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  • We did not observe inhibition of vCPH by any of

    2020-09-22

    We did not observe inhibition of vCPH by any of the TCA cycle intermediates (apart from the metal chelators citrate and isocitrate), nor by the factor inhibiting HIF (FIH; a 2OG-dependent asparaginyl hydroxylase) inhibitor N-oxalyl d-phenylalanine (NOF) or the γ-butyrobetaine dioxygenase inhibitor mildronate (moldonium). The inhibition of vCPH by GSKJ1 (IC50 = 15.9 ± 6.3 µM), which was originally developed as a functional probe for Paprotrain mg the JmjC KDMs, is notable, indicating the range of 2OG oxygenases that this compound targets may be wider than initially anticipated.39, 42, 43 Interestingly, Paprotrain mg treated with GSKJ4, a prodrug of GSKJ1, manifest a significant reduction in collagen production. The observed inhibition of vCPH by GSKJ1 suggests that the apparent inhibition of collagen biosynthesis by GSKJ4 may, at least in part, be due to inhibition of human C-P4Hs. There is also evidence that application of a PHD inhibitor can lead to an anti-fibrotic response, though whether this is facilitated by HIF upregulation, inhibition of 2OG oxygenases other than the PHDs, or another mechanism is not known (note the analysis of such results is complicated because some 2OG oxygenases, including some procollagen modifying 2OG oxygenases, are themselves HIF target genes). Finally, the results reveal the viability of inhibiting viral/microbial prolyl hydroxylases, including by PHD inhibitors in ongoing clinical trials, or close variants thereof. This result is of particular interest given the importance of prolyl hydroxylases in animal parasite proliferation, including Toxoplasma gondii, which is the cause of the widespread human parasitic disease, toxoplasmosis.
    Acknowledgements GWL was funded by a studentship from the Engineering and Physical Sciences Research Council and UCB through the University of Oxford’s Systems Approaches to Biomedical Science Industrial Doctorate Centre (EP/G037280/1). We thank the Biotechnology and Biological Sciences Research Council, British Heart Foundation, the Wellcome Trust, and Cancer Research UK for funding. We also thank A. Carr, S. Dakin, and T. Johnson (UCB) for their support during the project, as well as R. Hopkinson, J. Gannon, S. Wilkins, L. Walport, and S. Markolovic for helpful discussions.
    Introduction Collagen is the most abundant protein in the extracellular matrix. Collagen not only provides structural support for cells, tissues, and organs, but also mediates many important biological processes via its interactions with a diverse array of binding partners [[1], [2], [3], [4], [5], [6], [7]]. Dysregulation of these interactions can lead to significant disease pathology. For example, the interaction between discoidin domain receptor 2 (DDR2) and collagen is aberrantly elevated in osteoarthritis, resulting in induction of matrix metalloproteinase 13 and leading to degradation of articular cartilage [8,9]. A molecular-level understanding of DDR–collagen interactions and how they lead to DDR activation would provide the critical information needed to design selective antagonists for basic biomedical research and potential therapeutics. Herein we review the current efforts of using synthetic peptides and recombinant collagen to study these important interactions.
    DDR and collagen
    Use of synthetic peptides to study DDR–collagen interactions Designing synthetic peptides to mimic the collagen triple helix has been a rapidly developing field of research over the past few decades. Synthetic collagen-like peptides have been used to study the structure, the stability, and the biological function of collagen. The earliest works focused on synthesizing simple triple-helical tripeptide repeats such as (PPG) [77] and (POG) [78]. As more techniques to prepare collagen-like peptides and methods to characterize triple helices are being developed, our understanding of collagen and its interactions with other proteins has grown dramatically.
    Use of recombinant collagen to study DDR–collagen interactions