Generic design principles for antibody-based tumour necrosis factor (TNF) receptor 2 (TNFR2) agonists with FcγR-independent agonism.

Background: Selective TNFR2 activation can be used to treat immune pathologies by activating and expanding regulatory T-cells (Tregs) but may also restore anti-tumour immunity by co-stimulating CD8+ T-cells. Oligomerized TNFR2-specific TNF mutants or anti-TNFR2 antibodies can activate TNFR2 but suffer either from poor production and pharmacokinetics or in the case of anti-TNFR2 antibodies typically from the need of FcγR binding to elicit maximal agonistic activity. Methods: To identify the major factor(s) determining FcγR-independent agonism of anti-TNFR2 antibodies, we systematically investigated a comprehensive panel of anti-TNFR2 antibodies and antibody-based constructs differing in the characteristics of their TNFR2 binding domains but also in the number and positioning of the latter. Results: We identified the domain architecture of the constructs as the pivotal factor enabling FcγR-independent, thus intrinsic TNFR2-agonism. Anti-TNFR2 antibody formats with either TNFR2 binding sites on opposing sites of the antibody scaffold or six or more TNFR2 binding sites in similar orientation regularly showed strong FcγR-independent agonism. The affinity of the TNFR2 binding domain and the epitope recognized in TNFR2, however, were found to be of only secondary importance for agonistic activity. Conclusion: Generic design principles enable the generation of highly active bona fide TNFR2 agonists from nearly any TNFR2-specific antibody.

Antibody-based soluble and membrane-bound TWEAK mimicking agonists with FcγR-independent activity.

Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK.

Neutrophil-intrinsic TNF receptor signaling orchestrates host defense against Staphylococcus aureus.

Staphylococcus aureus is the leading cause of skin and soft tissue infections and is a major health burden due to the emergence of antibiotic-resistant strains. To address the unmet need of alternative treatments to antibiotics, a better understanding of the protective immune mechanisms against S. aureus skin infection is warranted. Here, we report that tumor necrosis factor (TNF) promoted protection against S. aureus in the skin, which was mediated by bone marrow-derived immune cells. Furthermore, neutrophil-intrinsic TNF receptor (TNFR) signaling directed immunity against S. aureus skin infections. Mechanistically, TNFR1 promoted neutrophil recruitment to the skin, whereas TNFR2 prevented systemic bacterial dissemination and directed neutrophil antimicrobial functions. Treatment with a TNFR2 agonist showed therapeutic efficacy against S. aureus and Pseudomonas aeruginosa skin infections, which involved increased neutrophil extracellular trap formation. Our findings revealed nonredundant roles for TNFR1 and TNFR2 in neutrophils for immunity against S. aureus and can be therapeutically targeted for protection against bacterial skin infections.

Design, Synthesis, and Anticancer Screening for Repurposed Pyrazolo[3,4-d]pyrimidine Derivatives on Four Mammalian Cancer Cell Lines.

The present study reports the synthesis of new purine bioisosteres comprising a pyrazolo[3,4-d]pyrimidine scaffold linked to mono-, di-, and trimethoxy benzylidene moieties through hydrazine linkages. First, in silico docking experiments of the synthesized compounds against Bax, Bcl-2, Caspase-3, Ki67, p21, and p53 were performed in a trial to rationalize the observed cytotoxic activity for the tested compounds. The anticancer activity of these compounds was evaluated in vitro against Caco-2, A549, HT1080, and Hela cell lines. Results revealed that two (5 and 7) of the three synthesized compounds (5, 6, and 7) showed high cytotoxic activity against all tested cell lines with IC50 values in the micro molar concentration. Our in vitro results show that there is no significant apoptotic effect for the treatment with the experimental compounds on the viability of cells against A549 cells. Ki67 expression was found to decrease significantly following the treatment of cells with the most promising candidate: drug 7. The overall results indicate that these pyrazolopyrimidine derivatives possess anticancer activity at varying doses. The suggested mechanism of action involves the inhibition of the proliferation of cancer cells.

The NEDD8-activating enzyme inhibition with MLN4924 sensitizes human cancer cells of different origins to apoptosis and necroptosis.

