Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance

Novel Therapeutic Strategies for the Treatment of Infections with Pestiviruses

The pestiviruses bovine viral diarrhoea viruses (BVDV), classical swine fever (CSFV) and border disease virus (BDV) are important pathogens for livestock. These viruses are worldwide responsible for significant economic losses. Infection of cattle with BVDV causes a spectrum of clinical signs, ranging from asymptomatic to severe disease potentially leading to death. The major losses due to BVDV infection include growth retardation and reduced milk production. BVDV crosses the placenta and infects the foetus, resulting, in persistently infected (PI) animals. PI animals continuously shed massive amount of virus during their life-time and serve as the reservoir of the virus in cattle herds. BVDV is endemic in bovine populations worldwide. In the last decades programs have been implemented to eliminate PI animals. The classic symptoms caused by infection with the CSFV in pigs are pyrexia, severe leucopoenia, haemorrhages and neurologic problems. This results in high morbidity and mortality. CSFV has been eradicated in parts of the world, i.e. the US, the EU and Australia, but it is still endemic in many other regions such as for example in Eastern European countries, Asia and South America. Vaccination and stamping out have been used for the control of CSFV. Since the control of CSFV in endemic regions remains problematic and since reintroduction of CSFV in virus-free regions results in a serious burden, new control measures for CSFV are needed. Moreover, given the enormous economic impact of BVDV, additional measures are needed for the control of this virus. Highly potent antiviral drugs may be an excellent tool to help control pestivirus infection. Efficient antiviral drugs have the benefit to result almost instantaneously in a protective effect (in contrast to vaccines where there is an immunity gap between vaccination and protection) and they may, in the case of CSFV control, be useful to close the immunity gap. Rapid control of outbreaks with CSFV will largely reduce the number of healthy animals that will need to be culled in the containment perimeter around the infected premises and help to minimize disruption of trade and thus to decrease the economic impact of the disease. Eliminating PI animals without prophylactic antivirals or vaccination of non-infected animals on a farm where a PI calf has been identified is currently regarded as an important control measure. In the last 10 to 15 years, our laboratory and others, identified different chemical classes of pestivirus inhibitors. The effect of two compounds (DB772 and BPIP) has been assessed in infected animals, DB772 in BVDV infected mini-zebu cows and the imidazopyridine BPIP (a compound discovered in our laboratory) in CSFV infected pigs. BPIP was shown to be effective in reducing viral load in infected pigs and to reduce transmission to sentinels. This provided the first proof-of-concept that a specific antiviral therapy has indeed great potential for the control of pestivirus infections in livestock. In a large scale screening effort aimed at identifying novel pestivirus inhibitors, we discovered several classes of potent and selective inhibitors of the in vitro replication of pestiviruses. To further optimize the antiviral activity hit-optimization programs were initiated for three different classes [2,6-bis(benzimidazol-2-yl)pyridine, BBP/CSFA-0; imidazolidine, BTB02541SC/CSFB-0; Quinolinecarboxamides, TO502-2403/CSFC-00 and TO505-6180/CSFC-0]. The structure-activity relationship (SAR) was carefully studied for these three chemical classes in an attempt to further improve the antiviral activity and selectivity. In parallel, the particular characteristics and mechanism of action of these (and yet some other) classes of compounds was studied. To this end, it was first attempted to select drug-resistant variants; which were next genotyped to identify in which gene (a) mutation(s) were accumulating. Surprisingly, for all classes of pestivirus inhibitors studied, the resistance-conferring mutations were located in the viral RNA-dependent RNA polymerase (RdRp). Moreover, the structurally very different classes of compounds proved all to be cross-resistant to each other as well as to earlier reported classes of pestivirus inhibitors (BPIP, AG110 and LZ37). All mutations identified were located in the fingertip of the RdRp. Remarkably, none of these compounds inhibited the activity of the purified BVDV RdRp but efficiently blocked the activity of viral replication complexes. Thus the different classes of pestivirus inhibitors all target a hotspot in the viral polymerase thereby inhibiting the functioning of the replication complex of which the RdRp is a component. Furthermore, our data demonstrate that this hotspot in the finger domain of the pestivirus RdRp is likely, and this in stark contrast to the situation for the related hepatitis virus, the only allosteric pocket in the polymerase. Inhibitors that bind to this pocket prevent the proper functioning of the RdRp (or interaction with other proteins or RNA) in the context of the viral replication complex. The findings reported in this thesis will contribute to the further development of additional control measures for pestiviral infections in livestock.status: publishe

