In Partial Fulfillment of the Requirements for the Degree of

Thanks to Dave VanderVelde for his incredible willingness to help with NMR experiments. To the men and women of the Barton lab: thank you for making my work environment supportive and enjoyable.

Introduction

Metal-Based Chemotherapeutics

However, the goal has also shifted to designing complexes with a new strategy based on selectivity by preparing transition metal complexes that are more selective than cisplatin due to a design strategy where the complex interacts with a specific biological target that is visible. in cancer cells. Establishing the cellular uptake and even the subcellular distribution of metal complexes is crucial for understanding and optimizing their activity.

DNA as a Target

• Covalent Interactions
• Intercalation
• Insertion

Indeed, when the complex is intercalated at the central TA/TA step of the oligonucleotide d(CCGGTACCGG)2, the complex is deeply, perpendicularly, and symmetrically intercalated into the base stack from the minor groove. The extruded bases (show i lue) π-stacking with the bpy rings of the intercalated metal complexes (interactions shown in black, metal complexes shown in red).

Figure 1.2 Crystal structure at 2.6-Å resolution of the intrastrand crosslink between cisplatin and the N7 atoms of adjacent guanines

Biological Activities of Metal Complexes

• Polypyridyl Complexes
• Ruthenium Arene Complexes
• Rhodium Metalloinsertors

Expulsion of the mismatched bases results in a large lesion that could be easily recognized in vivo. In one of the earliest studies, Novakova and co-workers studied four chloropolypyridylruthenium complexes (Figure 1.6) in murine and human tumor cell lines.

Figure 1.6 Chemical structures of four ruthenium polypyridyl complexes studied by Novakova and coworkers

Cellular Uptake of Metal Complexes

• Methods to Monitor Cellular Uptake
• Relationships Between Drug Uptake and Activity
• Mechanisms of Uptake

The uptake of positively charged molecules, like many inorganic therapeutics, can be driven by the cell's plasma membrane potential. In addition, the uptake of the compound increased significantly in the presence of valinomycin (which will increase membrane potential).

Figure 1.11 Chemical structures of Rh(III) polypyridyl complexes synthesized and studied to monitor uptake

Subcellular localization of the Metal Complex

• Methods to monitor Localization
• Peptide Conjugation

One method for visualizing the subcellular localization of metal-based therapeutic agents while maintaining the structural integrity of the cell is electron microscopy. After conjugation of the cobalt complex to the nuclear localization peptide, uptake of the complex into the nucleus of HT-29 cells increased significantly.97.

Figure 1.18 The rhodium metalloinsertor-peptide conjugate synthesized in order to accelerate uptake

Targeted Therapeutics

• Proteins as Targets
• Organelles as Targets
• DNA Lesions as Targets

However, the remaining ligands on each complex were designed to form a unique set of hydrogen-bonding interactions with the glycine-rich loop, ATP-binding pockets of six different kinases (Figure 1.21).104 In vivo studies have shown that anti-angiogenic properties of one of these types of compounds in zebrafish embryos illustrating their potential.105. The pyridocarbazole ligand, common to all complexes, binds to the hinge region (where the adenine part of ATP binds) of the ATP-binding pocket. The remaining ligands A, B, C, and D form a series of hydrogen bond interactions with the glycine-rich loop (where the ribose triphosphate moiety of ATP binds) of the ATP-binding pocket, each of which is unique to a particular kinase.

Figure 1.20 Chemical structures of targeted chemotherapeutics: (top, left to right) The octasporine complex OS1, a potent inhibitor of the protein kinase GSK3; General architecture of RAPTA cathepsin B inhibitors; Ruthenocene anal

Conclusion

Mismatches in genomic DNA occur naturally as a consequence of replication, but if not corrected can lead to mutations.115,116 The mismatch repair (MMR) pathway serves as a checkpoint to increase the fidelity of DNA replication ~1000-fold.117 Important is that deficiencies in the mismatch repair machinery have been associated with several types of cancer, as well as increased resistance to classical chemotherapeutics such as cisplatin.118 Therefore, the development of a targeted treatment for MFR-deficient cancers would be invaluable in the clinic. Due to the unique DNA mismatch binding properties of rhodium metalloinsertors, their biological properties in MMR-deficient cells were investigated. The unique reactivity and coordination geometry of the metal complexes make them the ideal scaffold for this new tailored design of targeted therapies.

