Exploration of Pyrido[3,4-d]pyrimidines as Antagonists of the Human Chemokine Receptor CXCR2.

Upregulated CXCR2 signalling is found in numerous inflammatory, autoimmune and neurodegenerative diseases, as well as in cancer. Consequently, CXCR2 antagonism is a promising therapeutic strategy for treatment of these disorders. We previously identified, via scaffold hopping, a pyrido[3,4-d]pyrimidine analogue as a promising CXCR2 antagonist with an IC50 value of 0.11 µM in a kinetic fluorescence-based calcium mobilization assay. This study aims at exploring the structure-activity relationship (SAR) and improving the CXCR2 antagonistic potency of this pyrido[3,4-d]pyrimidine via systematic structural modifications of the substitution pattern. Almost all new analogues completely lacked the CXCR2 antagonism, the exception being a 6-furanyl-pyrido[3,4-d]pyrimidine analogue (compound 17b) that is endowed with similar antagonistic potency as the original hit.

Optimization of triazolo[4,5-d]pyrimidines towards human CC chemokine receptor 7 (CCR7) antagonists.

CCR7 signaling directs the migration of both immune cells and cancer cells to the lymph nodes, is involved in numerous chronic inflammatory disorders and lymph node metastases. Despite the therapeutic promise of CCR7 antagonists, no potent and selective small molecule CCR7 antagonists have been reported to date. Since most human chemokine G protein-coupled receptors (GPCRs) share a conserved intracellular allosteric binding site, new CCR7 antagonist chemotypes may be identified by screening small molecules that are known to target this site in other chemokine GPCRs. In this work, our previously prepared series of 14 scaffold-modified analogues of a known thiazolo[4,5-d]pyrimidine CXCR2 antagonist were screened as potential CCR7 antagonists. This resulted in the discovery of a triazolo[4,5-d]pyrimidine analogue with an IC50 of 2.43 µM against CCR7 and 0.66 µM against CXCR2. Exploration of the structure-activity relationship (SAR) for the 3-, 5- and 7-position substituents of this triazolo[4,5-d]pyrimidine resulted in improved potency and selectivity, with an IC50 of 0.43 µM and 11.02 µM against CCR7 and CXCR2, respectively, for the most selective derivative. Molecular docking showed that the binding mode of these triazolo[4,5-d]pyrimidines in CCR7 and CXCR2 corresponds with those of previously co-crystallized ligands.

Systemic cytokines and GlycA discriminate disease status and predict corticosteroid response in HTLV-1-associated neuroinflammation.

BACKGROUND: HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is an incapacitating neuroinflammatory disorder for which no disease-modifying therapy is available, but corticosteroids provide some clinical benefit. Although HAM/TSP pathogenesis is not fully elucidated, older age, female sex and higher proviral load are established risk factors. We investigated systemic cytokines and a novel chronic inflammatory marker, GlycA, as possible biomarkers of immunopathogenesis and therapeutic response in HAM/TSP, and examined their interaction with established risk factors. PATIENTS AND METHODS: We recruited 110 People living with HTLV-1 (PLHTLV-1, 67 asymptomatic individuals and 43 HAM/TSP patients) with a total of 946 person-years of clinical follow-up. Plasma cytokine levels (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF) and GlycA were quantified by Cytometric Bead Array and 1NMR, respectively. Cytokine signaling and prednisolone response were validated in an independent cohort by nCounter digital transcriptomics. We used multivariable regression, machine learning algorithms and Bayesian network learning for biomarker identification. RESULTS: We found that systemic IL-6 was positively correlated with both age (r = 0.50, p < 0.001) and GlycA (r = 0.45, p = 0.00049) in asymptomatics, revealing an 'inflammaging" signature which was absent in HAM/TSP. GlycA levels were higher in women (p = 0.0069), but cytokine levels did not differ between the sexes. IFN-γ (p = 0.007) and IL-17A (p = 0.0001) levels were increased in untreated HAM/TSP Multivariable logistic regression identified IL-17A and proviral load as independent determinants of clinical status, resulting in modest accuracy of predicting HAM/TSP status (64.1%), while a machine learning-derived decision tree classified HAM/TSP patients with 90.7% accuracy. Pre-treatment GlycA and TNF levels significantly predicted clinical worsening (measured by Osame Motor Disability Scale), independent of proviral load. In addition, a poor prednisolone response was significantly correlated with higher post-treatment IFN-γ levels. Likewise, a transcriptomic IFN signaling score, significantly correlated with previously proposed HAM/TSP biomarkers (CASP5/CXCL10/FCGR1A/STAT1), was efficiently blunted by in vitro prednisolone treatment of PBMC from PLHTLV-1 and incident HAM/TSP. CONCLUSIONS: An age-related increase in systemic IL-6/GlycA levels reveals inflammaging in PLHTLV-1, in the absence of neurological disease. IFN-γ and IL-17A are biomarkers of untreated HAM/TSP, while pre-treatment GlycA and TNF predict therapeutic response to prednisolone pulse therapy, paving the way for a precision medicine approach in HAM/TSP.

