClARE.

PDF file, 41KB, MEL-3 does not affect AR mRNA levels in LAPC4 and LNCaP cells.

PDF file, 54KB.

PDF file, 58KB, MEL-3 does not induce apoptosis via Caspase-3 activation.

PDF file, 25KB, AR-level in ClARE and SelARE.

The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the β2-adrenergic receptor (β2-AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the β2-AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I125-cyanopindolol, a β2-AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a β2-AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the β2-AR. Oral administration of glucose with a non-bioavailable β2-AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical β2-ARs in stimulating gut glucose uptake, and suggesting enterocyte β2-AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether β2-ARs mediate glucose sensing also in other tissues.

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passagedin vitroin the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting.Abstract ImportancePaxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovidin vitro. The characteristics ofin vitroantiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors.

Many cancers harbor mutations in the Hippo pathway that lead to constitutive activation of the transcriptional co-activators YAP/TAZ that then bind the transcription factor TEAD and drive aberrant transcription of target genes involved in cell proliferation and tumor progression. Hyperactivation of YAP/TAZ has also been associated with resistance to targeted therapies, including MAPK pathway inhibitors. To target cancers that bear mutations in the Hippo pathway or are resistant to therapies due to YAP/TAZ activation, we developed SW-682, a pan-TEAD small molecule inhibitor that blocks TEAD-dependent transcription by binding to the palmitoylation pocket of all TEAD isoforms. In vitro, SW-682 inhibited the proliferation of human Hippo-mutant mesothelioma cells with nanomolar potency, with little to no effect on Hippo wild-type tumor cells. SW-682 down-regulated TEAD-dependent reporter gene expression in a dose-dependent manner, while having no effect on reporters monitoring other pathways. In vivo, daily oral administration of SW-682 to adult mice resulted in tumor regression in Hippo-mutant mesothelioma models and caused down-regulation of expression of the TEAD-dependent genes CCN1 and CCN2 and a YAP gene signature, as measured by qPCR or RNA-seq analysis. SW-682 has a favorable PK profile with good oral bioavailability in the mouse and was well tolerated with no signs of body weight loss. To test the hypothesis that TEAD inhibition can overcome YAP-driven resistance mechanisms, we explored SW-682 in combination with MEK inhibitors in several in vitro and ex vivo patient-derived tumor models including BRAF and NRAS mutated melanoma. Moreover, to identify new indications that may benefit from TEAD inhibition, we screened patient-derived 3D organoid tumor cells and matching patient-derived xenograft models that have been molecularly profiled. In summary, SW-682 is a potent and selective investigational TEAD inhibitor which demonstrated anti-tumor effects in models harboring aberrant expression of the Hippo pathway, suggesting therapeutic potential in multiple Hippo-mutant solid tumors. Citation Format: Lei Chen, Paula Milani de Marval, Kendall Powell, Mark Johnson, Greg Falls, Brian Lawhorn, Aurélie Candi, Amuri Kilonda, Bart Vanderhoydonck, Arnaud Marchand, Matthias Versele, Georg Halder, Stephen L. Gwaltney, Adeela Kamal. SW-682: A novel TEAD inhibitor for the treatment of cancers bearing mutations in the Hippo signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4964.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) has emerged as a promising cancer immunotherapy target. Previously, we reported on the functional role of PTPN2 in the pathogenesis of colorectal carcinoma (CRC). We demonstrated increased PTPN2 phosphatase activity correlates with disease progression and decreased immune responses in tumor tissues, while loss of PTPN2 in T-cells or in dendritic cells (DCs) reduces tumor burden in several CRC models and potentiates anti-PD1 efficacy (Katkeviciute et al., 2021). Others reported that loss of PTPN2 in tumor cells sensitizes to immune-mediated tumor killing (Manguso et al., 2017; Goh et al., 2022). Thus, PTPN2 was proposed as a critical node to modulate anti-tumor immunity, and PTPN2 inhibition is expected to enhance anti-tumor immunity by sensitizing tumor cells and by activating immune cells. Recently, also the closely related phosphatase PTPN1 (PTP1B) was demonstrated to restrain T-cell mediated tumor killing (Wiede et al., 2022). Inhibition of Protein Tyrosine Phosphatases has historically been a demanding area for drug discovery. However, both allosteric PTP inhibitors (eg targeting SHP2/PTPN11) and catalytic-site inhibitors (targeting PTPN2/N1) have recently progressed to clinical studies. We now identified a chemical series of novel dual PTPN2/N1 inhibitors. These inhibitors bind to the catalytic site of PTPN2, as confirmed by crystallography. The best molecules of the series are low nM inhibitors of PTPN2 and PTPN1 enzymes with excellent selectivity across other phosphatases. As an immediate consequence of PTPN2/N1 inhibition, these compounds augment phosphorylation of STAT1 and/or STAT5 isoforms in myeloid and T-cell lines, and in primary T-cells, macrophages and DCs. This results in immune-cell activation as evidenced by increased production of effector cytokines (e.g. IFNγ), immune-activation, levels of cytotoxicity markers in T-cells (e.g. granzyme B) as well as upregulation of antigen presentation and co-stimulatory markers in macrophages and DCs. Furthermore, inhibition of PTPN2/N1 sensitizes murine and human cancer cell lines to IFNγ, and enhances immune-mediated tumor killing in co-culture assays in vitro. In vivo evaluation of selected examples is in progress and results will be presented at the conference. Our data indicate that inhibition of PTPN2/N1 phosphatase activity is a powerful pharmacologic strategy to promote anti-tumor immunity, with strong potential to be exploited for cancer immunotherapy both as a single agent treatment in anti-PD1 refractory cancers, and in combination with anti-PD1 therapy. Citation Format: Kalliopi Pervolaraki, Egle Katkeviciute, Dominique Lambin, Sandro Boland, Amuri Kilonda, Vincent Pericolle, Marnik Nijs, Wanda Haeck, Kristine Metzger, Hugo Klaassen, Arnaud Marchand, Patrick Chaltin, Matthias Versele, Marianne Spalinger, Michael Scharl. Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4038.

