Abstract
Collagen prolyl 4-hydroxylase 1 (C-P4H1) is an α-ketoglutarate (α-KG)-dependent dioxygenase that catalyzes 4-hydroxylation of proline on collagen. C-P4H1-induced prolyl hydroxylation is required for proper collagen deposition and cancer metastasis. Therefore, targeting C-P4H1 is considered a potential therapeutic strategy for collagen-related cancer progression and metastasis. However, no C-P4H1 inhibitors are available for clinical testing, and the high content assay is currently not available for C-P4H1 inhibitor screening. In the present study, we developed a high-throughput screening assay by quantifying succinate, a byproduct of C-P4H-catalyzed hydroxylation. C-P4H1 is the major isoform of collagen prolyl 4-hydroxylases (CP4Hs) that contributes the majority prolyl 4-hydroxylase activity. Using C-P4H1 tetramer purified from the eukaryotic expression system, we showed that the Succinate-GloTM Hydroxylase assay was more sensitive for measuring C-P4H1 activity compared with the hydroxyproline colorimetric assay. Next, we performed high-throughput screening with the FDA-approved drug library and identified several new C-P4H1 inhibitors, including Silodosin and Ticlopidine. Silodosin and Ticlopidine inhibited C-P4H1 activity in a dose-dependent manner and suppressed collagen secretion and tumor invasion in 3D tissue culture. These C-P4H1 inhibitors provide new agents to test clinical potential of targeting C-P4H1 in suppressing cancer progression and metastasis.
Highlights
Collagen is the most abundant extracellular matrix (ECM) protein in the human body [1]
We developed a new method to quantify the Collagen prolyl 4-hydroxylase 1 (C-P4H1) activity by measuring succinate levels
Using the high-throughput screening assay based on this method, we identified several new C-P4H1 inhibitors, including Silodosin and Ticlopidine, from the FDA-approved drug library
Summary
Collagen is the most abundant extracellular matrix (ECM) protein in the human body [1]. Increased collagen expression and deposition is associated with fibrosis and tumor progression, and reducing collagen production is sufficient to inhibit the progression of these diseases [2,3,4]. Collagen is synthesized and forms the triple helix structure in the ER [5]. The basic unit of the triple-helical structure is Gly-pro-X, and the proline is often hydroxylated. Prolyl hydroxylation is required for the sharp twisting of collagen helix and secretion of collagen protein [6]. The proline hydroxylation pathway is considered a promising target to halt collagen expression and deposition
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