Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening

Abstract

Fibroproliferative diseases are characterized by excessive accumulation of extracellular matrix (ECM) components leading to organ dysfunction. This process is characterized by an increase in myofibroblast content and enzyme activity of xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan (PG) biosynthesis. Therefore, the inhibition of XT-I could be a promising treatment for fibrosis. We used a natural product-inspired compound library to identify non-substrate-based inhibitors of human XT-I by UPLC-MS/MS. We combined this cell-free approach with virtual and molecular biological analyses to confirm and prioritize the inhibitory potential of the compounds identified. The characterization for compound potency in TGF-β1-driven XYLT1 transcription regulation in primary dermal human fibroblasts (key cells in ECM remodeling) was addressed by gene expression analysis. Consequently, we identified amphotericin B and celastrol as new non-substrate-based XT-I protein inhibitors. Their XT-I inhibitory effects were mediated by an uncompetitive or a competitive inhibition mode, respectively. Both compounds reduced the cellular XYLT1 expression level and XT-I activity. We showed that these cellular inhibitor-mediated changes involve the TGF-β and microRNA-21 signaling pathway. The results of our study provide a strong rationale for the further optimization and future usage of the XT-I inhibitors identified as promising therapeutic agents of fibroproliferative diseases.

The Human Myofibroblast Marker Xylosyltransferase-I: A New Indicator for Macrophage Polarization

“…The relative quantification of the extracellular XT-I activity from cell culture supernatants and the intracellular XT-I activity from cell lysates was performed using a mass spectrometric enzyme activity assay, following a previously published procedure [ 31 , 32 ]. This relative in silico XT-I activity measurement relied on XT-I-catalyzed xylose incorporation into an XT-I-selective acceptor peptide after a defined period.…”

“…The XT-I activity that was determined from the cell-free setups was as a result of the FBS-supplementation of the growth medium was used for background subtraction from the extracellular activity values of the vessels that contained macrophages. A prediluted xylosylated-peptide standard of known concentration was prepared for the mass spectrometric determination of XT-I sample activity, as previously described [ 31 ], and was included in every mass spectrometric run that was performed. An aliquot of a positive control sample, consisting of recombinant Chinese hamster ovary-derived XT-I protein, was included in every mass assay for inter-assay correction purposes, such as varying reaction mixture compositions.…”

“…An aliquot of a positive control sample, consisting of recombinant Chinese hamster ovary-derived XT-I protein, was included in every mass assay for inter-assay correction purposes, such as varying reaction mixture compositions. The determined cellular XT-I activity was expressed in enzyme units U (μmol/min), and was normalized to the total protein content of the respective lysate, that was quantified via a bicinchonic acid assay, as specified previously [ 31 ].…”

UHPLC-QTOF-MS/MS based characterization of anti-tumor constituents in Ceratocarpus arenarius L. and identification of EGFR-TK inhibitors by virtual screening

“…Docking study was carried out for the virtual screening of potential active compounds using docking model in the MOE software. The crystallographic structures of EGFR tyrosine kinase domain with erlotinib (PDB ID: 4HJO) was obtained from protein data bank PDB, and the recognized constituents were subjected to docking study together with erlotinib as a reference molecule to evaluate the free energies and binding mode of the compounds against EGFR-TK ( Ly et al 2020).…”

Understanding of arthrofibrosis: New explorative insights into extracellular matrix remodeling of synovial fibroblasts

“…Regarding arthrofibrosis, the XYLT1 expression was used to control the efficiency of an anti-fibrotic treatment in a rat model of knee implant surgery or demonstrate the myofibroblast differentiation rate in human arthrofibrotic tissues [33,34]. Since fibroblast-to-myofibroblast transition was determined by the increased expression and activity of XT-I [9,35], the inhibition of XT could be a promising approach toward fibroproliferative diseases by modulating a downstream mediator of the TGF-β signaling [36].…”