Despite recent advancements in the development of vaccines and monoclonal antibody therapies for Ebola virus disease, treatment options remain limited. Moreover, management and containment of Ebola virus outbreaks is often hindered by the remote nature of the locations in which the outbreaks originate. Small-molecule compounds offer the advantage of being relatively cheap and easy to produce, transport and store, making them an interesting modality for the development of novel therapeutics against Ebola virus disease. Furthermore, the repurposing of small-molecule compounds, previously developed for alternative applications, can aid in reducing the time needed to bring potential therapeutics from bench to bedside. For this purpose, the Medicines for Malaria Venture provides collections of previously developed small-molecule compounds for screening against other infectious diseases. In this study, we used biologically contained Ebola virus to screen over 4,200 small-molecule drugs and drug-like compounds provided by the Medicines for Malaria Venture (i.e., the Pandemic Response Box and the COVID Box) and the Centre for Drug Design and Discovery (CD3, KU Leuven, Belgium). In addition to confirming known Ebola virus inhibitors, illustrating the validity of our screening assays, we identified eight novel selective Ebola virus inhibitors. Although the inhibitory potential of these compounds remains to be validated in vivo, they represent interesting compounds for the study of potential interventions against Ebola virus disease and might serve as a basis for the development of new therapeutics. ispartof: ANTIVIRAL RESEARCH vol:200 ispartof: location:Netherlands status: published

Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue1,2. There are no antiviral agents available to prevent or treat dengue. Here, we describe a highly potent dengue virus inhibitor (JNJ-A07) that exerts nanomolar to picomolar activity against a panel of 21 clinical isolates that represent the natural genetic diversity of known genotypes and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus revealing a previously undescribed mechanism of antiviral action. JNJ-A07 has a favourable pharmacokinetic profile that results in outstanding efficacy against dengue virus infection in mouse infection models. Delaying start of treatment until peak viraemia results in a rapid and significant reduction in viral load. An analogue is currently in further development. The small molecule JNJ-A07 interferes with the interaction between the NS3 and NS4B proteins of dengue virus and reduces the viral load in mice even when first administered at peak viraemia.