Selective Cytotoxicity of Rhodium Metalloinsertors in

Introduction

Confocal microscopy and flow cytometry studies with [Ru(L)2dppz]2+. dppz=dipyrido[3,2-a:2',3'-c]phenazine analogues have demonstrated that cellular accumulation occurs via passive diffusion, facilitated by a negative potential change across the cell membrane.19,20 Taken together , the observations that (i) rhodium metal inserters are able to recognize mismatches with high selectivity and. ii) ruthenium analog complexes accumulate inside cells and nuclei form a sound basis for the hypothesis that rhodium metal internalizers recognize misfolded DNA inside cells and predict that these complexes must selectively target MMR-deficient cells, which contain ~1000 times more mismatches than MMR-competent cells. The HCT116N cell line is transfected with human chromosome 3 (ch3), which restores MMR ability, while the HCT116O cell line is transfected with human chromosome 2 (ch2), leaving it MMR deficient. inhibit DNA synthesis in the MMR-deficient HCT116O cell line over the MMR-proficient HCT116N cell line,22 as measured by a DNA synthesis ELISA assay.23 Significantly, the binding affinities of the metalloinserters were found to be directly assocd. with the selectivity of the rhodium complexes for the MMR-deficient cell line, lending credence to the idea that these complexes target DNA mismatches in cell as well as in vitro.24. Here, we show that rhodium metalloinserts are preferentially cytotoxic to the MMR-deficient HCT116O cell line.

Figure 2.1 Chemical structures of rhodium metalloinsertors [Rh(HDPA) 2 chrysi] 3+ and [Rh(MeDPA) 2 chrysi] 3+

Experimental Protocols

• Materials
• Synthesis of Metal Complexes
• N-methyl-N-(pyridin-2-yl)pyridin-2-amine (MeDPA)
• Cell Culture
• ICP-MS Assay for Cellular Rhodium Levels
• Cellular Proliferation ELISA
• MTT Cytotoxicity Assay
• Cell Cycle Distribution Flow Cytometry Assay
• Cell Death Mode Flow Cytometry Assay

Cells were grown in tissue culture flasks and dishes (Corning Costar, Acton, MA) at 37°C under 5% CO 2 and humidified atmosphere. Ribonuclease was added to a final concentration of 30 g/ml and the cells were incubated overnight at 4 C. The next day, propidium iodide was added to a final concentration of 20 g/ml and the cells were analyzed by flow cytometry.

Results

• ICP-MS of whole cell lysates
• MTT cytotoxicity assay
• Cell cycle distribution
• Mode of cell death
• Caspase inhibition
• PARP inhibition

While the first generation complex [Rh(bpy)2chrysi]3+ is non-toxic for up to 72 hours, the dipyridylamine derivatives [Rh(HDPA)2chrysi]3+ and [Rh(MeDPA)2chrysi]3+ exhibit toxicity, specifically in the MMR-deficient HCT116O cell line after 48 hours. Furthermore, the HCT116O cell line also shows a significant increase in both the G2/M population and the G1 population. Rhodium treatment causes a sharp decrease in the living population of the HCT116O cell line with a corresponding increase in the necrotic and dead cell populations.

Figure 2.2 ICP-MS assay for rhodium accumulation. Normalized rhodium counts for whole cell lysates treated with 0 or 10 µM [Rh(bpy) 2 chrysi] 3+ , [Rh(HDPA) 2 chrysi] 3+ , or [Rh(NH 3 ) 4 chrysi] 3+ for 48 hours

Discussion

• Uptake
• Cytotoxicity
• Cell Cycle Distribution
• Necrotic Mode of Cell Death
• General Implications

It is important that the concentration ranges and incubation times of the treatments are applied in the MTT tests for [Rh(HDPA)2chrysi]3+. Flow cytometry analysis shows that cell death is preceded by disruption of the cell cycle. In the case of the HCT116O line, the G2/M-phase population also increases in response to rhodium treatment.

Figure 2.7 PARP inhibition assay. HCT116N and HCT116O cells were plated in 96-well format at densities of 5 x 10 4 cells ell an treate ith 0 µM “-” r 0 µM “+”

Conclusions

During the first 12 hours, the rhodium complex accumulates in cells and binds to mitochondrial or genomic DNA mismatches. These biological effects are more pronounced at each response stage in the MMR-deficient HCT116O cell line compared to the MMR-proficient HCT116N cell line, strongly suggesting that DNA mismatches are indeed cellular targets of rhodium metalloinsertors. Finally, PARP activation and necrotic cell death are observed 48 to 72 hours after rhodium treatment (Figure 2.5 and Figure 2.7).

