Abstracts
Background Pulmonary and hepatic fibrosis are intractable and fatal human disorders. In our previous study, we found that eupatilin, a chromone-scaffold (CS) derivative (CSD), has a strong anti-fibrotic effect on in vitro fibrogenesis as well as a murine lung fibrosis model. Through structure-activity relationship studies, ONG41008 was identified as a more potent anti-fibrotic and anti-inflammatory CS-analog.
Methods A set of cell imaging studies, transcriptomic analyses, and in vivo mouse model involving bleomycin-induced lung fibrosis (BLM) or a murine nonalcoholic steatohepatitis (NASH) model were conducted to explore how ONG41008 exhibits both anti-fibrotic and anti-inflammatory capabilities. In vitro anti-inflammatory activity of ONG41008 was also investigated in RAW264.7 cells, a mouse monocytic cell line stimulated with LPS.
Findings ONG41008 dismantled latent transforming growth factor complex (LTC), limiting TGFβ to TGFβ receptor via depolymerization of F-actin. ONG41008 was able to reverse pathogenic myofibroblasts into non-pathogenic cell types confirmed by both cell shape changes and global transcriptomics analyses. Expression of macrophages homing chemokines in mouse hepatic stellate cells (HSCs) was significantly mitigated by ONG41008. Orally administered ONG41008 significantly improved onset of lung fibrosis. Oral administration of ONG41008 significantly diminished nonalcoholic fatly liver disease (NFALD) scores. ONG41008 substantially inhibited the production of proinflammatory cytokines, including TNFα and several important chemokines affecting T cells or macrophages. TNFα was situated at the central nod in the LPS-ONG41008-macrophages interactome. ONG41008 inhibited endocytosis of CD14, a LPS coreceptor
Interpretation ONG41008 could be a potent drug for a broad range of fibrotic diseases or hyper-inflammatory diseases.
Funding an Osteoneurogen intramural fund
Evidence before this study We previously showed that eupatilin containing a chromone-scaffold (CS) exclusively found in the plant kingdom effectively prevented in vitro fibrogenesis as well as in bleomycin-induced lung fibrosis model. Importantly, cell reversion representing pathogenic myofibroblasts (pMFB) to an intermediate cell type occurred in vitro, and restimulation of the intermediate cell type was trans-differentiated by long exposure to TGFβ, strongly suggesting that this kind of flexible cell reversion would exist in vivo and shed into light on regenerative capacity in fibrotic diseases. One notable feature associated with eupatilin was able to inhibit epithelial-mesenchymal transition (EMT), which plays a central role in initiation and perpetuation of tissue scarring.
Added value of this study Furthering these intriguing therapeutic capabilities, a more effective and stable analog of eupatilin, called ONG41008, was generated and subjected to ameliorating fibrogenesis and inhibiting the various disease activities associated with pMFB. We found that ONG41008 was a potent inhibitor of TGFβ biogenesis such that binding of active TGFβ to TGF receptor (TGFR) was limited, resulting in cessation of TGFR signaling and EMT was completely inhibited, leading to lesser fibrogenesis. Interestingly, ONG41008-treated pMFB underwent generation of a hybrid interactome comprising of a major nuclear reprogramming one and a minor residual fibrogenic interactome, suggesting that ONG41008 is a driver of cell reversion as we saw in the previous study of eupatilin. Macrophages have been well appreciated for liver inflammation and innate immunity. It turned out that ONG41008 was able to block macrophages-chemoattracting proteins called chemokines and directly acted on macrophages, resulting in blocking gene expression of various proinflammatory and inflammatory cytokines.
Implications of all the available evidence All data considered, we believe that ONG41008 is a potent anti-fibrotic drug candidate. And its effectiveness seems likely related to inhibition of macrophage activation. Accordingly, ONG41008 could be used for dysfunctional immunometabolism like NASH as well as blocking the macrophages-mediated innate immunity like rheumatoid arthritis.
Competing Interest Statement
H-S Kim, I-K Kim, and B-S Youn retain the shares of Osteoneurogen, M-K Meang retains a stock option, and SB Kim is employed by OsteoNeuroGen. The current contents of the ONG41008 data have been granted as the subject of a Korean patent and a US patent and has been subjected to USPTO.
Footnotes
bioRxiv Dear Editor, We would like to revise the existing preprint entitling discovery of a small molecule having both potent anti-fibrotic and anti-inflammatory capabilities to bioRxiv (https://www.biorxiv.org/content/10.1101/770404v3). The major reason is we have enormously fortified the preprint with macrophage functions and detailed transcriptomic analysis with depth and width, associated with our drug candidate called ONG41008. The title is unchanging. We have reliably replicated the existing experiments and the addition of the macrophage regulatory functions associated with ONG41008 is a new and daunting discovery. We believe that you shall find what we meant by reading the revised preprint manuscript. We look forward to hearing from your reply. Sincerely, Byung-Soo Youn, Ph.D. CEO (Founder) #411, 128, Gasan digital 1-ro, Geumcheon-gu, Seoul, 08507 Republic of Korea Tel) 822- 6267-2739 Fax) 822-6267-2740 Web) www.osteoneurogen.com Web) www.osteoneurogen.com
