Identification of an in vivo orally active dual-binding protein-protein interaction inhibitor targeting TNFα through combined in silico/in vitro/in vivo screening.

TNFα is a homotrimeric pro-inflammatory cytokine, whose direct targeting by protein biotherapies has been an undeniable success for the treatment of chronic inflammatory diseases. Despite many efforts, no orally active drug targeting TNFα has been identified so far. In the present work, we identified through combined in silico/in vitro/in vivo approaches a TNFα direct inhibitor, compound 1, displaying nanomolar and micromolar range bindings to TNFα. Compound 1 inhibits the binding of TNFα with both its receptors TNFRI and TNFRII. Compound 1 inhibits the TNFα induced apoptosis on L929 cells and the TNFα induced NF-κB activation in HEK cells. In vivo, oral administration of compound 1 displays a significant protection in a murine TNFα-dependent hepatic shock model. This work illustrates the ability of low-cost combined in silico/in vitro/in vivo screening approaches to identify orally available small-molecules targeting challenging protein-protein interactions such as homotrimeric TNFα.

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

Identification of a ligand for tumor necrosis factor receptor from Chinese herbs by combination of surface plasmon resonance biosensor and UPLC-MS.

Identification of bioactive compounds directly from complex herbal extracts is a key issue in the study of Chinese herbs. The present study describes the establishment and application of a sensitive, efficient, and convenient method based on surface plasmon resonance (SPR) biosensors for screening active ingredients targeting tumor necrosis factor receptor type 1 (TNF-R1) from Chinese herbs. Concentration-adjusted herbal extracts were subjected to SPR binding assay, and a remarkable response signal was observed in Rheum officinale extract. Then, the TNF-R1-bound ingredients were recovered, enriched, and analyzed by UPLC-QTOF/MS. As a result, physcion-8-O-ß-D-monoglucoside (PMG) was identified as a bioactive compound, and the affinity constant of PMG to TNF-R1 was determined by SPR affinity analysis (K D = 376 nM). Pharmacological assays revealed that PMG inhibited TNF-α-induced cytotoxicity and apoptosis in L929 cells via TNF-R1. Although PMG was a trace component in the chemical constituents of the R. officinale extract, it had considerable anti-inflammatory activities. It was found for the first time that PMG was a ligand for TNF receptor from herbal medicines. The proposed SPR-based screening method may prove to be an effective solution to analyzing bioactive components of Chinese herbs and other complex drug systems. Graphical abstract Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them. Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them.

Pharmacogenomics of biological treatment in rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) demonstrates a high heterogeneity in clinical responses to treatment. Although the efficacy of biological therapy has undoubtedly been established, the response differs considerably between individuals. This variability between individuals has aroused the research for biomarkers predictive of treatment response. Pharmacogenomics underlying individual responses to drugs is rapidly developed and has the potential of realizing the personalized therapy in RA. This review will summarize the pharmacogenomics of biological therapies approved for clinical RA treatment. AREAS COVERED: The pharmacogenomics underlies individual responses to biological drugs in RA. Current studies on pharmacogenomics of biological therapy in RA are presented. EXPERT OPINION: The personalized treatment in RA according to pharmacogenomics is promising, but the available pharmacogenomic data on biological treatment in RA are not adequate and consistent and still require further larger sample studies to corroborate.

Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity.

The interaction between 4-1BB ligand (CD137L), a member of the tumor necrosis factor superfamily, and its receptor 4-1BB provides a co-stimulatory signal for T lymphocyte proliferation and survival. However, the structure of 4-1BBL has not been thoroughly investigated, and none of the human recombinant 4-1BBL molecules available have been described as capable of co-stimulating T cells. The present work provides a model of the three-dimensional structure of the tumor necrosis factor homology domain of 4-1BBL and describes the production of a recombinant human soluble 4-1BBL whose originality lies in that it contains the whole extracellular tail preceding the tumor necrosis factor homology domain and an AviTag peptide (AviTag-4-1BBL) allowing enzymatic biotinylation and multimerization via streptavidin. We provide evidence that this chimeric protein exists as a homotrimer, whereas commercial FLAG-tagged 4-1BBL does not. This resulted in a much higher affinity for 4-1BB (1.2 nM) as compared with FLAG-4-1BBL (55.2 nM). We demonstrate that the single extracellular cysteine residue in the tail (Cys-51) could form a disulfide bond, both in our recombinant protein and in physiologically expressed 4-1BBL. The mutation of this cysteine residue exerted no effect on trimerization but increased the dissociation rate of AviTag-4-1BBL from 4-1BB. In its soluble form, AviTag-4-1BBL did not stimulate purified T cells but dramatically inhibited proliferation of peripheral blood mononuclear cells stimulated with anti-CD3 mAb. In contrast, a very significant co-stimulatory effect was observed on purified T cells once AviTag-4-1BBL was immobilized onto streptavidin beads. In addition, we show that the cross-linking of two trimeric AviTag-4-1BBL molecules was the minimum step required to elicit significant costimulatory activity.