Can we attenuate ischaemia-reperfusion injury of allografts in a porcine left lung transplant models by adsorption of cytokines?

OBJECTIVES: Primary graft dysfunction resulting from ischaemia-reperfusion injury remains a major obstacle after lung transplantation (LTx) and is associated with morbidity and mortality. Continuous release of inflammatory cytokines, due to the process of ischaemia and reperfusion, triggers a complex cascade of apoptosis and necrosis resulting in graft dysfunction. Previous studies demonstrated successful graft improvement by cytokine filtration during ex vivo lung perfusion. We hypothesize that plasma cytokine filtration with CytoSorb® during in vivo graft perfusion immediately after implantation may attenuate ischaemia-reperfusion injury after left LTx in a porcine model. METHODS: Left porcine LTx was performed with allografts preserved for 24 h at 4°C. In the treatment group [T] (n = 7), a veno-venous shunt was created to insert the cytokine filter (CytoSorbents, Berlin, Germany). In the sham group [S] (n = 4), the shunt was created without the filter. Haemodynamic parameters, lung mechanics, blood gases and plasma cytokines were assessed during 6 h in vivo reperfusion. RESULTS: During 6 h of reperfusion, significant differences in plasma pro-inflammatory cytokine [interferon (IFN)-α, IFN-γ and interleukin (IL)-6] concentrations were observed between [T] and [S], but surprisingly with higher plasma levels in the [T] group. Plasma concentrations of other pro-inflammatory cytokines (IL-1ß, IL-12p40, IL-4, IL-6, IL-8, IFN-α, IFN-γ and tumour necrosis factor-α) and anti-inflammatory cytokines (IL-10) did not find any evidence for a difference. Furthermore, our study failed to show meaningful difference in haemodynamics and blood gases. Also, no statistically significant differences were found between [T] and [S] in biopsies and wet-to-dry ratio at the end of the experiment. CONCLUSIONS: In our porcine left LTx model cytokine filtration did not achieve the intended effect. This is in contrast to previous studies with CytoSorb use during ex vivo lung perfusion as a surrogate LTx model. Our findings might highlight the fact that the theoretical benefit of inserting an additional cytokine adsorber to improve graft function in clinical practice should be critically evaluated with further studies.

SARS-CoV-2 Virion Infectivity and Cytokine Production in Primary Human Airway Epithelial Cells.

The emergence of new SARS-CoV-2 variants and the replacement of preceding isolates have been observed through B.1.1.7, B.1.351, B.1.617.2, and B.1.1.529 lineages (corresponding to alpha, beta, delta, and omicron variants of concern (VoC), respectively). However, there is still a lack of biological evidence to which extent those VoC differ from the ancestral lineages. By exploiting human airway epithelial cell (HAEC) cultures, which closely resemble the human airway architecture and physiology, we report distinctive SARS-CoV-2 tropism in different respiratory tissues. In general, SARS-CoV-2 VoC predominantly infect and replicate in HAEC better than the progenitor USA-WA1 isolate or the BavPat1 isolate, which contains the D614G mutation, even though there is little to no difference between variants regarding their infectivity (i.e., virion-per-vRNA copy ratio). We also observe differential tissue-specific innate immunity activation between the upper and lower respiratory tissues in the presence of the virus. Our study provides better comprehension of the behavior of the different VoC in this physiologically relevant ex vivo model.

