Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor.

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

Down-regulation of A20 promotes immune escape of lung adenocarcinomas.

Inflammation is a well-known driver of lung tumorigenesis. One strategy by which tumor cells escape tight homeostatic control is by decreasing the expression of the potent anti-inflammatory protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20. We observed that tumor cell intrinsic loss of A20 markedly enhanced lung tumorigenesis and was associated with reduced CD8+ T cell-mediated immune surveillance in patients with lung cancer and in mouse models. In mice, we observed that this effect was completely dependent on increased cellular sensitivity to interferon-γ (IFN-γ) signaling by aberrant activation of TANK-binding kinase 1 (TBK1) and increased downstream expression and activation of signal transducer and activator of transcription 1 (STAT1). Interrupting this autocrine feed forward loop by knocking out IFN-α/ß receptor completely restored infiltration of cytotoxic T cells and rescued loss of A20 depending tumorigenesis. Downstream of STAT1, programmed death ligand 1 (PD-L1) was highly expressed in A20 knockout lung tumors. Accordingly, immune checkpoint blockade (ICB) treatment was highly efficient in mice harboring A20-deficient lung tumors. Furthermore, an A20 loss-of-function gene expression signature positively correlated with survival of melanoma patients treated with anti-programmed cell death protein 1. Together, we have identified A20 as a master immune checkpoint regulating the TBK1-STAT1-PD-L1 axis that may be exploited to improve ICB therapy in patients with lung adenocarcinoma.