The transient receptor potential (TRP) channel TRPV2 is widely expressed in a variety of different cell types and tissues. However, elucidating the exact biological functions of TRPV2 is significantly hampered by the lack of selective pharmacological tools to modulate channel activity in vitro and in vivo. This study aimed to identify new compounds that modify TRPV2 activity via the use of a plate-based calcium imaging approach to screen a drug repurposing library. Three antihistaminic drugs, loratadine, astemizole and clemizole were identified to reduce calcium-influx evoked by the TRPV2 agonist tetrahydrocannabivarin in HEK293 cells expressing murine TRPV2. Using single-cell calcium-microfluorimetry and whole-cell patch clamp recordings, we further confirmed that all three compounds induced a concentration-dependent block of TRPV2-mediated Ca

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 μM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors., Graphical abstract Image 1

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50= 7 nM) without affecting the activity of human cathepsin L (IC50> 10 μM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10(RNA copies/mg) reduction of the viral RNA copies and 3.7 log10(TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

Background: The Hippo pathway is an evolutionarily conserved signaling cascade whose deregulation can promote excessive cell proliferation and tumor development. Pathway output is mediated by the YAP and TAZ transcriptional co-activators, which bind to TEAD family transcription factors to drive target gene expression. Genomic aberrations in Hippo pathway components result in constitutive activation of YAP/TAZ, as seen with NF2 mutations in subsets of mesothelioma and other cancers. Hyperactivation of YAP/TAZ has also been associated with resistance to a variety of targeted agents, including EGFR and CDK4/6 inhibitors, suggesting that targeting the pathway may have utility as part of rationally selected combinations, in addition to genomically-informed monotherapy applications. Activity of the YAP/TAZ-TEAD complex thus represents a compelling pharmacologic target, due to its essential role in the pathway, and the presence of a conserved druggable site in TEAD that is required for transcriptional function. Results and Discussion: Using biophysical techniques, we identified novel small molecules that bind to the TEAD auto-palmitoylation pocket. Initial hits were optimized for antagonism of TEAD-based transcription and drug-like properties, ultimately producing highly potent and orally bioavailable TEAD inhibitors. These compounds selectively inhibited the proliferation of cancer cell lines harboring genomic alterations in the Hippo pathway with low nM potency. In vivo models of Hippo pathway-altered xenografts showed consistent monotherapy activity, with dose-dependent and durable tumor regressions achieved at well-tolerated doses. Further characterization of these compounds as monotherapies and as part of rationally-designed combination regimens is ongoing. Citation Format: Adeela Kamal, Aurélie Candi, Matthias Versele, Bart Vanderhoydonck, Arnaud Marchand, Ron de Jong, Thuy Hoang, Georg Halder, Patrick Chaltin, Stephen L. Gwaltney, Mike Burgess. Novel antagonists of TEAD palmitoylation inhibit the growth of Hippo-altered cancers in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3945.

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. The Sec preprotein export pathway is an attractive potential alternative target. It is essential for bacterial viability and includes components that are absent from eukaryotes. Here we used a new high throughputin vivoscreen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, fourteen HTS secretion inhibitors (HSI), belonging to 8 structural classes, were identified (IC50−and Gram+bacterial species (including those from the WHO top pathogens list). Seven of them, HSI#6, 9; HSI#1, 5, 10 and HSI#12, 14 representing three structural families were microbicidals. HSI#6 was the most potent (IC50of 0.4-8.7 μM), against 13 species of both Gram−and Gram+bacteria. HSI#1, 5, 9 and 10 inhibited viability of Gram+bacteria with IC50~6.9-77.8 μM. HSI#9, 12 and 14 inhibited viability ofE. colistrains with IC50E. colistrain missing TolC to improve permeability with IC504-14 μM, indicating their inability to penetrate the outer membrane.In vitroassays revealed that antimicrobial activity was not related to inhibition of the SecA component of the translocase and hence HSI molecules may target new unknown components that affect secretion. The results provide proof of principle for our approach, and new starting compounds for optimization.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2–driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life–derived B1a cells, can be an effective therapeutic strategy to treat MCL.

International audience; Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVBres) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve similar to 18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVBres was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.

Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue

3-Acyl-indole derivative 1 was identified as a novel dengue virus (DENV) inhibitor from a DENV serotype 2 (DENV-2) phenotypic antiviral screen. Extensive SAR studies led to the discovery of new derivatives with improved DENV-2 potency as well as activity in nanomolar to micromolar range against the other DENV serotypes. In addition to the potency, physicochemical properties and metabolic stability in rat and human microsomes were improved during the optimization process. Chiral separation of the racemic mixtures showed a clear preference for one of the two enantiomers. Furthermore, rat pharmacokinetics of two compounds will be discussed in more detail, demonstrating the potential of this new series of pan-serotype-DENV inhibitors.

OBJECTIVES: We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. METHODS: HuAECs were infected with RSV-A Long strain (0.01 CCID50/cell, where CCID50 represents 50% cell culture infectious dose in HEp2 cells) on the apical compartment of the culture. At the time of infection or at 1 or 3 days post-infection, selected inhibitors were added and refreshed daily on the basal compartment of the culture. Viral shedding was followed up by apical washes collected daily and quantifying viral RNA by RT-qPCR. RESULTS: RSV-A replicates efficiently in HuAECs and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post-infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleoside) elicited a marked antiviral effect even when the start of treatment was delayed until 1 day or even 3 days after infection. Levels of the inflammation marker RANTES (mRNA) increased ∼200-fold in infected, untreated cultures (at 3 weeks post-infection), but levels were comparable to those of uninfected cultures in the presence of PC786, an RSV replication inhibitor, suggesting that an efficient antiviral treatment might inhibit virus-induced inflammation in this model. CONCLUSIONS: Overall, HuAECs offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds. ispartof: JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY vol:73 issue:7 pages:1823-1829 ispartof: location:England status: published

To suppress serious influenza infections in persons showing insufficient protection from the vaccines, antiviral drugs are of vital importance. There is a need for novel agents with broad activity against influenza A (IAV) and B (IBV) viruses and with targets that differ from those of the current antivirals. We here report a new small molecule influenza virus inhibitor referred to as CPD A (chemical name: N-(pyridin-3-yl)thiophene-2-carboxamide). In an influenza virus minigenome assay, this non-nucleoside compound inhibited RNA synthesis of IAV and IBV with EC50 values of 2.3 μM and 2.6 μM, respectively. Robust in vitro activity was noted against a broad panel of IAV (H1N1 and H3N2) and IBV strains, with a median EC50 value of 0.20 μM, which is 185-fold below the 50% cytotoxic concentration. The action point in the viral replication cycle was located between 1 and 5 h p.i., showing a similar profile as ribavirin. Like this nucleoside analogue, CPD A was shown to cause strong depletion of the cellular GTP pool and, accordingly, its antiviral activity was antagonized when this pool was restored with exogenous guanosine. This aligns with the observed inhibition in a cell-based IMP dehydrogenase (IMPDH) assay, which seems to require metabolic activation of CPD A since no direct inhibition was seen in an enzymatic IMPDH assay. The combination of CPD A with ribavirin, another IMPDH inhibitor, proved strongly synergistic. To conclude, we established CPD A as a new inhibitor of influenza A and B virus replication and RNA synthesis, and support the potential of IMPDH inhibitors for influenza therapy with acceptable safety profile. ispartof: ANTIVIRAL RESEARCH vol:196 ispartof: location:Netherlands status: published