Figure 2.8 Model for the cellular response to rhodium metalloinsertors. Within 12 h of treatment with [Rh(HDPA) 2 chrysi] 3+ , cellular accumulation is observed (Figure 2.2)

Cell-Selective Biological Activity of Rhodium

Introduction

However, this process of adding a fluorescent molecule to a drug has itself been shown to alter the subcellular localization of the compound.7 There have been cases where the addition of an organelle-specific peptide to a therapeutic agent resulted in drastically altered activity of the agent. , which was attributed to altered subcellular localization.8,9 Fluorescent organic therapeutics, as well as metal-based therapeutics, contain spectroscopic or spectrometric handles for detection and can therefore be mapped inside the cell. Specifically, the subcellular distribution of the drug shifted from nuclear to cytosolic as drug resistance increased.10 Moreover, it was observed in a study by Liu and colleagues of a series of Au(I) and Ag(I) bidentate pyridylphosphine anticancer agents. that increased lipophilicity resulted in increased potency of the drug, with preferential accumulation of the drug in the mitochondria.11 In a final study, ICP-MS was used to track the uptake and subcellular localization of cisplatin as well as two ruthenium-based chemotherapeutics pt. in clinical trials, NAMI-A and KP1019. Here, we correlate the selectivity of a variety of rhodium metalloinsertors targeting MMR-deficient cells with the subcellular localization of the complexes.

Experimental Protocols

• Materials
• Oligonucleotide Synthesis
• Synthesis and Characterization of Metal Complexes
• N-alkyl-N-(pyridin-2-yl)pyridin-2-amine
• Photocleavage Competition Titrations
• Binding Constant Determination
• Cell Culture
• Cellular Proliferation ELISA
• MTT Cytotoxicity Assay
• Nuclear Isolation Protocol
• Mitochondrial Isolation Protocol
• ICP-MS Assay for Whole-Cell Rhodium Levels
• ICP-MS Assay for Nuclear Rhodium Levels
• ICP-MS Assay for Mitochondrial Rhodium Levels

The mean value obtained (ie, the logarithm of the [rhodium complex] at the inflection point of the curve) was converted to concentration units ([Rh50%]). 50 μL of the saturated drink was then combined with 50 μL of a 2% HNO3 solution (v/v), while proteins in the remaining lysate were quantified by the bicinchoninic acid assay (BCA).21 The resulting. 750 μL of the saturated liquor was then combined with 50 μL of a 2% HNO3 solution (v/v), while proteins in the remaining lysate were quantified by the bicinchoninic acid assay (BCA).21 The resulting 1% HNO3 solution was analyzed for rhodium content on an HP unit -4500 ICP-MS.

Figure 3.1 Binding affinities determined through DNA photocleavage. The DNA hairpin sequence is 5*’-GGCAGGCATGGCTTTTTGCCATCCCTGCC-3’ (un e line enotes the mismatch, asterisk denotes the radiolabel)

Results

• Binding Affinities for Metal Complexes at Single Base Mismatches
• Quantitation of Inhibition of Cellular Proliferation using an Enzyme-
• MTT Cytotoxicity Assay
• ICP-MS Assay for Whole-Cell Rhodium Levels
• ICP-MS Assay for Nuclear Rhodium Levels
• ICP-MS Assay for Mitochondrial Rhodium Levels

HCT116N and HCT116O cells were plated and treated with the various rhodium complexes at the concentrations indicated in Figure 3.7 for 24, 48 or 72 hours. The compounds showing delayed activity in the ELISA assay ([Rh(chrysi)(phen)(MeDPA)]3+ and [Rh(bpy)2(chrysi)]3+) do not show significant cytotoxicity against either the cell lines in the MTT assay, consistent with previous observations.1 The remaining compounds ([Rh(chrysi)(phen)(HDPA)]3+, [Rh(chrysi)(phen)(PrDPA)]3+, [Rh( PrDPA)2( chrysi)]3+ and [Rh(DIP)2(chrysi)]3+ exhibit either little or no selective cytotoxicity against the HCT116O cell line, consistent with their activities in the ELISA assay. Briefly, HCT116O cells were treated with the different rhodium complexes for 24 h, the nuclei were isolated, and rhodium concentrations of the different samples were determined by ICP-MS and normalized to the number of nuclei.

Figure 3.3 Chemical structures, binding affinities for CC mismatches, and approximated nuclear concentration of all compounds studied

Discussion

• Variations in Complexes Synthesized
• Metalloinsertor Uptake and Nuclear Accumulation
• Mitochondrial Accumulation of Rhodium Metalloinsertors
• General Implications for Design

This altered subcellular localization may be the reason for the lack of selectivity of the compound for one cell line over the other. The only difference between the two cell lines is the presence of a functional copy of the MLH1 gene in the HCT116N. Since the only difference between the two cell lines is a functional copy of the MLH1 gene, a gene encoding a nuclear MMR protein, the cell-selective behavior of our metalloinsertors must be related to this MMR deficiency.