OBJECTIVES: MLN4924 is an inhibitor of NEDD8-activating enzyme (NAE) that interferes with the cullin-RING ubiquitin ligase complexes formation and the nuclear factor kappa B (NF-κB) activation. Here, we investigated the cytotoxic effect of MLN4924 and its ability to sensitize a broad range of cancer cells of different origins to tumour necrosis factor-α (TNF)-induced cell death alongside unravelling its mechanism of action. MATERIALS AND METHODS: Cell viability and caspases processing were determined after MLN4924 treatment either alone or with zVAD-fmk (pan caspase inhibitor), necrostatin-1 (nec-1, RIPK1 inhibitor) and necrosulfonamide (NSA, MLKL inhibitor). Moreover, MLN4924 ability to potentiate TNF-induced cell death was evaluated in 24 cell lines of different cancer origins. The impact of NAE inhibition with MLN4924 on TNF-induced apoptosis and necroptosis was evaluated using zVAD-fmk and nec-1, respectively. RESULTS: MLN4924 alone was able to induce cell death in different cell lines that was attributed to apoptosis induction. Also, MLN4924 sensitized different cancer cell lines to TNF-induced cell death. MLN4924/TNF-induced cell death was apoptosis and necroptosis dependent that may be attributed to MLN4924 inhibition of NF-κB pathway activation. CONCLUSIONS: Targeting NAE and NF-κB pathway with MLN4924 represents a substantial approach to enhance the sensitivity of diverse types of cancer cells. Moreover, the broad in vitro screening of MLN4924 anticancer activity provides a valuable guidance for elucidating the susceptible cancer types for the prospective clinical application of MLN4924.

A Near-Complete Series of Four Atropisomeric Jozimine A2-Type Naphthylisoquinoline Dimers with Antiplasmodial and Cytotoxic Activities and Related Alkaloids from Ancistrocladus abbreviatus.

Three new naphthylisoquinoline dimers, jozibrevines A-C (1a-c), were isolated from the West African shrub Ancistrocladus abbreviatus, along with the known dimer jozimine A2 (1d). The two molecular moieties of 1a-d are coupled via the sterically constrained 3',3″-positions of their two naphthalene units, so that the central biaryl linkage is rotationally hindered. With the two outer axes also being chiral, 1a-d possess three consecutive stereogenic axes. The four isolated dimers all have the same constitutions and identical absolute configurations at the four stereogenic centers, but differ by their axial chirality. They belong to the extremely small class of Dioncophyllaceae-type naphthylisoquinoline dimers, i.e., being devoid of oxygen functions at C-6 and bearing the R-configuration at C-3 in their isoquinoline portions. Besides these dimers, the plant produces predominantly typical Ancistrocladaceae-type monomeric compounds, i.e., with the S-configuration at C-3 and an oxygen function at C-6, such as the new ancistrobrevines K (5) and L (6). Furthermore, a new hybrid-type (i.e., mixed Ancistrocladaceae/Dioncophyllaceae-type) alkaloid was identified, named ancistrobrevine M (7), which is 3R-configured and 6-oxygenated. Remarkable was the discovery of its "inverse hybrid-type" counterpart, dioncoline A (8). It is the as yet only known 3S-configured naphthylisoquinoline lacking an O-functionality at C-6. The new jozibrevines A-C (1a-c) exhibited pronounced antiplasmodial activities in the submicromolar range, with 1a being the most potent compound (IC50, 0.012 µM). Furthermore, jozimine A2 (1d) showed cytotoxicity against human colon carcinoma (HT-29), fibrosarcoma (HT1080), and multiple myeloma (MM.1S) cancer cells, displaying IC50 values of 12.0, 9.0, and 5.0 µM, respectively, whereas jozibrevines A (1a) and B (1b) were nontoxic in this concentration range.

Bioactive Brominated Oxindole Alkaloids from the Red Sea Sponge Callyspongia siphonella.

In the present study, LC-HRESIMS-assisted dereplication along with bioactivity-guided isolation led to targeting two brominated oxindole alkaloids (compounds 1 and 2) which probably play a key role in the previously reported antibacterial, antibiofilm, and cytotoxicity of Callyspongia siphonella crude extracts. Both metabolites showed potent antibacterial activity against Gram-positive bacteria, Staphylococcus aureus (minimum inhibitory concentration (MIC) = 8 and 4 µg/mL) and Bacillus subtilis (MIC = 16 and 4 µg/mL), respectively. Furthermore, they displayed moderate biofilm inhibitory activity in Pseudomonas aeruginosa (49.32% and 41.76% inhibition, respectively), and moderate in vitro antitrypanosomal activity (13.47 and 10.27 µM, respectively). In addition, they revealed a strong cytotoxic effect toward different human cancer cell lines, supposedly through induction of necrosis. This study sheds light on the possible role of these metabolites (compounds 1 and 2) in keeping fouling organisms away from the sponge outer surface, and the possible applications of these defensive molecules in the development of new anti-infective agents.

Soluble TNF-like weak inducer of apoptosis (TWEAK) enhances poly(I:C)-induced RIPK1-mediated necroptosis.

