ABSTRACT
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.
Subject(s)
DNA Repair Enzymes/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Neoplasms/drug therapy , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Pyrimidines/administration & dosage , 8-Hydroxy-2'-Deoxyguanosine , Animals , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/isolation & purification , Deoxyguanosine/metabolism , Humans , Mice , Neoplasms/genetics , Neoplasms/pathology , Nucleotides/metabolism , Oxidation-Reduction , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Proto-Oncogene Proteins B-raf/genetics , RNA, Small Interfering/genetics , Xenograft Model Antitumor AssaysABSTRACT
Semicarbazide-sensitive amine oxidase (SSAO) activity in plasma is increased in diabetes, and in particular, in diabetic patients with vascular complications. It has been speculated that SSAO is involved in the development of such complications due to the production of cytotoxic compounds. In this work, we have induced diabetes in a previously described mouse-model, overexpressing SSAO in smooth muscle cells. SSAO activity was estimated as well as expression of the endogenous mouse gene and human transgene using real-time PCR. Diabetes induced an increase in SSAO activity in serum, kidney, and adipose tissue of transgenic animals. An inverse correlation between SSAO activity and mouse SSAO mRNA levels was observed in transgenic animals with diabetes. These results further support the suggestion of a negative feedback control of the SSAO gene expression. The increased SSAO activity in diabetes is most likely dependent on post-transcriptional modifications or activation of existing inactive enzyme molecules.
Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Cell Adhesion Molecules/metabolism , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/enzymology , Mice, Transgenic/metabolism , Muscle, Smooth/enzymology , Adipose Tissue/enzymology , Alloxan , Amine Oxidase (Copper-Containing)/genetics , Animals , Cell Adhesion Molecules/genetics , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/genetics , Humans , Kidney/enzymology , Lung/enzymology , Mice , Organ Specificity , Recombinant Proteins/metabolism , Tissue DistributionABSTRACT
Patients with diabetes mellitus and with vascular complications in particular, exhibit higher plasma activities of semicarbazide-sensitive amine oxidase (SSAO) compared to control subjects. It has been speculated that production of cytotoxic products of SSAO may cause endothelial damage and thus contribute to the development of diabetic vascular complications such as retino-, nephro-, and neuropathies as a result of SSAO activity.In order to explore the possibility that high SSAO activity contributes to the development of vascular complications in diabetes, we have performed two studies in patients with Type-2 diabetes quantifying plasma SSAO activity, HbA(1c), and urinary levels of the SSAO substrate, methylamine. We also examined the prevalence of retinopathy in these patients. Additionally, we have studied a model of transgenic mice expressing human SSAO in smooth muscle cells. The transgenic mice have an increased SSAO activity as well as mRNA expression. Histological studies revealed a specific aorta phenotype with a condensed and rigid vessel wall in some of the transgenic mice. No wild-type animals displayed this phenotype.In conclusion, we suggest that this transgenic mouse model may be of great value for increasing the knowledge about to what extent human SSAO contributes to vascular complications in diabetes, and also to which extent inhibition of SSAO can prevent the development of such complications.
