Small-molecule-mediated OGG1 inhibition attenuates pulmonary inflammation and lung fibrosis in a murine lung fibrosis model.

Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by persistent micro-injuries to alveolar epithelial tissues accompanied by aberrant repair processes. IPF is currently treated with pirfenidone and nintedanib, compounds which slow the rate of disease progression but fail to target underlying pathophysiological mechanisms. The DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) has significant roles in the modulation of inflammation and metabolic syndromes. Currently, no pharmaceutical solutions targeting OGG1 have been utilized in the treatment of IPF. In this study we show Ogg1-targeting siRNA mitigates bleomycin-induced pulmonary fibrosis in male mice, highlighting OGG1 as a tractable target in lung fibrosis. The small molecule OGG1 inhibitor, TH5487, decreases myofibroblast transition and associated pro-fibrotic gene expressions in fibroblast cells. In addition, TH5487 decreases levels of pro-inflammatory mediators, inflammatory cell infiltration, and lung remodeling in a murine model of bleomycin-induced pulmonary fibrosis conducted in male C57BL6/J mice. OGG1 and SMAD7 interact to induce fibroblast proliferation and differentiation and display roles in fibrotic murine and IPF patient lung tissue. Taken together, these data suggest that TH5487 is a potentially clinically relevant treatment for IPF but further study in human trials is required.

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli.

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.

Synthesis and secretion of a fibrinolytically active tissue-type plasminogen activator variant in Escherichia coli.

A gene encoding a variant (lacking amino acids 6-173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.