Extracellular 2',3'-cyclic GMP-AMP (cGAMP) has a critical immune-transmitter role in the tumor microenvironment (TME). Tumor cells produce and secrete cGAMP, which primes immune cells for tumor rejection through STING (stimulator of interferon genes) signalling (Marcus et al., Immunity 2018; Carozza et al., Nature Cancer 2020). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) is the only enzyme known to hydrolyze extracellular cGAMP. In concert with the ectoATPase CD39 and the 5'-nucleotidase CD73, ENPP1 also contributes to the generation of an immunosuppressive TME by converting extracellular ATP into adenosine. ENPP1 is overexpressed in a number of tumor types, including breast cancer, liver cancer, thyroid cancer and sarcomas, and has been associated with poor outcome. Hence, inhibition of ENPP1 is an emerging strategy to augment anti-tumor immunity by stabilizing extracellular cGAMP and ATP, thereby turning cold tumors into immunologically hot tumors. Here, we report on the identification and characterization of novel chemical series of ENPP1 inhibitors. A drug-like small-molecule library (~160,000 compounds) was screened to identify inhibitors of ENPP1-mediated cGAMP hydrolysis. Hits were confirmed to also inhibit ATP hydrolysis by ENPP1, but not nucleotide hydrolysis mediated by the closely related ENPP2 (autotaxin) enzyme. A selection of hits, chemically distinct from previously reported ENPP1 inhibitors (such as those based on QS1; Carroza et al, Cell Chem Biol, 2020), were subjected to hit-to-lead optimization supported by structure-based guidance. This led to the identification of ENPP1 inhibitors with sub-nM potency on ENPP1 in biochemical assays, which maintained a >1000x selectivity window with respect to ENPP2 and to other phosphodiesterases. These compounds translated well to stabilization of nucleotides in the presence of ENPP1-overexpressing cancer cell lines, with IC50 values in the nM range. A selection of compounds was profiled for DMPK (drug metabolism and pharmacokinetic) parameters, and compounds with a suitable profile were prioritized for in vivo evaluation. Orally bio-available, metabolically stable compounds were assessed in mouse syngeneic tumor models, selected on the basis of high ENPP1 and high cGAS (cGAMP synthase) expression. Stabilization of cGAMP, activation of a STING-mediated cytokine response and immune-cell infiltration/activation in the TME were used as pharmacodynamic endpoints. An update on in vivo efficacy data in a range of ENPP1-positive tumor models will be provided during the presentation. These novel orally bioavailable ENPP1 inhibitors unleash local, TME-restricted, innate immune activation, and hold the promise to overcome the current limitations of direct STING agonists. Citation Format: Matthias Versele, Javier del Pino Garcia, Ilse Vandecaetsbeek, Karolien Castermans, Ranie Kellens, Wanda Haeck, Hugo Klaassen, Arnaud Bourin, Dries De Clercq, Sara Allasia, Sandro Boland, Arnaud Marchand, Patrick Chaltin, Mathieu Bollen. Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 53.

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 &lt, 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9, HSI#1, 5, 10, and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 &lt, 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

The Hippo pathway is a highly conserved signaling pathway across higher-order vertebrates and a key modulator of developmental biology. Both genetic aberrations as well as non-genetic dysregulation of the pathway lead to constitutive nuclear localization and transcriptional activity of the YAP/TAZ-TEAD complex in multiple solid tumor types, including mesothelioma, uveal melanoma, squamous cell cancer, liver cancer, lung cancer, etc. Genetic aberrations are manifested as gene amplifications and gene fusions of the core transcriptional YAP/TAZ-TEAD complex subunits, and, more commonly, as deletions or loss-of-function mutations in the upstream negative regulators of the Hippo pathway such as NF2, LATS1/2 or FAT1. More recently, constitutive activation of YAP/TAZ-TEAD has been implicated in cancer therapy resistance and in immune evasion. Multiple efforts have been devoted to identify small-molecule inhibitors of the YAP/TAZ-TEAD protein-protein interaction, yet with limited success reported to date. Based on the identification of an auto-palmitoylation pocket centrally located in TEAD, and its reported role to sustain YAP/TAZ-TEAD transcriptional activity, we set up a biophysical assay to detect selective small-molecule binding into the palmitoylation pocket of TEAD1. Based on screening a rationally designed compound collection in this assay and iterations of analoging, we identified several novel chemical series of TEAD-palmitoylation pocket binders. Hits were confirmed as specific allosteric inhibitors of YAP/TAZ-TEAD transcription in cell-based assays (Q-PCR and reporter gene assays). Soaking compounds in TEAD crystals revealed structural information enabling hit-to-lead optimization of two different chemical series. Best allosteric inhibitors in the series display single-digit nM potency in transcriptional assays, and translate to low nM inhibition of Hippo mutant (but not WT, >1000x selectivity window) mesothelioma proliferation. These molecules are well suited to probe for additional Hippo-dependent solid cancer types using in vitro cancer cell panels, selected based on genetics and/or a YAP/TAZ-TEAD gene signature. Furthermore, optimization towards orally bioavailable compounds is in progress and an update on in vivo efficacy in various solid tumor models will be presented. Citation Format: Matthias Versele, Aurélie Candi, Marnik Nijs, Wanda Haeck, Hugo Klaassen, Wim Smets, Stéphane Spieser, Bart Vanderhoydonck, Arnaud Marchand, Patrick Chaltin, Leticia Sansores, Georg Halder. Discovery of novel potent allosteric inhibitors of YAP/TAZ-TEAD transcription for the treatment of multiple solid tumor types addicted to Hippo signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5229.

