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Antagonism of mTOR Activity by a Kinetically Inert Rhodium(III) Complex [ChemPlusChem](ChemPlusChem Via Acquire Media NewsEdge) Kinetically inert Group 9 metal complexes have found emerging use as inhibitors of protein kinases or as modulators of protein-protein interactions. A series of cyclometalated rhodium(III) and iridium(III) complexes was investigated as inhibitors of mammalian target of rapamycin (mTOR) activity. Cell-free and cell-based experiments revealed rhodium(III) complex 1 to be a potent mTOR inhibitor (IC50 =0.01 µm in the cell-free system), with potency comparable to that of rapamycin. The inhibition by complex 1 was found to be dependent on FK506-binding protein 12 (FKBP12), which suggests that complex 1 may behave as a modulator of the mTOR-FKBP12 interaction. Preliminary structure-activity relationships indicated that the donor ligand and the nature of the metal center are important determinants for mTOR inhibitory activity. Rhodium(III) complex 1 represents the first metal-based inhibitor of mTOR activity, and demonstrates the potential of kinetically inert Group 9 complexes as protein-protein interaction modulators. Keywords : bioorganometallic chemistry · Group 9 metal complexes · inhibitors · rhodium · structure-activity relationships The clinical success of the anticancer compound cisplatin has inspired extensive investigation into the utility of transitionmetal complexes as therapeutic agents.[1] Although most early platinum-based analogues mainly targeted DNA, advancing knowledge in molecular biology has uncovered the possibility of metal complexes acting as molecularly targeted agents against particular proteins or enzymes.[2] Compared to organic molecules, octahedral metal complexes possess several advan- tages that render them suitable as inhibitors of specific biomo- lecular targets. For example, octahedral metal complexes have a unique geometry for the precise arrangement of coligands, whereas organic molecules are largely restricted to the linear, trigonal planar, and tetrahedral geometries offered by the carbon skeleton. In addition, the steric and electronic proper- ties of metal complexes can be easily adjusted without the need for lengthy synthetic protocols, owing to the modular nature of transition-metal complex synthesis. These features may allow octahedral metal complexes to explore regions of the chemical space in protein binding pockets that are inacces- sible to purely organic molecules. In recent years, a number of kinetically inert ruthenium(II),[3] iridium(III),[4] and rhodium(III)[5] complexes have been reported as inhibitors of protein kinases or as modulators of protein-protein interactions.[6] The mammalian target of rapamycin (mTOR) is a serine/ threonine kinase that plays an important role in controlling fundamental cellular processes, such as cell growth, prolifera- tion, survival, and metabolism.[7] In the PI3K/Akt/mTOR path- way (PI3K = phosphoinositide 3-kinase, Akt = serine/threonine kinase), mTOR integrates upstream signaling molecules such as insulin, nutrients, and growth factors.[8] The PI3K/Akt/mTOR sig- naling pathway is often dysregulated in human tumors and has therefore attracted considerable attention as a possible therapeutic target.[9] Rapamycin (sirolimus), an immunosuppressant and antiproli- ferative drug, forms a complex with FK506-binding protein 12 (FKBP12) that inhibits mTOR activity.[10] Intriguingly, the majori- ty of the contacts in the crystal structure of the rapamycin- FKBP12-mTOR ternary complex are formed between the small molecule and the two protein partners, with only two minor interactions being formed between FKBP12 and mTOR.[11] Bind- ing of rapamycin to FKBP12 is proposed to create a new inter- action surface that can be recognized by the FKBP12-rapamy- cin-binding domain of mTOR, which is located N-terminally to the kinase catalytic domain.[12] Additionally, FKBP12 exhibits a low affinity for mTOR in the absence of rapamycin. Hence, ra- pamycin can be considered as a protein-protein interaction stabilizer of the mTOR-FKBP12 interaction. Second-generation mTOR inhibitors target the kinase domain, though they may also show some affinity for PI3K and other PI3K-related protein kinases.[13] However, to the best of our knowledge, no metal- based inhibitor of mTOR has been reported in the literature. Herein, we present the discovery of cyclometalated rhodium- (III) complexes as metal-based modulators of mTOR activity. An in-house panel of rhodium(III) complexes containing vari- ous C^N and N^N ligands was screened in a preliminary enzyme-linked immunosorbent assay (ELISA) to evaluate their ability to inhibit mTOR kinase activity. In this assay, recombi- nant mTOR is preincubated with FKBP12 and the metal com- plexes, and transferred to wells containing the mTOR substrate p70S6K. Active mTOR phosphorylates p70S6K at Thr389 in the presence of adenosine 5'-triphosphate, and the kinase activity of mTOR is detected using anti-p70S6K-T389 antibody. From the preliminary results, the novel rhodium(III) complex 1 (Scheme 1) emerged as a top candidate. Complex 1 was syn- thesized according to a modified literature protocol,[14] and characterization of the complex is given in the Supporting In- formation. A dose-response experiment was then conducted to investigate the potency of complex 1 against mTOR activity. The results