Cell-Selective Cytotoxicity of a Fluorescent Rhodium Metalloinsertor Conjugate Results from Irreversible DNA Damage at Base Pair Mismatches

Abstract

Up to twenty percent of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability (MSI) that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate and accumulate DNA mismatches. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)] 2+ (RhPPO) is the most potent, selective compound in this class and acts by targeting DNA mismatches, resulting in preferential cytotoxicity to MMR-deficient cancers. To explore further the cellular mechanism of action of RhPPO, we conjugated the metal complex to a fluorescent probe, cyanine 3 (Cy3). RhPPO-Cy3 binds DNA mismatches and retains the selectivity and potent cytotoxic activity of RhPPO for MMR-deficient cell lines. RhPPO-Cy3 forms discrete foci in the cell nucleus that overlap with sites of DNA damage, suggesting that the lesions occur at or near DNA mismatch sites. RhPPO-Cy3 foci persist over time, despite initial processing of the lesion and recruitment of repair proteins, consistent with the idea that the complex binding to a mismatch prevents repair. RhPPO-Cy3 binding does not lead to activation of p53 and the apoptotic pathway. Together, these findings support the idea that RhPPO-Cy3 binding leads to irreversible DNA damage at DNA mismatches that enables selective cytotoxicity to MMR-deficient cells.

In vivo anticancer activity of a rhodium metalloinsertor in the HCT116 xenograft tumor model

“…We have designed and characterized rhodium metalloinsertors which bind specifically to DNA mismatches and as a result induce selective toxicity within MMR-deficient cancer cell lines ( 16 , 22 , 23 , 31 – 33 ). In addition, various ruthenium complexes have been designed and synthesized as chemotherapeutics, although with different biological targets ( 21 , 34 – 36 ).…”

Rhodium Complexes Targeting DNA Mismatches as a Basis for New Therapeutics in Cancers Deficient in Mismatch Repair

“…We found that RhPPO-Cy3 displayed selective luminescence upon binding to mismatched DNA in vitro , with a binding affinity, K B (CC), of 1.8 × 10 6 M –1 , comparable to that of unconjugated RhPPO (5.4 × 10 6 M –1 ) . RhPPO-Cy3 also maintained high potency and selectivity for MMR-deficient cells . Thus, the properties of RhPPO-Cy3 enabled further study of rhodium metalloinsertor activity at DNA mismatches in cells.…”

“…RhPPO-Cy3 formed nuclear foci in MMR-deficient cells, demonstrating for the first time that a rhodium complex localizes to genomic DNA (Figure ). Furthermore, the RhPPO-Cy3 foci showed partial overlap with DNA damage-induced foci containing pH2AX and Rad51 . In addition to H2AX phosphorylation, RhPPO-Cy3 binding also induced phosphorylation of the checkpoint protein kinase Chk1.…”

“…Cisplatin-induced DNA lesions may be repaired or induce cell death by apoptosis (Figure ). Interestingly, RhPPO and its conjugate, RhPPO-Cy3, do not activate p53, and cells die by necrosis rather than by apoptosis. , We considered whether the MMR pathway itself may be required to induce apoptosis after RhPPO treatment, as is the case for cisplatin. It may be that the chromatin context of the DNA lesions generated by RhPPO binding is not recognized by the appropriate DNA repair factors, or that the cells are blocked in a cell cycle stage where the necessary repair pathway is not active.…”

Cyanine Conjugate‐Based Biomedical Imaging Probes

“…For selectively detection of DNA mismatches, Barton and colleagues conjugated Cy3 to two slightly different rhodium metalloinsertors, i.e., [Rh(phen)(chrysi)(HDPA)] 3+ ( 58 ) and [Rh(PPO)(chrysi)(HDPA)] 2+ ( 59 ) (Figure 10h). [ 87 ] Both probes displayed an enhanced fluorescence emission toward single‐mismatched DNA and were able to differentiate the MMR‐deficient versus MMR‐proficient phenotype in genomic DNA extraction and living cells, respectively. Additionally, 59 could induce irreversible DNA damage at or near mismatched site (Figure 10h) and possessed limited cytotoxicity for MMR‐deficient HCT‐116 O cells (EC 50 = 1.0 × 10 −6 ± 0.1 × 10 −6 m ).…”