Table 3.1 Qualitative nuclear a and mitochondrial b uptake properties, as well as the presence or absence of cell-selective biological activity c for all ten metalloinsertors

Conclusion

This work supports the hypothesis that nuclear DNA mismatch binding is responsible for the unique cell-selective biological activity of our rhodium metalloinsertors. Furthermore, the fact that the three compounds that are not selective for the MMR-deficient cell line do not have mitochondrial accumulation suggests that mitochondrial mismatch DNA targeting is not responsible for cell-selective behavior (Figure 3.10). This genomic mismatch binding is thought to be responsible for the unique preferential targeting of MMR-deficient cells by rhodium metalloinsertors.

Figure 3.10 Model for the requirements for MMR-deficient cell-selectivity by rhodium metalloinsertors

However, the ELISA data reported here were obtained in parallel under the same conditions for all ten metal inserters to verify trends.

An Inducible, Isogenic Cancer Cell Further Validates

Introduction

A completely isogenic cell line system is described in which expression of the MMR gene MLH1 can be switched on or off using shRNA. In this study, MMR deficiency is induced in the NCI-H23 cell line system, resulting in MSI and increased resistance to DNA damaging agents. As a potential step toward developing a drug that targets the end state of MMR deficiency, this cell line system is being used to further validate rhodium metalloinserts as chemotherapeutic agents.

Experimental Protocols

• Antibodies and Western Blots
• Cell Viability Assays
• Statistical Analysis
• Synthesis of Chemical Compounds

Cells cultured in the absence or presence of doxycycline were plated with cells per well in 96-well plates and allowed to incubate overnight. Cells cultured in the absence or presence of doxycycline were plated at 500-2,000 cells per well. well in a 6-well plate and allowed to incubate overnight. Cells were treated with compounds in a dose-response fashion for 24 hours, and then media were aspirated and replaced with fresh media containing no compound.

Results

• Inhibition of MLH1 by shRNA Reduces MLH1 Protein Levels
• MLH1-Deficient NCI-H23 Subclones Exhibit Increased Resistance to
• MLH1-deficient NCI-H23 Subclones Display Increased Sensitivity to

Cells were treated with etoposide and cell viability was assessed after 4 days using a Cell Titer Glo assay. Top) Cells were treated with [Rh(DPE)(phen)chrysi]2+ and cell viability was assessed after 4 days using a Cell Titer Glo assay. Bottom) Cells were treated with [Rh(HDPA) 2chrysi]3+ and cell viability was assessed after 4 days using a Cell Titer Glo assay.

Figure 4.1 MLH1 shRNA expression is inducible. NCI-H23 subclones 4-10 and 4-13 were induced for MLH1 shRNA by splitting into two cultures, one maintained in inducing conditions (+ Dox) and the other grown in the absence of doxycycl

Discussion

• Generation of Isogenic Cell Lines
• Implications for Chemotherapy

The differential sensitivity of MLH1-deficient subclones to rhodium metalloinserter compounds is consistent with the hypothesis that these compounds exert their effects via binding to DNA mismatches present in MMR-deficient cells. It provides a model for studying questions about induced MMR deficiency and enables the identification of molecules that may offer therapeutic benefits in MMR-deficient cancers. MEFs may be less sensitive to defects in DNA repair than cancer cells and are reported to exhibit a much lower mutation rate than MMR-deficient cancer cells.25,29.

Figure 4.9 Treatment of NCI-H23 subclones with rhodium metalloinsertor compounds does not alter MSI status

Conclusion

Introduction

Experimental Protocols

• Materials
• Oligonucleotide Synthesis
• Synthesis and Characterization of Ligands and Metal Complexes
• X-Ray Structure Determination
• Metalloinsertor pH Titrations
• Photocleavage Competition Titrations
• Binding Constant Determination
• Covalent DNA binding assay
• Circular Dichroism Study of -[Rh(chrysi)(phen)(DPE)] 2+ Bound to
• Cell Culture
• Cellular Proliferation ELISA
• MTT Cytotoxicity Assay
• Cell Death Mode Flow Cytometry Assay
• ICP-MS Assay for Whole-Cell Rhodium Levels

The coordinates of the hydrogen atoms bonded to N1, N2, O1W and O2W were located in the differential Fourier synthesis and refined semi-freely using a distance constraint. The coordinates of the hydrogen atoms bound to N1, N2 and O1M were located in the differential Fourier synthesis and refined semi-freely using a distance constraint. The coordinates of the hydrogen atoms bonded to N1, N2 and O1T were located in the differential Fourier synthesis and refined semi-freely using a distance constraint.

Results

• Synthesis and Characterization of Compounds
• Binding Affinities for Metal Complexes at Single Base Mismatches
• Covalent DNA Binding
• Quantitation of Inhibition of Cellular Proliferation using an Enzyme-
• MTT Cytotoxicity Assay
• ICP-MS Assay for Whole-Cell Rhodium Levels
• Mode of cell death

Discussion

• In Vitro Characterization
• In Cellulo Characterization

Conclusion

Conclusion