Programmed death-ligand 1 is a glycoprotein expressed on antigen presenting cells, hepatocytes, and tumors which upon interaction with programmed death-1, results in inhibition of antigen-specific T cell responses. Here, we report a mechanism of inhibiting programmed death-ligand 1 through small molecule-induced dimerization and internalization. This represents a mechanism of checkpoint inhibition, which differentiates from anti-programmed death-ligand 1 antibodies which function through molecular disruption of the programmed death 1 interaction. Testing of programmed death ligand 1 small molecule inhibition in a humanized mouse model of colorectal cancer results in a significant reduction in tumor size and promotes T cell proliferation. In addition, antigen-specific T and B cell responses from patients with chronic hepatitis B infection are significantly elevated upon programmed death ligand 1 small molecule inhibitor treatment. Taken together, these data identify a mechanism of small molecule-induced programmed death ligand 1 internalization with potential therapeutic implications in oncology and chronic viral infections.

BACKGROUND AND PURPOSEThe chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3.EXPERIMENTAL APPROACHAs both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined.KEY RESULTSThe CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the ‘30s loop’ (residues 30–40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues.CONCLUSION AND IMPLICATIONSWe provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.LINKED ARTICLEThis article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x

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The dosimeter system exhibits a small angular dependence of approximately 2% which needs to be considered for measurements involving other than normal incident beams angles. This applies in particular to clinical in vivo measurements where the orientation of the dosimeter is dictated by clinical circumstances and cannot be optimized as otherwise suggested here. When measuring in a phantom, the proposed new setup should be considered. It changes the orientation of the dosimeter so that a coplanar beam arrangement always hits the disk shaped detector material from the thin side and thereby reduces the angular dependence of the response to within the measurement uncertainty of about 1%. This improvement makes the dosimeter more attractive for clinical measurements with multiple coplanar beams in phantoms, as the overall measurement uncertainty is reduced. Similarly, phantom based postal audits can transition from the traditional TLD to the more accurate and convenient OSLD.

Objective. All ␥-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2.Methods. JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22-and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice.Results. In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6-or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-␥ production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models.Conclusion. In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.

AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC] = 0.08 to 0.27 μM; EC = 0.33 to 1.32 μM) with no significant cytotoxicity (50% cytotoxic concentration > 10 μM). Addition of 40% human serum resulted in a 5-fold increase in the ECs. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.

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