Alyson G. Weidmann

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA.

Construction and Application of a Rh–Pt DNA Metalloinsertor Conjugate

We report the synthesis and characterization of a bimetallic conjugate (RhPt) in which an oxaliplatin derivative is tethered to a rhodium metalloinsertor through an aminomalonate leaving group ligand. The complex interacts with DNA through metalloinsertion at a base pair mismatch followed by formation of a covalent Pt–DNA adduct. Characterization of RhPt in mismatch repair-deficient HCT116O cells reveals increased cytotoxicity compared to cisplatin and oxaliplatin as well as relative to the unconjugated rhodium and platinum counterparts. Caspase and poly-ADP ribose polymerase inhibition assays indicate that RhPt induces apoptotic cell death. Inductively coupled plasma mass spectrometry (ICP-MS) experiments reveal that RhPt exhibits enhanced cellular uptake properties that contribute to its increased efficacy.

Targeted Chemotherapy with Metal Complexes

Classical chemotherapeutics, such as cisplatin and its analogues, have been highly successful in the clinic, yet improvements can certainly be made, given the significant side effects associated with the killing of healthy cells. Recent advances in the field of chemotherapy include the development of targeted anticancer agents, compounds that are directed towards a specific biomarker of cancer, with the hopes that such targeted therapies might have reduced side effects given their greater selectivity. Here we discuss several transition metal complexes that are tailored towards various biomolecules associated with cancer. Most notably, the success of rhodium metalloinsertors, which specifically bind to nucleic acid base mismatches in DNA, highlight the enormous potential of this exciting new strategy. GRAPHICAL ABSTRACT

A Monofunctional Platinum Complex Coordinated to a Rhodium Metalloinsertor Selectively Binds Mismatched DNA in the Minor Groove

We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh-O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA nonclassically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and it triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors.

Biological effects of simple changes in functionality on rhodium metalloinsertors

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors–bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex–our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure–function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus.

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells

Significance We have developed a family of rhodium compounds that bind DNA mismatches and are cytotoxic to mismatch repair (MMR)-deficient cancer cells. This study describes the cellular mechanism of action of the compounds that enables this selective cytotoxicity. The compound binding mode, metalloinsertion, displaces the mismatched base pairs from the DNA helix and creates a DNA lesion that activates the DNA damage response. This lesion is unable to be repaired in MMR-deficient cells. This mechanism is distinct from other compounds that target DNA and provides a therapeutic strategy for treatment of cancers with MMR deficiency. The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)]2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.