TNF-like weak inducer of apoptosis (TWEAK) and inhibition of protein synthesis with cycloheximide (CHX) sensitize for poly(I:C)-induced cell death. Notably, although CHX preferentially enhanced poly(I:C)-induced apoptosis, TWEAK enhanced primarily poly(I:C)-induced necroptosis. Both sensitizers of poly(I:C)-induced cell death, however, showed no major effect on proinflammatory poly(I:C) signaling. Analysis of a panel of HeLa-RIPK3 variants lacking TRADD, RIPK1, FADD, or caspase-8 expression revealed furthermore similarities and differences in the way how poly(I:C)/TWEAK, TNF, and TRAIL utilize these molecules for signaling. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for this response in TNF and TRAIL signaling. TRADD-RIPK1-double deficiency differentially affected poly(I:C)-triggered gene induction but abrogated gene induction by TNF completely. FADD deficiency abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis, whereas TRADD elicited protective activity against all three death inducers. A general protective activity against poly(I:C)-, TRAIL-, and TNF-induced cell death was also observed in FLIPL and FLIPS transfectrants.

New Cytotoxic Cyclic Peptide from the Marine Sponge-Associated Nocardiopsis sp. UR67.

A new cyclic hexapeptide, nocardiotide A (1), together with three known compounds-tryptophan (2), kynurenic acid (3), and 4-amino-3-methoxy benzoic acid (4)-were isolated and identified from the broth culture of Nocardiopsis sp. UR67 strain associated with the marine sponge Callyspongia sp. from the Red Sea. The structure elucidation of the isolated compounds were determined based on detailed spectroscopic data including ¹D and ²D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while the absolute stereochemistry of all amino acids components of nocardiotide A (1) was deduced using Marfey's method. Additionally, ten known metabolites were dereplicated using HR-ESI-MS analysis. Nocardiotide A (1) displayed significant cytotoxic effects towards the murine CT26 colon carcinoma, human HeLa cervix carcinoma, and human MM.1S multiple myeloma cell lines. The results obtained revealed sponge-associated Nocardiopsis as a substantial source of lead natural products with pronounced pharmacological activities.

Vascular Endothelial Growth Factor Expression in Hepatitis C Virus-Induced Liver Fibrosis: A Potential Biomarker.

The major complication of hepatitis C virus (HCV) infection is the induction of hepatic fibrosis. In this study, we investigated the correlation between the expression level of vascular endothelial growth factor (VEGFA) at mRNA and protein levels and the progression of HCV-related liver fibrosis. One hundred twenty subjects were selected for this study: 15 controls and 105 chronic HCV patients with different fibrosis grades (44 F0-F1 and 61 F2-F4). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure VEGFA mRNA in peripheral blood mononuclear cells, while enzyme-linked immunosorbent assay (ELISA) was used to measure the secreted VEGFA protein in serum. Both qRT-PCR and ELISA results showed that HCV patients have significantly higher VEGFA expression than that of controls (P = 0.036 and 0.043, respectively). Moreover, patients with late fibrotic stages (F2-F4) exhibited the highest levels of VEGFA mRNA and protein (P = 0.008 and 0.041, respectively) when compared with controls. An area under the receiver operating characteristic curve (AUC of the ROC) for the circulatory VEGFA protein between HCV patients with fibrosis and healthy controls was 0.92 (P = 0.043). Our data suggest that VEGFA protein is a promising noninvasively diagnostic biomarker for HCV-induced liver fibrosis.

Impact of OAS1 Exon 7 rs10774671 Genetic Variation on Liver Fibrosis Progression in Egyptian HCV Genotype 4 Patients.

The aim of this study was to assess the impact of genetic variants of oligoadenylate synthetase 1 (OAS1) single-nucleotide polymorphism (SNP) rs10774671 at the exon 7 splice acceptor site on liver fibrosis progression and hepatitis C virus (HCV) outcome in Egyptian HCV genotype 4 patients. In this study, 195 subjects were enrolled; 60 controls and 135 chronic HCV genotype 4 patients with different fibrosis grades. All subjects were genotyped for OAS1 SNP rs10774671 polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. There was an increasing trend of liver fibrosis progression as 52.9% GG, 73.6% GA, and 83.3% AA genotypes were detected in late fibrosis patients (p = 0.025). The AA genotype was higher in the late fibrosis group than in the early fibrosis group (83.3% vs. 16.7%) (p = 0.001). The A allele was significantly affecting the liver fibrosis progression rate, more than the G allele (p = 0.001). The multivariate analysis showed that the OAS1 GA and AA genotypes were independent factors associated with liver progression (p = 0.009, odds ratio [OR] 3.467, 95% confidence interval [CI] 1.273-7.584). In addition, the A allele was associated with liver fibrosis progression (p = 0.014, OR 2.525, 95% CI 1.157-4.545). The polymorphism at OAS1 exon 7 rs3741981 might be a potential genetic marker and can be useful in the assessment of liver fibrosis progression and disease outcome in HCV-infected patients.