Osteoclastic function is accelerated in male patients with type 2 diabetes mellitus: the preventive role of osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) on the decrease of bone mineral density.

To clarify the pathogenesis of altered bone metabolism in diabetic state and its underlying mechanisms, the bone mineral content and fasting levels of serum intact parathyroid hormone (i-PTH), intact osteocalcin (i-OC), tartrate-resistant acid phosphatase (TRAP) and osteoclastgenesis inhibitory factor/osteoprotegerin (OCIF/OPG) were measured in male type 2 diabetic patients and their age-matched controls. In addition, urine levels of osteoclastic markers, C-telopeptide of type I collagen (CTx), deoxypyridinoline (DPD), and N-telopeptide of type I collagen (NTx) were simultaneously determined. Serum levels of i-PTH and i-OC in diabetic patients were significantly lower than those in the controls. Conversely, serum concentrations of TRAP were significantly elevated in diabetic patients. However, no clear correlation was observed between serum i-OC and TRAP. It was also observed that urinary excretion of CTx, DPD, and NTx was significantly increased in the diabetics as compared with the controls. Unexpectedly, serum levels of OCIF/OPG tended to be higher in the diabetic group, and these values exhibited a significantly positive correlation with those of serum TRAP. There was found a significantly negative correlation between serum TRAP and bone mineral density (BMD) and also between serum OCIF/OPG and bone mineral density. It seems probable that OCIF/OPG has a suppressive role on the increased bone resorption to prevent further loss of the skeletal bone mass in type 2 diabetic patients.

Biologic agents for the treatment of juvenile rheumatoid arthritis: current status.

Biologic therapies, primarily anticytokine therapies, are being increasingly used in patients with juvenile rheumatoid arthritis (JRA). Levels of a variety of proinflammatory cytokines have been shown to be elevated in the peripheral blood and synovial fluid and tissue in children with JRA. In a blinded, randomized, controlled trial in children with severe, long-standing, polyarticular-course JRA not responsive to standard therapies, etanercept showed a statistically significantly greater response rate than placebo. Approximately 75% of these children responded to etanercept. Etanercept has been efficacious in 50-60% of children with active systemic JRA in open clinical trials with acceptable tolerance. Adverse events seen in children treated with etanercept have been similar in type and frequency to those reported in adults. Infliximab has been studied in several open clinical trials in both polyarticular and systemic JRA and found to, overall, have demonstrated efficacy in approximately 60% of patients. Approximately 3-5% of patients have demonstrated infusion reactions or frank allergic reactions and 9% developed new autoantibodies. Anakinra has been studied in children with polyarticular JRA. Approximately 65% of patients developed injection-site reactions and 68% demonstrated a response to the medication. Anakinra may have increased efficacy in systemic JRA. Interleukin (IL)-6 is highly related to the systemic disease manifestations in systemic JRA and two patients treated with a monoclonal antibody to the IL-6 receptor have demonstrated significant improvement with prolonged clinical control with continued treatment. A particular pediatric concern is the effect of immunosuppressive biologics in children who are exposed to or develop varicella. These children should be treated, both in terms of prophylaxis and aggressive antivaricella treatment, as for other immunosuppressed children. Anticytokine biologics have demonstrated great promise in the treatment of JRA and a variety of other pediatric rheumatic diseases, although at this time the randomized, placebo-controlled data are limited only to etanercept in children with polyarticular JRA. Randomized trials are ongoing to better define both the efficacy and safety of these novel treatments for children with JRA and other rheumatic diseases.

Identification and characterization of a shared TNFR-related receptor for subgroup B, D, and E avian leukosis viruses reveal cysteine residues required specifically for subgroup E viral entry.