Effect of Particle Carriers for Intraperitoneal Drug Delivery on the Course of Ovarian Cancer and Its Immune Microenvironment in a Mouse Model.

Novel treatment strategies are needed to provide a better prognosis for ovarian cancer. For this purpose, the current study was designed to evaluate the effects of different types of particle drug carriers on tumor response and on the tumor immune microenvironment (TME) after intraperitoneal (IP) administration in a murine tumor model. Mice with ID8-fLuc ovarian cancer were injected IP with pegylated liposomes, hydroxyapatite, polystyrene, poly(lactic-co-glycolic acid) (PLGA) and calcium carbonate (CaCO3) microparticles to evaluate the effect of the candidate carriers without drugs. Our results show that several types of microparticle drug carriers caused hyperproliferation of the tumor when injected IP, as reflected in a reduced survival or an accelerated onset of ascites. Alterations of the product formulation of CaCO3 microparticles could result in less hyperproliferation. The hyperproliferation caused by CaCO3 and PLGA was largely driven by a strong innate immune suppression. A combination with chemotherapy was not able to sufficiently counteract the tumor progression caused by the drug carriers. This research points towards the importance of evaluating a drug carrier before using it in a therapeutic setting, since drug carriers themselves can detrimentally influence tumor progression and immune status of the TME. However, it remains to be determined whether the hyperproliferation in this model will be of relevance in other cancer models or in humans.

Neo-Adjuvant Chemotherapy Reduces, and Surgery Increases Immunosuppression in First-Line Treatment for Ovarian Cancer.

In monotherapy, immunotherapy has a poor success rate in ovarian cancer. Upgrading to a successful combinatorial immunotherapy treatment implies knowledge of the immune changes that are induced by chemotherapy and surgery. METHODOLOGY: Patients with a new d ovarian cancer diagnosis underwent longitudinal blood samples at different time points during primary treatment. RESULTS: Ninety patients were included in the study (33% primary debulking surgery (PDS) with adjuvant chemotherapy (ACT), 61% neo-adjuvant chemotherapy (NACT) with interval debulking surgery (IDS), and 6% debulking surgery only). Reductions in immunosuppression were observed after NACT, but surgery reverted this effect. The immune-related proteins showed a pronounced decrease in immune stimulation and immunosuppression when primary treatment was completed. NACT with IDS leads to a transient amelioration of the immune microenvironment compared to PDS with ACT. CONCLUSION: The implementation of immunotherapy in the primary treatment schedule of ovarian cancer cannot be induced blindly. Carboplatin-paclitaxel seems to ameliorate the hostile immune microenvironment in ovarian cancer, which is less pronounced at the end of primary treatment. This prospective study during primary therapy for ovarian cancer that also looks at the evolution of immune-related proteins provides us with an insight into the temporary windows of opportunity in which to introduce immunotherapy during primary treatment.

Synthesis and Anti-HIV Activity of a Novel Series of Isoquinoline-Based CXCR4 Antagonists.

An expansion of the structure-activity relationship study of CXCR4 antagonists led to the synthesis of a series of isoquinolines, bearing a tetrahydroquinoline or a 3-methylpyridinyl moiety as head group. All compounds were investigated for CXCR4 affinity and antagonism in competition binding and calcium mobilization assays, respectively. In addition, the anti-HIV activity of all analogues was determined. All compounds showed excellent activity, with compound 24c being the most promising one, since it displayed consistently low nanomolar activity in the various assays.

Labyrinthopeptin A1 inhibits dengue and Zika virus infection by interfering with the viral phospholipid membrane.

To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC50 around 1 µM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses.

Discovery of HIV entry inhibitors via a hybrid CXCR4 and CCR5 receptor pharmacophore-based virtual screening approach.

