A combined in vitro/in silico approach to identifying off-target receptor toxicity

Many xenobiotics can bind to off-target receptors and cause toxicity via the dysregulation of downstream transcription factors. Identification of subsequent off-target toxicity in these chemicals has often required extensive chemical testing in animal models. An alternative, integrated in vitro/in silico approach for predicting toxic off-target functional responses is presented to refine in vitro receptor identification and reduce the burden on in vivo testing. As part of the methodology, mathematical modelling is used to mechanistically describe processes that regulate transcriptional activity following receptor-ligand binding informed by transcription factor signalling assays. Critical reactions in the signalling cascade are identified to highlight potential perturbation points in the biochemical network that can guide and optimise additional in vitro testing. A physiologically-based pharmacokinetic model provides information on the timing and localisation of different levels of receptor activation informing whole-body toxic potential resulting from off-target binding

Histidine Tag-Specific PEGylation Improves the Circulating Half-Life of TIMP2

An overarching limitation of therapeutic biologics is the limited half-life these proteins often exhibit once in circulation. PEGylation, the chemical conjugation of proteins to poly(ethylene glycol) (PEG), is a common strategy to improve protein pharmacokinetics (PK) by enhancing stability, reducing immunogenicity, and decreasing renal clearance. Tissue Inhibitor of Metalloproteinases 2 (TIMP2) is a 22 kDa matrisome protein that exhibits therapeutic potential across a range of human disease models yet possesses a short serum halflife. To advance the therapeutic development of recombinant His-tagged TIMP2(TIMP2), we utilized primary amine conjugation (1 kDa) and site-specific histidine conjugation (10 kDa) to improve its circulating half-life. Primary amine conjugation of PEG molecules to TIMP2 (TIMP2-a-PEG(n)) is efficient, yet it produces multiple positional isomers that are difficult to purify. Furthermore, high levels of conjugation can affect the MMP-inhibitory activity of TIMP2. Despite this, TIMP2-a-PEG(n) displays a significant improvement (11.5-fold) in serum half-life versus unconjugated TIMP2. In contrast, site-specific histidine conjugation targets the histidine tag, enabling the purification of mono-PEGylated (TIMP2-H-PEG(1)) and di-PEGylated (TIMP2-H-PEG(2)) forms. Our findings demonstrate that TIMP2-H-PEG(1) exhibits improved PK with enhanced stability and a 6.2-fold increase in circulating halflife while maintaining MMP-inhibitory activity. These results suggest that site-specific PEGylation at a C-terminal His6 tag is a promising approach for further preclinical development of TIMP2 as a therapeutic biologi

TIMP-2 suppresses tumor growth and metastasis in murine model of triple-negative breast cancer

Abstract Metastasis is the primary cause of treatment failures and mortality in most cancers. Triple-negative breast cancer (TNBC) is refractory to treatment and rapidly progresses to disseminated disease. We utilized an orthotopic mouse model that molecularly and phenotypically resembles human TNBC to study the effects of exogenous, daily tissue inhibitor of metalloproteinase-2 (TIMP-2) treatment on tumor growth and metastasis. Our results demonstrated that TIMP-2 treatment maximally suppressed primary tumor growth by ~36–50% and pulmonary metastasis by &amp;gt;92%. Immunostaining assays confirmed disruption of the epithelial to mesenchymal transition (EMT) and promotion of vascular integrity in primary tumor tissues. Immunostaining and RNA sequencing analysis of lung tissue lysates from tumor-bearing mice identified significant changes associated with metastatic colony formation. Specifically, TIMP-2 treatment disrupts periostin localization and critical cell-signaling pathways, including canonical Wnt signaling involved in EMT, as well as PI3K signaling, which modulates proliferative and metastatic behavior through p27 phosphorylation/localization. In conclusion, our study provides evidence in support of a role for TIMP-2 in suppression of triple-negative breast cancer growth and metastasis through modulation of the epithelial to mesenchymal transition, vascular normalization, and signaling pathways associated with metastatic outgrowth. Our findings suggest that TIMP-2, a constituent of the extracellular matrix in normal tissues, may have both direct and systemic antitumor and metastasis suppressor effects, suggesting potential utility in the clinical management of breast cancer progression.</jats:p