Background/Objectives: Bilirubin is a hydrophobic molecule that binds the carrier protein albumin for transport through systemic circulation. Bilirubin is cleared from the body through the liver and excreted into the intestines, where the microbiota modifies the chemical structure, forming urobilin, which can be reabsorbed into circulation by the hepatic portal vein. Urobilin has no known function. It is also unknown whether urobilin binds albumin for transport in circulation. We hypothesized that because of the likeness of their chemical structures, urobilin would also bind albumin like bilirubin does. Methods: First, we used in silico docking to predict if urobilin would bind to albumin and compared it to the bilirubin binding sites. To test this binding in vitro, we applied bilirubin’s fluorescent property, which occurs when it is bound to a protein, including albumin, and exposed to light. We also used this method to determine if urobilin could exhibit autofluorescence when protein bound. Results: We found that bilirubin was predicted to bind albumin at amino acids E208, K212, D237, and K240 through hydrogen bonds. However, urobilin was predicted to bind albumin using different hydrogen bonds at amino acids H67, K240, and E252. We found that urobilin has a fluorescent property that can be quantified when bound to albumin. We performed a concentration response for urobilin–albumin fluorescent binding and observed a direct relationship between the urobilin level and the fluorescence intensity. Conclusions: The in silico docking analysis and autofluorescence results demonstrate that urobilin binds to albumin and might compete with bilirubin. This is the first study to identify a urobilin-binding protein and the important aspects of its physiological function and transport in circulation.

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.