Chemokine receptors are key regulators of cell migration in terms of immunity and inflammation. Among these, CCR5 and CXCR4 play pivotal roles in cancer metastasis and HIV-1 transmission and infection. They act as essential co-receptors for HIV and furnish a route to the cell entry. In particular, inhibition of either CCR5 or CXCR4 leads very often the virus to shift to a more virulent dual-tropic strain. Therefore, dual receptor inhibition might improve the therapeutic strategies against HIV. In this study, we aimed to discover selective CCR5, CXCR4, and dual CCR5/CXCR4 antagonists using both receptor- and ligand-based computational methods. We employed this approach to fully incorporate the interaction attributes of the binding pocket together with molecular dynamics (MD) simulations and binding free energy calculations. The best hits were evaluated for their anti-HIV-1 activity against CXCR4- and CCR5-specific NL4.3 and BaL strains. Moreover, the Ca2+ mobilization assay was used to evaluate their antagonistic activity. From the 27 tested compounds, three were identified as inhibitors: compounds 27 (CCR5), 6 (CXCR4) and 3 (dual) with IC50 values ranging from 10.64 to 64.56 µM. The binding mode analysis suggests that the active compounds form a salt bridge with the glutamates and π-stacking interactions with the aromatic side chains binding site residues of the respective co-receptor. The presented hierarchical virtual screening approach provides essential aspects in identifying potential antagonists in terms of selectivity against a specific co-receptor. The compounds having multiple heterocyclic nitrogen atoms proved to be relatively more specific towards CXCR4 inhibition as compared to CCR5. The identified compounds serve as a starting point for further development of HIV entry inhibitors through synthesis and quantitative structure-activity relationship studies.

Early protein expression profile in bronchoalveolar lavage fluid and clinical outcomes in primary graft dysfunction after lung transplantation.

OBJECTIVES: Primary graft dysfunction (PGD) remains a major post-transplant complication and is associated with increased morbidity and mortality. Mechanisms evoking PGD are not completely clear, but inflammation plays a central role. We investigated the association between PGD and inflammatory proteins present in immediate postoperative bronchoalveolar lavage. METHODS: All double-lung recipients transplanted at our institution from 2002 to 2018 were included in our study. We retrospectively selected 80 consecutive lung transplant recipients with different PGD grades (n = 20 for each PGD grades 0-1 to 2-3). In bronchoalveolar lavage performed within the first 24 h after donor aortic cross-clamping following lung transplantation, concentrations of 30 cytokines, chemokines and growth factors were assessed by enzyme-linked immunosorbent assay (ELISA) and correlated with donor and recipient demographics and outcomes. For analysis, 2 groups were defined: 'mild' PGD (grade 0-1) and 'severe' PGD (grades 2-3). RESULTS: Significant differences between mild and severe PGD were found in 8 biomarkers [interleukin (IL)-6, IL-10, IL-13, eotaxin, granulocyte colony-stimulating factor, interferon γ, macrophage inflammatory protein 1α, surfactant protein D (SP-D); P < 0.05]. Increased IL-10 and IL-13, but none of the other proteins, were associated with short-term outcome (longer time to extubation; P = 0.005 and P < 0.0001; increased intensive care unit stay; P = 0.012 and P < 0.0001; and hospital stay; P = 0.041 and P = 0.002). There were no significant differences in donor and recipient characteristics between the groups. CONCLUSIONS: Expression profiles of key inflammatory mediators in bronchoalveolar lavage fluid differed significantly between lung transplant recipients with severe versus mild PGD and correlated with clinical outcome variables. Further research should focus on the early mechanisms leading to PGD.

A unique class of lignin derivatives displays broad anti-HIV activity by interacting with the viral envelope.