Attempted suicide is a strong risk factor for subsequent suicidal behavior. In recent years, a particular interest has been given to follow-up interventions as a potential effective strategy in preventing recurrent suicidal behavior. We developed a follow-up intervention program called OSTA (organization of a suitable monitoring for suicide attempters) aimed at addressing this issue and tested its effectiveness in a 1-year randomized controlled trial. Individuals who attempted suicide and were admitted to the emergency department (ED) of Bicêtre Hospital (n=320) were randomly allocated to receive either the OSTA program or a control treatment. On an intention to treat basis, the proportion of patients who reattempted suicide did not differ significantly between the interventional group (IG) 14.5% (22/152) and the control group (CG) 14% (21/150). There were also no significant differences, between the two arms, in the number of suicide attempts. Although no significant difference has been found between the OSTA program and the control treatment concerning the rate of suicide reattempts, we believe that further studies should be conducted to test the effectiveness of more standardized follow-up studies in suicide prevention.

We report the use of reconstituted 3D-human airway epithelium cells of bronchial origin (HuAEC) in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. RSV-A replicates efficiently in HuAEC and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleosides) elicited a marked antiviral effect even when start of treatment was delayed until one or even three days after infection. Levels of the inflammation marker RANTES (mRNA) increased ∼200-fold in infected-untreated cultures (at three weeks post infection), but levels were comparable to those of uninfected cultures in the presence of PC-876, a RSV-replication inhibitor, demonstrating that an efficient antiviral treatment inhibits virus induced inflammation in this model. Overall, HuAEC offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds.

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies. ispartof: Frontiers in Microbiology vol:9 issue:FEB ispartof: location:Switzerland status: published

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition., Tumor cells can fuel their metabolism with lactate. Here the authors show that inhibition of mitochondrial pyruvate carrier (MPC) blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation and inhibits oxidative metabolism, ultimately resulting in cytotoxicity and radiosensitization.

Antibiotics typically fail to completely eradicate a bacterial population, leaving a small fraction of transiently antibiotic-tolerant persister cells intact. Persisters are therefore seen to be a major cause of treatment failure and greatly contribute to the recalcitrant nature of chronic infections. The current study focused on Pseudomonas aeruginosa , a Gram-negative pathogen belonging to the notorious ESKAPE group of pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species) and, due to increasing resistance against most conventional antibiotics, posing a serious threat to human health. Greatly contributing to the difficult treatment of P. aeruginosa infections is the presence of persister cells, and elimination of these cells would therefore significantly improve patient outcomes. In this study, a small-molecule library was screened for compounds that, in combination with the fluoroquinolone antibiotic ofloxacin, reduced the number of P. aeruginosa persisters compared to the number achieved with treatment with the antibiotic alone. Based on the early structure-activity relationship, 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009) was selected for further characterization. Combination of SPI009 with mechanistically distinct classes of antibiotics reduced the number of persisters up to 10 6 -fold in both lab strains and clinical isolates of P. aeruginosa . Further characterization of the compound revealed a direct and efficient killing of persister cells. SPI009 caused no erythrocyte damage and demonstrated minor cytotoxicity. In conclusion, we identified a novel antipersister compound active against P. aeruginosa with promising applications for the design of novel, case-specific combination therapies in the fight against chronic infections.

