Evaluation of Computationally Designed Peptides against TWEAK, a Cytokine of the Tumour Necrosis Factor Ligand Family.

The tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumour necrosis factor ligand family and has been shown to be overexpressed in tumoral cells together with the fibroblast growth factor-inducible 14 (Fn14) receptor. TWEAK-Fn14 interaction triggers a set of intracellular pathways responsible for tumour cell invasion and migration, as well as proliferation and angiogenesis. Hence, modulation of the TWEAK-Fn14 interaction is an important therapeutic goal. The targeting of protein-protein interactions by external agents, e.g., drugs, remains a substantial challenge. Given their intrinsic features, as well as recent advances that improve their pharmacological profiles, peptides have arisen as promising agents in this regard. Here, we report, by in silico structural design validated by cell-based and in vitro assays, the discovery of four peptides able to target TWEAK. Our results show that, when added to TWEAK-dependent cellular cultures, peptides cause a down-regulation of genes that are part of TWEAK-Fn14 signalling pathway. The direct, physical interaction between the peptides and TWEAK was further elucidated in an in vitro assay which confirmed that the bioactivity shown in cell-based assays was due to the targeting of TWEAK. The results presented here are framed within early pre-clinical drug development and therefore these peptide hits represent a starting point for the development of novel therapeutic agents. Our approach exemplifies the powerful combination of in silico and experimental efforts to quickly identify peptides with desirable traits.

Clinical efficacy of sublingual allergen-specific immunotherapy in 22 cats with atopic dermatitis.

BACKGROUND: Sublingual immunotherapy (SLIT) has been deployed in humans and dogs; to the best of the authors' knowledge, there are no published studies about the use of SLIT in cats. OBJECTIVES: Evaluate the clinical efficacy of SLIT in atopic cats sensitized to dust and storage mites, assessing immunological changes associated with SLIT treatment. ANIMALS: Twenty-two client-owned cats with clinical signs compatible with feline atopic dermatitis (fAD) and serum allergen-specific immunoglobulin (Ig)E against house dust and storage mites. METHODS AND MATERIALS: Prospective, multicentre, open-label clinical trial. Individualized mite-specific SLIT was administered orally for 12 months. All cats underwent clinical examination to record SCORing feline allergic dermatitis (SCORFAD), pruritus Visual Analog Scale (pVAS) and serum allergen-specific IgE and IgG, every three months for 12 months. RESULTS: Sixteen of 22 cats (73%) completed the study and three of six cats withdrawn from the study were included in an intention-to-treat analysis. SCORFAD and pVAS values decreased significantly from baseline (T0) to the third month of treatment (P = 0.0004 and P = 0.0013, respectively), with median total values ranging from 19 (6-44) (T0) to 2.5 (0-17) (T12) (P = 0.0001), and from 8 (6-10) (T0) to 2.3 (0-8) (T12) (P = 0.0001), respectively. Allergen-specific IgE values decreased significantly from the ninth month (T9) of treatment (P = 0.0032), with median scores decreasing from 56 (12-729) (T0) to 34 (0-158) (T12) (P = 0.0208). No significant differences in allergen-specific IgG values were observed throughout the study. No adverse effects related to the use of SLIT were reported. CONCLUSIONS AND CLINICAL IMPORTANCE: Sublingual immunotherapy should be considered a rapid, effective, safe and well-tolerated treatment in cats with feline atopic dermatitis fAD.

GRP94 promotes brain metastasis by engaging pro-survival autophagy.

BACKGROUND: GRP94 is a glucose-regulated protein critical for survival in endoplasmic reticulum stress. Expression of GRP94 is associated with cellular transformation and increased tumorigenicity in breast cancer. Specifically, overexpression of GRP94 predicts brain metastasis (BM) in breast carcinoma patients with either triple negative or ErbB2 positive tumors. The aim of this study was to understand if microenvironmental regulation of GRP94 expression might be a hinge orchestrating BM progression. METHODS: GRP94 ablation was performed in a BM model BR-eGFP-CMV/Luc-V5CA1 (BRV5CA1) of breast cancer. In vitro results were validated in a dataset of 29 metastases in diverse organs from human breast carcinomas and in BM tissue from tumors of different primary origin. BM patient-derived xenografts (PDXs) were used to test sensitivity to the therapeutic approach. RESULTS: BMs that overexpress GRP94 as well as tumor necrosis factor receptor-associated factor 2 are more resistant to glucose deprivation by induction of anti-apoptotic proteins (B-cell lymphoma 2 and inhibitors of apoptosis proteins) and engagement of pro-survival autophagy. GRP94 ablation downregulated autophagy in tumor cells, resulting in increased BM survival in vivo. These results were validated in a metastasis dataset from human patients, suggesting that targeting autophagy might be strategic for BM prevention. Indeed, hydroxychloroquine treatment of preclinical models of BM from PDX exerts preventive inhibition of tumor growth (P < 0.001). CONCLUSIONS: We show that GRP94 is directly implicated in BM establishment by activating pro-survival autophagy. Disruption of this compensatory fueling route might prevent metastatic growth.

FN14 and GRP94 expression are prognostic/predictive biomarkers of brain metastasis outcome that open up new therapeutic strategies.

Brain metastasis is a devastating problem in patients with breast, lung and melanoma tumors. GRP94 and FN14 are predictive biomarkers over-expressed in primary breast carcinomas that metastasized in brain. To further validate these brain metastasis biomarkers, we performed a multicenter study including 318 patients with breast carcinomas. Among these patients, there were 138 patients with metastasis, of whom 84 had brain metastasis. The likelihood of developing brain metastasis increased by 5.24-fold (95%CI 2.83-9.71) and 2.55- (95%CI 1.52-4.3) in the presence of FN14 and GRP94, respectively. Moreover, FN14 was more sensitive than ErbB2 (38.27 vs. 24.68) with similar specificity (89.43 vs. 89.55) to predict brain metastasis and had identical prognostic value than triple negative patients (p < 0.0001). Furthermore, we used GRP94 and FN14 pathways and GUILD, a network-based disease-gene prioritization program, to pinpoint the genes likely to be therapeutic targets, which resulted in FN14 as the main modulator and thalidomide as the best scored drug. The treatment of mice with brain metastasis improves survival decreasing reactive astrocytes and angiogenesis, and down-regulate FN14 and its ligand TWEAK. In conclusion our results indicate that FN14 and GRP94 are prediction/prognosis markers which open up new possibilities for preventing/treating brain metastasis.