Quinolinecarboxamides Inhibit the Replication of the Bovine Viral Diarrhea Virus by Targeting a Hot Spot for the Inhibition of Pestivirus Replication in the RNA-Dependent RNA Polymerase

The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a "hot spot" for the inhibition of pestivirus replication.status: publishe

3-Biphenylimidazo[1,2-a]pyridines or [1,2-b]pyridazines and analogues, novel Flaviviridae inhibitors

International audienceUsing Ttou 84 as starting point, a novel class of biphenyl derivatives of imidazo[1,2-a]pyridine and imidazo[1,2-b]pyridazine was designed to optimize the inhibitory properties on the replication of the bovine viral diarrhoea virus (BVDV) and hepatitis C virus (HCV). Three sites of pharmacomodulation were chosen i.e. positions 2, 3 and 6 on the central heterocyclic core structure. From the 49 analogues tested, only compound 18j (3-(2'-hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-b]pyrida zine) showed antiviral activity in the HCV replicon system reminiscent of selective inhibition (60-70% inhibition). Compound 4f (3-(biphen-3-yl)-2-(4-fluorophenyl)-6-phenylthioimidazo[1,2-a]pyridine) proved to be the most selective inhibitor of BVDV replication and showed no or only marginal cross-resistance with known inhibitors of pestivirus replication. The cross-resistance profile of 4f might indicate that 4f does not interact with the same binding site as BPIP, VP32947, AG110 or LZ37. From 42 analogues tested against both viruses, QSAR studies were discussed in regard to BVDV antiviral activity

Substituted 2,6-bis(benzimidazol-2-yl)pyridines: A novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase

2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1μM, 0.05±0.01μM and 0.3±0.04μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1μM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication.publisher: Elsevier articletitle: Substituted 2,6-bis(benzimidazol-2-yl)pyridines: A novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase journaltitle: Antiviral Research articlelink: http://dx.doi.org/10.1016/j.antiviral.2014.03.010 content_type: article copyright: Copyright © 2014 Elsevier B.V. All rights reserved.status: publishe

Highly potent and selective inhibition of bovine viral diarrhea virus replication by γ-carboline derivatives

Several novel γ-carboline derivatives were identified as selective inhibitors of bovine viral diarrhea virus (BVDV) replication in cell cultures. Among them, 3,4,5-trimethyl-γ-carboline (SK3M4M5M) was the most active against BVDV (Nose strain) in MDBK cells, with a 50% effective concentration of 0.017±0.005μM and a selectivity index of 435. The compound inhibited viral RNA synthesis in a dose-dependent fashion. In a time of drug-addition experiment during a single viral replication cycle, SK3M4M5M lost its antiviral activity when first added at 8h or later after infection, which coincides with the onset of viral RNA synthesis. When selected γ-carboline derivatives, including SK3M4M5M, were examined for their inhibitory effect on the mutant strains resistant to some classes of nonnucleoside BVDV RNA-dependent RNA polymerase inhibitors, all of which target the top of the finger domain of the polymerase, the strains displayed cross-resistance to the γ-carboline derivatives. These results indicate that the γ-carboline derivatives may possibly target a hot spot of the RNA-dependent RNA polymerase. Although SK3M4M5M was highly active against BVDV, the compound proved inactive against hepatitis C virus (HCV) in HCV RNA replicon cells.status: publishe

Baricitinib restrains the immune dysregulation in severe COVID-19 patients

COVID-19 patients develop pneumonia generally associated to lymphopenia and severe inflammatory response due to uncontrolled cytokine release. These mediators are transcriptionally regulated by the JAK-STAT signaling pathways, which can be disabled by small molecules

Immune checkpoint blockade therapy mitigates systemic inflammation and affects cellular FLIP-expressing monocytic myeloid-derived suppressor cells in non-progressor non-small cell lung cancer patients

ABSTRACTCancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1β-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients’ outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1β in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression

Fatal cytokine release syndrome by an aberrant FLIP/STAT3 axis

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-CoV-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homolog in myeloid cells triggered a STAT3-linked, progressive, and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury, and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state