br Unantimycin A has a
Unantimycin A has a chemical structure similar to that of SW-163A, which is in the neoantimycin class of compounds. Neoantimycin and antimycin A are best known to inhibit complex III by specifically binding to the quinone reduction site (Qi) of this complex, thus blocking electron transfer from heme bH to ubiquinone. There are several neoantimycin analogs with diﬀerent benzoic BODIPY 493/503 moieties. They include JBIR-04, which contains a non-substituted benzene, and JBIR-05, which lacks a formyl group . There is ongoing research on the structure-activity-relationship (SAR) of antimycin/neoantimycin. It has been suggested that the 2-hydroxy-3-formylamino group in these
Y. Futamura et al.
compounds are essential for their inhibitory eﬀects . In contrast, UK-2A, another antimycin analog without the functional group, ex-hibits a biological activity similar to that of antimycin A . There-fore, it is still important to conduct SAR studies on new analogs of such compounds. Unantimycin A is a unique neoantimycin analog with a 3-hydroxybenzoic acid group instead of the key functional group. How-ever, its inhibitory eﬀect on complex III was approximately 30 times less than that of antimycin A or SW-163A (Fig. 6 and Fig. S6). This data is important in conducting detailed SAR studies and developing more potent analogs.
Besides, we also found that NPL40330, a thiourea containing com-pound, inhibits mitochondria complex I activity. Although many mi-tochondria complex I inhibitors, such as rotenoids, piericidins, acet-ogenins, capsaicins, amilorides, biguanides, and IACS-10759 have been reported [47–49]; however, NPL40330 is not chemically similar to the other complex I inhibitors at all. Furthermore, there is few biological activity reports regarding NPL40330 and analogues thereof, indicating that this compound may be a new class of complex I inhibitors. Thus, the screening methodology based on bioenergetics and proteomic profiling is promising for the discovery of unique antimetabolic com-pounds.
In order to find novel compounds that aﬀect cancer metabolism, we constructed a screening system based on bioenergetics and proteomic profiling of cancer cells treated with the compounds. Using the screening system, we found that unantimycin A and NPL40330 inhibit mitochondrial respiration. In order to elucidate the mechanisms of ac-tion of the target compounds, we set up an in vitro reconstitution assay for mitochondrial ETC using semi-intact cells. The results showed that the targets of NPL40330 and unantimycin A are mitochondrial com-plexes I and III, respectively.
We thank Ms. H. Kondo, Ms. K. Noda, Ms. Y. Nakata, Ms. Y. Hirata, and Ms. M. Tanaka for conducting the proteome analysis; Dr. T. Suzuki and Dr. N Dohmae for their help with the protein identification study; Ms. A. Okano and Dr. C. L. Lim for isolating the natural products; Mr. H. Hirano, Mr. Y. Iwai, and Ms. A. Yoshioka for providing the chemical library and hit compounds; and Dr. J. Otaka and Mr. K. Yamamoto for performing the biological activity tests. This work was supported in part by JSPS KAKENHI Grant Numbers JP17H06412, JP18H03945, JP17K07783, JP17K01970, JP16K01941, JP17K07784, and a Grant-in-Aid for the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from AMED.
 M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg eﬀect: the metabolic requirements of cell proliferation, Science 324 (2009)
clastogenesis inhibitor through the inhibition of glyoxalase I, Proc. Natl. Acad. Sci.
Identification of a small-molecule inhibitor of DNA topoisomerase II by proteomic profiling, Chem. Biol. 18 (2011) 743–751.
K. Lemire, J. Orrell, J. Teich, S. Chomicz, D.A. Ferrick, Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic func-tion and enhanced glycolysis dependency in human tumor cells, Am. J. Physiol. Cell Physiol. 292 (2007) C125–C136.  D.A. Ferrick, A. Neilson, C. Beeson, Advances in measuring cellular bioenergetics using extracellular flux, Drug Discov. Today 13 (2008) 268–274.
Y. Futamura et al.
 A. Subedi, Y. Futamura, M. Nishi, A. Ryo, N. Watanabe, H. Osada, High-throughput screening identifies artesunate as selective inhibitor of cancer stemness: involve-ment of mitochondrial metabolism, Biochem. Biophys. Res. Commun. 477 (2016) 737–742.
 H. Miyoshi, N. Tokutake, Y. Imaeda, T. Akagi, H. Iwamura, A model of antimycin A binding based on structure-activity studies of synthetic antimycin A analogues, Biochim. Biophys. Acta 1229 (1995) 149–154.  K. Machida, H. Takimoto, H. Miyoshi, M. Taniguchi, UK-2A,B,C and D, novel an-tifungal antibiotics from Streptomyces sp.517.02. V. Inhibition mechanism of bo-vine heart mitochondrial cytochrome bc1 by the novel antibiotic UK-2A, J.