We have previously employed high-throughput virtual screening to identify 6,699-biapigenin as only the second inhibitor of NEDD8-activating enzyme from a natural product and natural product-like database [44]. While transition metal complexes have been widely utilized for the treatment of cancer [45?7], their activity against NEDD8-activating enzyme has not been explored. Inspired by the above findings as well as pioneering works from the Meggers’s group on the design of structurally rigid octahedral ruthenium(II) [48?3] and iridium(III) [53?5] complexes as shape-complementary inhibitors of protein kinases, we sought to investigate the biological effects of a series of cyclometallated rhodium(III) complexes on the NEDD8 pathway. Cyclometallated rhodium(III) complexes containing the dipyrido[3,2-a:29,39-c]phenazine dipyridophenazine (dppz) scaffold were chosen because of the following reasons: 1) the rhodium(III) complex adopts an octahedral geometry rather than a square planar or tetrahedral symmetry, thus allowing much larger structural complexity for potential use in drug design; 2) the octahedral geometry of the rhodium complex provides a globular and rigid scaffold with limited conformational freedoms of the coligands that may interact with the previously inaccessible regions of chemical space in NAE; 3) the synthetic route for 1 is modular and convenient, thus allowing structural modification without the need for lengthy synthetic protocols; and 4) the extended aromatic dppz ligand structurally resembles the planar nature of NAE inhibitor 6,699-biapigenin [44], potentially functioning as the recognition arm for NAE. We report herein the synthesis and characterization of the racemic mixture of rhodium(III) complex [Rh(ppy)2(dppz)]+ (1) and its analogues (2?). Complex 1 was found to inhibit NAE activity in vitro and in cellulo. We then investigated the structureactivity relationship of the Rh(III) complexes against NAE activity in vitro. Furthermore, complex 1 inhibited downstream CRLregulated substrate degradation and NF-kB signaling in cellulo, and was also found to exhibit prominent anti-proliferative activity against a human cancer cell line. Molecular modeling analysis revealed that 1 occupied the same binding pocket as MLN4924, the most potent NAE inhibitor to date.
Results and Discussion
The precursor complexes [Rh(ppy)2(OH2)2]+ (ppy = 2-phenylpyridine) and [Rh(fppy)2(OH2)2]+ (fppy = 4-(2-pyridyl)benzaldehyde), dppz, and dppz analogues were prepared according to a modified literature method [56?8]. Complex 1 was prepared by treating the precursor complex [Rh(ppy)2(OH2)2]+ with dppz in refluxing MeCN, and was precipitated as the CF3SO3?salts. Complexes 2? were synthesized in a similar fashion using the corresponding precursor complexes and ligands (Figure S1). The structures and the purities of complex 1? were determined using NMR spectroscopy and high resolution mass spectrometry. The impact of 1 on the NAE activity was first evaluated by a cell-free assay that measures the formation of the Ubc12NEDD8 conjugation product. Recombinant human NAE was incubated with Ubc12 and NEDD8 in the presence of vehicle (DMSO) or 1. NAE promotes NEDDylation of Ubc12 which leads to the formation of the Ubc12-NEDD8 thioester product. We observed that this process was suppressed in the presence of 1. Western blot analysis revealed a dose-dependent reduction of Ubc12-NEDD8 formation by 1 with an IC50 value of ca. 1.5 mM and complete inhibition at 12.5 mM (Figure 2a). We next evaluated the ability of 1 to inhibit Ubc12-NEDD8 formation inside human epithelial colorectal adenocarcinoma (Caco-2) cells. A dose-dependent decrease in the level of the Ubc12-NEDD8 conjugate was observed upon treatment with 1 for 16 hours (Figure 2b). However, no significant inhibition of Ubc9SUMO formation was observed at the same concentrations tested. The potency of 1 against NAE activity in cells was comparable to that of known inhibitor MLN4924 under the conditions employed. This result shows that complex 1 was able to suppress the activity of NAE in a cellular system but not the closely related SAE, which is consistent with the result from cell-free western blotting. We then screened three analogues of complex 1 in order to establish a brief structure-activity relationship for the observed in vitro effects against NAE activity in the cell-free assay (Figure S2). Interestingly, the results showed that functionalizing the dppz ligand with methyl groups (complex 2) or the ancillary ppy ligands with aldehyde groups (complex 3) yielded less active analogues (IC50 = ca. 6.25 mM). However, the incorporation of an electronwithdrawing nitro group on the dppz motif together with aldehyde functionalities at the ppy ligands (complex 4) resulted in the weakest NAE inhibitory activity (IC50 = ca. 13 mM). Furthermore, the uncoordinated dppz ligand was found to be inactive against Ubc12-NEDD8 conjugation at a relative high concentration tested
Figure 1. Chemical structures of MLN4924, [Rh(ppy)2(dppz)]+ (1) and Rh(III) analogues (2?). Figure 2. Complex 1 inhibits NAE activity in a dose-dependent manner. Western blots show dose-dependent inhibition of a) Ubc-12-NEDD8 formation in a cell-free system and b) cellular Ubc12-NEDD8 levels by 1. MLN4924 was included for comparison. (data not shown). Taken together, these data suggest that the metal center plays an important role in the arrangement of ligands for optimal recognition of the protein binding site, and that the observed in vitro potency of these analogues are sensitive to steric and/or electronic properties of the metal complexes. NAE promotes ubiquitination and the subsequent degradation of a subset of proteins regulated by CRLs such as IkBa [59,60] and p27 [61,62]. The IkBa protein plays a central role in repressing the activity of the transcription factor NF-kB [63,64], which is involved in important cellular processes including the immune response, programmed cell death, as well as cancer initiation and progression [65,66]. The inhibition of NAE is therefore a potential approach to block the degradation of IkBa thus preventing NF-kB activation. On the other hand, the p27 is a cell cycle regulator which controls cell cycle progression during the G1 phase [67].