Genetic and receptor interference data have indicated the presence of one or more cellular receptors for subgroup B, D, and E avian leukosis viruses (ALV) encoded by the s1 allele of the chicken tvb locus. Despite the prediction that these viruses use the same receptor, they exhibit a nonreciprocal receptor interference pattern: ALV-B and ALV-D can interfere with infection by all three viral subgroups, but ALV-E only interferes with infection by subgroup E viruses. We identified a tvb(s1) cDNA clone which encodes a tumor necrosis factor receptor-related receptor for ALV-B, -D, and -E. The nonreciprocal receptor interference pattern was reconstituted in transfected human 293 cells by coexpressing the cloned receptor with the envelope (Env) proteins of either ALV-B or ALV-E. This pattern of interference was also observed when soluble ALV surface (SU)-immunoglobulin fusion proteins were bound to this cellular receptor before viral challenge. These data demonstrate that viral Env-receptor interactions can account for the nonreciprocal interference between ALV subgroups B, D, and E. Furthermore, they indicate that a single chicken gene located at tvb(s1) encodes receptors for these three viral subgroups. The TVB(S1) protein differs exclusively at residue 62 from the published subgroup B- and D-specific receptor, encoded by the s3 allele of tvb. Residue 62 is a cysteine in TVB(S1) but is a serine in TVB(S3), giving TVB(S1) an even number of cysteines in the extracellular domain. We present evidence for a disulfide bond requirement in TVB(S1) for ALV-E infection but not for ALV-B infection. Thus, ALV-B and ALV-E interact in fundamentally different ways with this shared receptor, a finding that may account for the observed biological differences between these two ALV subgroups.

TNFR80-dependent enhancement of TNFR60-induced cell death is mediated by TNFR-associated factor 2 and is specific for TNFR60.

Costimulation of TNFR80 can strongly enhance TNFR60-induced cell death. In this study, we show that this enhancement is TNFR60 selective, as neither TNF-related apoptosis-inducing ligand/Apo2 ligand-, Apo1/Fas-, ceramide-, nor daunorubicin-mediated cell death was affected by costimulation of TNFR80. We further demonstrate that TNFR-associated factor 2 (TRAF2) is critically involved in both negative and positive regulation of TNF-induced cell death. Overexpression of TRAF2 and of a TRAF2 mutant, deficient in nuclear factor-kappaB activation, selectively desensitized and enhanced, respectively, TNFR60-induced cell death in HeLa cells. However, upon costimulation of TNFR80, which mediates activation of nuclear factor-kappaB and the c-Jun amino-terminal kinase via TRAF2, TNF-induced cell death is drastically enhanced in parental and TRAF2-transfected, but not in TRAF2 (87-501)-transfected cells. These data point to a critical role of TRAF2 in the apoptotic TNFR cross talk, whereby the TNFR80-dependent enhancement of TNFR60-induced cell death is due to TNFR80-mediated negative regulation of TRAF2 function(s). An interference with TRAF2 function was confirmed independently by analysis of c-Jun amino-terminal kinase activation via TNFR60 upon prestimulation of TNFR80. We propose that the apoptotic TNFR cross talk is based on TNFR80-mediated abrogation of antiapoptotic TRAF2-dependent signaling pathways initiated by TNFR60, but not Apo1/Fas or the apoptotic TNF-related apoptosis-inducing ligand receptors.

Specific induction of 80-kDa tumor necrosis factor receptor shedding in T lymphocytes involves the cytoplasmic domain and phosphorylation.

The 80-kDa TNFR (TNFR80) expressed by activated human T cells is constitutively phosphorylated and undergoes limited proteolytic cleavage (shedding) at the cell surface releasing a 40-kDa soluble TNF-binding protein. Triggering of activated T cells through the TCR rapidly increased the rate of TNFR80 shedding > 20-fold more than nonstimulated cells, demonstrating that shedding is a specific, inducible process. The protein kinase inhibitor staurosporine inhibited constitutive phosphorylation and blocked inducible shedding of TNFR80, suggesting that phosphorylation may be important for cleavage of the extracellular domain. However, a deletion mutation of the entire cytoplasmic domain of human TNFR80 was shed when expressed in murine L929 cells, albeit relatively poorly compared with full length receptor. This demonstrates that the cytoplasmic domain is important but not essential for cleavage of the extracellular domain of TNFR80. Moreover, a requirement for phosphorylation of proteins other than the receptor was revealed by the finding that staurosporine completely blocked cleavage of the cytoplasmic deletion mutant. Collectively, these results demonstrate that protein phosphorylation is essential and the cytoplasmic domain is important for regulating the inducible production of soluble TNF-binding proteins by activated effector T cells.