Translation of a Protease Turnover Assay for Clinical Discrimination of Mucinous Pancreatic Cysts

Abstract

The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 µL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care.

A Prototype Assay Multiplexing SARS-CoV-2 3CL-Protease and Angiotensin-Converting Enzyme 2 for Saliva-Based Diagnostics in COVID-19

“…The peptide substrate is adapted from a previously identified 3CL protease cleavage sequence before NSP7 in the polyprotein (ATLQAIAS), an N-terminal dimeric dye to enable assay readout, and a C-terminal biotinylated lysine for immobilization to magnetic beads [33,36]. The lysine-biotin residue is separated from the rest of the peptide by serine-diethylene glycol spacer to preserve the biotin-streptavidin complex [35,40]. After immobilization on streptavidin-conjugated blocked magnetic beads, the unbound substrate is washed away using an excess Buffer I.…”

“…Synthetic procedures for the dimeric rhodamine 6G dye used in the present study have been reported previously [34,35]. This dye was coupled to the N-terminus of resinbound biotinylated peptides, prepared using a liberty blue synthesizer (CEM, Matthews, NC, USA).…”

“…This duplex assay is designed to run on saliva samples, though it will likely tolerate a variety of other biofluids [32]. A magnetic bead platform with a novel labeled peptide substrate, capable of both fluorescence and surface-enhanced resonance Raman spectroscopy (SERS) readouts, is reported [33][34][35]. This new 3CL protease assay can be run on the same sample immediately after ACE2 assay with readouts of both enzymes in 30 min using minimal sample volumes at concentrations below the dimer dissociation constant.…”

Addressing the unmet clinical need for low-volume assays in early diagnosis of pancreatic cancer

“…The authors of this review have translated assays using Raman-active dyes that have shown detection limits below those of traditional fluorescence measurements ( 71 ). Furthermore, Suresh et al have published the first example of translating a SERS assay for protease activity of gastricsin to a clinically ready platform with a small cohort of human pancreatic cyst fluid samples ( 72 ). Pancreatic cyst fluid is a notoriously tricky medium with which to run assays or optically quantify results, and the simplest solution is significant sample dilution to diminish interfering substances.…”

Molecular Docking Investigation of Dioscorea alata Compounds Binding to CCKBR, CHRM3, CHRM5, and H2R for Gastric Ulcer Treatment

“…PASSOnline is a database for predicting the bioactivity of organic compounds based on the analysis of the structure-activity relationships of more than 300,000 organic compounds as a training set, which has an average accuracy of above 95 % [11]. The bioactivities selected were gastrin inhibition, gastritis treatment [12], inhibition of gastric secretion [13], and gastricsin inhibition [14].…”