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.

KIND-a non-redundant protein database.

SUMMARY: KIND (Karolinska Institutet Nonredundant Database) is a protein database where identical sequences, both full length and partial, have been removed. The database contains nearly 274 900 sequences, half of which originate from the protein sequence databases Swissprot and PIR, while the other half come from translated open reading frames in GenPept and TrEMBL. AVAILABILITY: KIND is downloadable from ftp://ftp.mbb.ki.se/pub/KIND.

Prediction of amyloid fibril-forming proteins.

In Alzheimer's disease and spongiform encephalopathies proteins transform from their native states into fibrils. We find that several amyloid-forming proteins harbor an alpha-helix in a polypeptide segment that should form a beta-strand according to secondary structure predictions. In 1324 nonredundant protein structures, 37 beta-strands with > or =7 residues were predicted in segments where the experimentally determined structures show helices. These discordances include the prion protein (helix 2, positions 179-191), the Alzheimer amyloid beta-peptide (Abeta, positions 16-23), and lung surfactant protein C (SP-C, positions 12-27). In addition, human coagulation factor XIII (positions 258-266), triacylglycerol lipase from Candida antarctica (positions 256-266), and d-alanyl-d-alanine transpeptidase from Streptomyces R61 (positions 92-106) contain a discordant helix. These proteins have not been reported to form fibrils but in this study were found to form fibrils in buffered saline at pH 7.4. By replacing valines in the discordant helical part of SP-C with leucines, an alpha-helix is found experimentally and by secondary structure predictions. This analogue does not form fibrils under conditions where SP-C forms abundant fibrils. Likewise, when Abeta residues 14-23 are removed or changed to a nondiscordant sequence, fibrils are no longer formed. We propose that alpha-helix/beta-strand-discordant stretches are associated with amyloid fibril formation.