In Gordts et al. (2015), we have shown that lignosulfonic acid, a commercially available lignin derivative, possesses broad antiviral activity against human immunodeficiency virus (HIV) and Herpes simplex virus (HSV) by preventing viral entry into susceptible target cells. Because of the interesting safety profile as potential microbicide, we now determined the antiviral activity of a series of lignosulfonates in order to understand better which molecular features can contribute to their antiviral activity. Here, 24 structurally different lignosulfonates were evaluated for their capacity to inhibit HIV and HSV transmission and replication in various cellular assays. These derivatives differ in origin (hardwood or softwood), counter-ion used during sulphite processing (Na+, Ca2+, or NH4+), sulphur content, carboxylic acid percentage, and molecular weight fraction, which allowed to determine structure-activity relationships. We demonstrate that the broad antiviral activity of lignosulfonates is mainly dependent on their molecular weight and that their mechanism of action is based on interactions with the viral envelope glycoproteins. This makes the lignosulfonates a potential low-cost microbicide that protects women from sexual HIV and HSV transmission and thus prevents life-long infection.

Prone Positioning During Ex Vivo Lung Perfusion Influences Regional Edema Accumulation.

BACKGROUND: Ex vivo lung perfusion (EVLP) is developed to increase the quantity and quality of suitable grafts for lung transplantation. Standardly, lungs are mounted supine with the risk of fluid accumulation in the dorsal regions. Therefore, we investigated the impact of experimental prone position on graft function during EVLP. MATERIALS AND METHODS: Porcine lungs were mounted on a normothermic EVLP for 6 h in supine [S], (n = 7) or prone position [P], (n = 7). Physiology during EVLP was recorded. After EVLP, biopsies were assessed for wet-to-dry weight (W/D) ratios and pathology, broncho-alveolar lavage was measured, and the left lung was computed tomography (CT) scanned. RESULTS: Physiological parameters were similar between both groups, despite a higher pulmonary vascular resistance in [P] (P = 0.0002). In [S], W/D ratios and CT density of dorsal areas were higher compared to ventral (P = 0.0017 and P = 0.053, respectively). In [P], W/D and CT density between ventral and dorsal regions were similar, meaning that pulmonary edema was distributed more homogeneously throughout the lung. Histology and cytokine levels in perfusate and broncho-alveolar lavage did not differ between both groups. CONCLUSIONS: Prone positioning during EVLP is feasible and leads to more homogenous distribution of interstitial fluid. Supine position resulted in more concentrated edema accumulation in lower dependent regions.

Immunoregulatory effects of multipotent adult progenitor cells in a porcine ex vivo lung perfusion model.

BACKGROUND: Primary graft dysfunction (PGD) is considered to be the end result of an inflammatory response targeting the new lung allograft after transplant. Previous research has indicated that MAPC cell therapy might attenuate this injury by its paracrine effects on the pro-/anti-inflammatory balance. This study aims to investigate the immunoregulatory capacities of MAPC cells in PGD when administered in the airways. METHODS: Lungs of domestic pigs (n = 6/group) were subjected to 90 minutes of warm ischemia. Lungs were cold flushed, cannulated on ice and placed on EVLP for 6 hours. At the start of EVLP, 40 ml of an albumin-plasmalyte mixture was distributed in the airways (CONTR group). In the MAPC cell group, 150 million MAPC cells (ReGenesys/Athersys, Cleveland, OH, USA) were added to this mixture. At the end of EVLP, a physiological evaluation (pulmonary vascular resistance, lung compliance, PaO2/FiO2), wet-to-dry weight ratio (W/D) sampling and a multiplex analysis of bronchoalveolar lavage (BAL) (2 × 30 ml) was performed. RESULTS: Pulmonary vascular resistance, lung compliance, PaO2/FiO2 and W/D were not statistically different at the end of EVLP between both groups. BAL neutrophilia was significantly reduced in the MAPC cell group. Moreover, there was a significant decrease in TNF-α, IL-1ß and IFN-γ in the BAL, but not in IFN-α; whereas IL-4, IL-10 and IL-8 were below the detection limit. CONCLUSIONS: Although no physiologic effect of MAPC cell distribution in the airways was detected during EVLP, we observed a reduction in pro-inflammatory cytokines and neutrophils in BAL in the MAPC cell group. This effect on the innate immune system might play an important role in critically modifying the process of PGD after transplantation. Further experiments will have to elucidate the immunoregulatory effect of MAPC cell administration on graft function after transplantation.