Association of Myxovirus Resistance Gene Promoter Polymorphism with Response to Combined Interferon Treatment and Progression of Liver Disease in Chronic HCV Egyptian Patients.

To evaluate the frequency of single-nucleotide polymorphism at the -88 myxovirus resistance (MxA) gene promoter region in relation to the status of hepatitis C virus (HCV) progression and response to combined interferon (IFN) in chronic HCV Egyptian patients. One hundred ten subjects were enrolled in the study; 60 HCV genotype 4-infected patients who underwent combined IFN therapy and 50 healthy individuals. All subjects were genotyped for -88 MxA polymorphism by the restriction fragment length polymorphism technique. There was an increasing trend of response to combined IFN treatment as 34.9% of GG, 64.3% of GT, and 66.7% of TT genotypes were sustained responders (P=0.05). The T allele was significantly affecting the response rate more than G allele (P=0.032). Moreover, the hepatic fibrosis score and hepatitis activity were higher in GG genotypes compared with the GT and TT genotypes. The multivariate analysis showed that the MxA GG genotype was an independent factor increasing the no response to IFN therapy (P=0.04, odds ratio [OR] 3.822, 95% confidence interval [CI] 1.056-11.092), also MxA G allele (P=0.0372, OR 2.905, 95% CI 1.066-7.919). MxA -88 polymorphism might be a potential biomarker to predict response to IFN and disease progression in chronic HCV-infected patients.

Human cytomegalovirus infection inhibits response of chronic hepatitis-C-virus-infected patients to interferon-based therapy.

BACKGROUND AND AIM: Cytomegalovirus (CMV) is a ubiquitous pathogen that infects the majority of humans. Co-infection of CMV and hepatitis C virus (HCV) may deteriorate the prognosis of HCV-infected patients. This study was conducted to examine the role of CMV reactivation in determining the response rate to treatment with interferon and ribavirin therapy in chronic HCV patients. METHODS: Viral loads and genotyping were assessed using reverse transcription polymerase chain reaction and Innolipa systems, respectively. Reactivation of CMV in HCV patients who were all positive for CMV immunoglobulin G antibodies was tested by amplification of the gB1 gene using the end-point dilution quantitative-nested polymerase chain reaction method. RESULTS: CMV DNA was detected in 89.7% of non-responders and in 34.6% of sustained virological responders. Patients with reactivated CMV had significantly higher fibrosis scores (72.7%) than those with undetectable CMV DNA (23.8%, P=0.002). Patients with positive CMV had higher rates of non-response and relapse (79.5%) than those with negative CMV DNA (19%). Chronic HCV patients with latent CMV had higher rates of response (81%) to treatment than those with reactivated CMV (20.5%, P<0.001). Therefore, HCV patients with reactivated CMV and advanced fibrosis were least likely to achieve a sustained virological response following interferon therapy. This possibility is reduced to 50% of its original value in patients with reactivated CMV without fibrosis. CONCLUSIONS: Besides the staging of liver fibrosis, CMV co-infection should be considered as an extremely important factor when designing predictive models for HCV response to interferon treatment.

Single nucleotide polymorphism at exon 7 splice acceptor site of OAS1 gene determines response of hepatitis C virus patients to interferon therapy.

BACKGROUND AND AIM: Response to interferon therapy and disease progression in hepatitis C virus (HCV) infected patients differs among individuals, suggesting a possibility of a contribution of host genetic factors. 2'-5'-oligoadenylate synthetase 1 (OAS1), an important component of the innate immune system with a proven antiviral function, may therefore have a relationship with the response to interferon therapy and clinical course of HCV disease. Our aim was to determine the frequency of single nucleotide polymorphism (SNP) at exon 7 splice acceptor site (SAS) of the OAS1 gene in relation to the interferon response and status of HCV infection. METHODS: A 203 bp fragment containing exon 7 SAS was amplified in 70 HCV chronic patients and 50 healthy controls. SNP was examined using restriction fragment length polymorphism (RFLP) genotyping method. Correlations of SNP genotypes with response to interferon and clinical status of patients were statistically analyzed. RESULTS: There was an increasing trend of response from AA to AG to GG genotypes (P = 0.007). Genotype AA was associated with non-response to interferon and higher degree of liver fibrosis (P = 0.05). Multivariate analysis showed this SNP as independent and a significant determinant of the outcome of interferon therapy (odds ratio 4.913 [95% confidence interval 1.365-8.2], P = 0.006). CONCLUSIONS: This is the first study to show a significant association between the functional SNP at exon 7 SAS of OAS1 gene and the viral response to interferon in chronic HCV patients. Patients with AA genotype were associated with progressive HCV disease and viral resistance to interferon therapy. This OAS SNP is a potential bio-marker to predict IFN response in chronic hepatitis C patients.