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), is an infection that is responsible for roughly 1.5 million deaths per year. The situation is further complicated by the wide-spread resistance to the existing first- and second-line drugs. As a result of this, it is urgent to develop new drugs to combat the resistant bacteria as well as have lower side effects, which can promote adherence to the treatment regimens. Targeting the de novo synthesis of thymidylate (dTMP) is an important pathway to develop drugs for TB. Although Mtb carries genes for two families of thymidylate synthases (TS), ThyA and ThyX, only ThyX is essential for its normal growth. Both enzymes catalyze the conversion of uridylate (dUMP) to dTMP but employ a different catalytic approach and have different structures. Also, ThyA is the only TS found in humans. This is the rationale for identifying selective inhibitors against ThyX. We exploited the NADPH oxidation to NADP+ step, catalyzed by ThyX, to develop a spectrophotometric biochemical assay. Success of the assay was demonstrated by its effectiveness (average Z'=0.77) and identification of selective ThyX inhibitors. The most potent compound is a tight-binding inhibitor with an IC50 of 710nM. Its mechanism of inhibition is analyzed in relation to the latest findings of ThyX mechanism and substrate and cofactor binding order. publisher: Elsevier articletitle: Discovery of a new Mycobacterium tuberculosis thymidylate synthase X inhibitor with a unique inhibition profile journaltitle: Biochemical Pharmacology articlelink: http://dx.doi.org/10.1016/j.bcp.2017.03.017 content_type: article copyright: © 2017 Published by Elsevier Inc. ispartof: Biochemical Pharmacology vol:135 pages:69-78 ispartof: location:England status: published

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 μg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Inhibition of mitochondrial metabolism for treatment-resistant tumors has attracted renewed attention. Pharmacologic inhibition of mitochondrial oxidative phosphorylation (OXPHOS) is efficacious in preclinical models of chemo-resistant AML, glycolysis-deficient glioma, and Swi/Snf mutant lung cancer, and is associated with an apparent robust safety index (Molina et al., 2018, Nat Med 24: 1036-1046; Lissanu Deribe et al., 2018, Nat Med 24: 1047-1057). In addition, immune-suppressive cell types in the tumor micro-environment depend on OXPHOS metabolism, including CD4+ regulatory T-cells (Tregs), whereas tumor-infiltrating effector T-cells instead rely on glycolytic metabolism (Angelin et al., 2017, Cell Metab. 25: 1282-1293). Here, we have used a phenotypic drug discovery approach to identify selective inhibitors of mitochondrial -but not glycolytic- tumor cell metabolism. Based on initial hits derived from a high-throughput screening campaign, a chemical series was optimized to achieve single digit nM potency (best IC50= 2 nM) in targeting OXPHOS-dependent cancer cells (grown on lactate as sole carbon source) but not glycolytic cells (grown on glucose; IC50 > 10 microM). Target deconvolution within this chemical series revealed 2 distinct mechanisms. One chemical subseries are direct inhibitors of the mitochondrial enzyme, dihydroorotate dehydrogenase (DHODH). Consistent with previous reports on DHODH inhibitors, these compounds potently impair AML cell proliferation (best IC50= 10nM) through induction of myeloid cell differentiation -a trait that can be rescued by providing exogenous uridine to the cell cultures (IC50 > 10 microM). A second chemically distinct subset of compounds inhibit mitochondrial OXPHOS, but does not inhibit DHODH. As expected, these compounds selectively target OXPHOS-dependent cancer cell lines, and display a robust selectivity window (determined in glycolysis-dependent cell lines). Moreover, these inhibitors do not affect a mixed-lymphocyte reaction (MLR) assay. In-depth metabolomic profiling in cancer cells fueled by glucose, lactate or glutamine, the precise molecular target of these compounds, and further in vivo characterization of these compounds will be presented. Finally, the unique opportunity to simultaneously inhibit both DHODH and OXPHOS using dual inhibitors will be evaluated in chemo-resistant AML models. Citation Format: Matthias Versele, Philippe Selhorst, Kristine Metzger, Marnik Nijs, Hugo Klaassen, Amuri Kilonda, Damien Marchand, Philippe Arzel, Dominique Lambin, Jean-Christophe Vanherck, Arnaud Marchand, Patrick Chaltin, Cyril Corbet, Olivier Feron. Discovery of novel DHODH and OXPHOS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3865.

FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and development of resistance has been reported. In this work we describe the identification of potent and novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This effort led to the identification of five compounds that were confirmed to be active on additional FLT3 dependent cell lines (cellular EC50 values between 35 and 700 nM), while having no significant effect on 24 other tyrosine kinases.