revealed that complex 1 inhibited mTOR activity with an IC50 value (concentration of compound required to in- hibit 50 % of kinase activity) of 0.01 mm in the ELISA (Figure 1). Significantly, the inhibitory effect of complex 1 was compara- ble to that of rapamycin in a side-by-side experiment. Given the promising activity exhibited by complex 1, we de- cided to synthesize several rhodium(III) and iridium(III) ana- logues (2-5 ; Scheme 1) to investigate the effect of structural modification on this type of complex. The characterization of the novel complexes 2-4 is given in the Supporting Informa- tion. The mTOR inhibitory activi- ty of these Group 9 complexes was evaluated by ELISA (Fig- ure 2). The rhodium(III) com- plexes 1-4 each contain two 2- (4-methylphenyl)pyridine C^N moieties, but differ in the nature of their N^N donor ligand. The most active complex, rhodium(III) complex 1, possess- es the 5,5'-dimethyl-2,2'-bipyri- dine N^N ligand. Rhodium(III) complexes 2 and 3 contain more bulky 4,4'-di-tert-butyl-2,2'-bipyri- dine and 4,4'-nonyl-2,2'-bipyri- dine N^N ligands, respectively, and showed lower mTOR inhibi- tory activity than complex 1. This suggests that excessively large N^N substituents may reduce inhibitory potency for this type of complex. The more polar 4,4'-dimethoxy-2,2'-bipyri- dine N^N ligand also did not en- hance mTOR inhibitory activity, as observed for the rhodium(III) complex 4. Finally, the iridium(III) congener 5, which differs from rhodium(III) complex 1 only in the nature of the metal center, also showed reduced potency. This indicates the importance of the rhodium(III) center in provid- ing an optimum arrangement of the coligands in the complex to achieve greater binding affinity for mTOR and/or FKBP12 proteins. To investigate the mechanism of rhodium(III) complex 1,we evaluated the inhibitory activity of the complex in the pres- ence or absence of FKBP12. The results showed that the inhibi- tory effect of complex 1 was FKBP12-dependent. The mTOR in- hibitory activity of complex 1 was strongly attenuated in the absence of FKBP12 (Figure 3). The kinase activity of mTOR im- munoprecipitates from treated human hepatocellular carcino- ma (HepG2) cells was also evaluated to determine whether complex 1 could inhibit mTOR activity in cells. Briefly, cells were treated with complex 1 or rapamycin. Cell lysates were precipitated with anti-mTOR/FRAP antibody (FRAP = FKBP12-ra- pamycin-associated protein) and the mTOR activity in the im- munoprecipitates was determined by ELISA. Encouragingly, complex 1 has similar mTOR inhibitory potency in cells to rapa- mycin (Figure 4). We observed that the immunoprecipitates of cells treated with 0.1, 1, or 10 mm of complex 1 showed 48, 61, or 78 % reduction in mTOR activity, respectively, with an esti- mated IC50 value of 0.1 mm. By comparison, rapamycin inhibited mTOR activity in immunoprecipitates by about 38, 54, and 73 % at a concentration of 0.1, 1, or 10 mm, respectively. Derivatives of rapamycin (rapalogues) have been extensively investigated for antiproliferative activity, with everolimus and temsirolimus being approved for the treatment of renal cell carcinoma and mantle cell lymphoma.[15] The anticancer mech- anism of the rapalogues is proposed to involve the inhibition of mTOR phosphorylation activity.[16] This leads to the downre- gulation of genes involved with cell-cycle progression, protein translation, angiogenesis, and the induction of G1 cell-cycle arrest and apoptosis in some cell lines.[17] Therefore, we investi- gated the cytotoxicity of complex 1 towards HepG2 cells. The results showed that complex 1 exhibited an IC50 value of 1 mm for cytotoxicity against HepG2 cells after incubation for 48 h. We presume that the cytotoxicity of complex 1 could be ex- plained, at least in part, by the inhibition of mTOR signaling by the rhodium(III) complex in the HepG2 cells. In conclusion, we have discovered the first metal-based in- hibitor of mTOR activity. The rhodium(III) complex 1 attenuated mTOR activity in a cell-free system and in mTOR immunopreci- pitates from treated cells. A brief structure-activity relationship analysis with related complexes suggested that the size and hydrophobicity of the N^N donor ligand and the nature of the metal center are important determinants for mTOR inhibitory activity. Further lead optimization of analogues of rhodium(III) complex 1 is currently under way in our laboratory. Our find- ings also suggest that complex 1 may exhibit a similar mecha- nism of action to rapamycin. Complex 1 may first bind to FKBP12 to create a protein-protein interface that is subse- quently recognized by the mTOR FKBP12-rapamycin-binding domain. Therefore, complex 1 may be tentatively considered as a protein-protein interaction stabilizer of the mTOR-FKBP12 interaction. 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China) Fax: (+852) 3411-7348 E-mail: [email protected] [b] H.-J. Zhong,+ L.-J. Liu, Dr. C.-H. Leung State Key Laboratory of Quality Research in Chinese Medicine Institute of Chinese Medical Sciences University of Macau, Macao (P. R. China) Fax: (+853) 2884-1358 E-mail: [email protected] [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cplu.201400014. mTOR=mammalian target of rapamycin. This article is part of the "Early Career Series". To view the complete series, visit : http://chempluschem.org/earlycareer. (c) 2014 Blackwell Publishing Ltd. |