Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor–induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival.

Osteoclasts, bone-resorptive multinucleated cells derived from hematopoietic stem cells, are associated with many bone-related diseases, such as osteoporosis. Osteoclast-targeting small-molecule inhibitors are valuable tools for studying osteoclast biology and for developing antiresorptive agents. Here, we have discovered that methyl-gerfelin (M-GFN), the methyl ester of the natural product gerfelin, suppresses osteoclastogenesis. By using M-GFNimmobilized beads, glyoxalase I (GLO1) was identified as an M-GFN-binding protein. GLO1 knockdown and treatment with an established GLO1 inhibitor in osteoclast progenitor cells interfered with osteoclast generation, suggesting that GLO1 activity is required for osteoclastogenesis. In cells, GLO1 plays a critical role in the detoxification of 2-oxoaldehydes, such as methylglyoxal. M-GFN inhibited the enzymatic activity of GLO1 in vitro and in situ. Furthermore, the cocrystal structure of the GLO1/M-GFN complex revealed the binding mode of M-GFN at the active site of GLO1. These results suggest that M-GFN targets GLO1, resulting in the inhibition of osteoclastogenesis.osteoclast ͉ small molecule ͉ crystal structure

Visual observation is a powerful approach for screening bioactive compounds that can facilitate the discovery of attractive druggable targets following their chemicobiological validation. So far, many high-content approaches, using sophisticated imaging technology and bioinformatics, have been developed. In our study, we aimed to develop a simpler method that focuses on intact cell images because we found that dynamic changes in morphology are informative, often reflecting the mechanism of action of a drug. Here, we constructed a chemical-genetic phenotype profiling system, based on the high-content cell morphology database Morphobase. This database compiles the phenotypes of cancer cell lines that are induced by hundreds of reference compounds, wherein those of well-characterized anticancer drugs are classified by mode of action. Furthermore, we demonstrate the applicability of this system in identifying NPD6689, NPD8617, and NPD8969 as tubulin inhibitors.

Anti-apoptotic oncoproteins Bcl-2 and Bcl-xL are overexpressed in many cancers, 1 resulting in the expansion of a transformed population and the advancement of the multidrug-resistant stage. Consequently, Bcl-2/Bcl-xL have stood out among molecular targets in oncology, and the functional blockade of these proteins will be an aid to novel anti-tumor therapies. Since these proteins are known to show an anti-apoptotic effect partly through forming a heterodimer with pro-apoptotic Bcl-2 members, such as Bax and Bak, 2 several researchers have rationally designed and synthesized compounds that target their binding pocket and have reported several compounds such as HA14-1 and ABT-737 as Bcl-2/BclxL inhibitors. 3 Currently, several lines of evidence indicate that Bcl-2/Bcl-xL clearly have other functions related to their abilities to interact physically with many other proteins; however, the underlying mechanisms for the regulation of apoptosis by Bcl-2/ Bcl-xL through interacting with such proteins still remain unclear. 4 For further understanding of the regulation of apoptosis by Bcl-2/ Bcl-xL, the development of a new class of chemical tools is required. Cell-based chemical-genetic screens have been used to help discover small, cell-permeable bioactive molecules that induce phenotypic changes, and through subsequent identification of their target proteins, they can contribute to reveal the molecular basis of biological processes. Therefore, we constructed a cell-based chemical-genetic screening system to discover small molecules that induce apoptosis in Bcl-xL-overexpressing human small cell lung carcinoma Ms-1 cells when combined with anti-tumor drugs. In the course of our screening, we isolated a structurally and functionally unique compound, named incednine (1), from the culture broth of Streptomyces sp. ML694-90F3. Here, we describe the isolation, structure elucidation, and biological activities of 1.Incednine (1) was obtained (18.9 mg/L) as a pale-yellow powder from the cultured broth of the producing strain by centrifugal liquid-liquid partition chromatography for two reasons: (1) this compound decomposes easily under acidic conditions or when exposed to light, and (2) the isolation using a solid carrier such as silica gel was inefficient. The molecular formula of 1 was found to be C 42 H 63 N 3 O 8 by HRESIMS. The characteristic UV absorption (λ max ) 294.5, 309.5, 322.5, 356.0 nm) was indicative a polyene moiety. The positive color reaction to GL reagent, negative reaction to ninhydrin, and the typical IR absorption at 1650, 1510 cm -1 suggested the presence of an amido group.The assignable NMR spectra were obtained when the sample was dissolved in CD 3 OH/H 2 O (3:1) and measured at -5°C. The 13 C NMR and DEPT spectra revealed that 1 contained 42 carbons, including one carbonyl, four quaternary sp 2 , one quaternary sp 3 , fourteen sp 2 methines, nine sp 3 methines, four methylenes, and nine methyl carbons. One carbonyl signal and eighteen sp 2 carbons require the presence of three rings from the unsaturatio...

Amino-group carrier proteins (AmCPs) mediate the biosynthesis of lysine and arginine in some bacteria and archaea. Here we demonstrate that an uncharacterized AmCP-mediated biosynthetic system functions to biosynthesize the previously uncharacterized and nonproteinogenic amino acid (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH) in Streptomyces sp. SANK 60404. DADH is incorporated into a novel peptide metabolite, vazabitide A, featuring an azabicyclo-ring structure, by nonribosomal peptide synthetases and successive modification enzymes in this bacterium. As the AmCP-mediated machinery for DADH biosynthesis is widely distributed in bacteria, further analysis of uncharacterized AmCP-containing gene clusters will lead to the discovery of novel bioactive compounds and novel biosynthetic enzymes.