DNA mismatch binding and antiproliferative activity of rhodium metalloinsertors

Abstract

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 10(4) to 10(8) M(-1) for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo.

Figure 1

Rh(L)₂chrysi³⁺ family of metalloinsertors.

Figure 2

Binding affinities determined through DNA photocleavage. The DNA hairpin sequence is 5′-GGCAGGXATGGCTTTTTGCCATCCCTGCC-3′ (X = C or A, underline denotes the mismatch). Samples were irradiated and electrophoresed through a 20% denaturing PAGE gel. A light control (LC, without rhodium) and dark control (DC, without irradiation) were included. A representative autoradiogram of a photocleavage titration with rac-Rh(bpy)₂chrysi³⁺ (A, arrows indicate positions of mismatched bases) and a representative sigmoidal curve fit of pooled data from photocleavage titrations for binding constant determination (B) are shown.

Figure 3

Inhibitory effects of Rh(NH₃)₄chrysi³⁺ as a function of incubation time on cellular proliferation. Shown are plots of BrdU incorporation (a measure of DNA synthesis and therefore cellular proliferation) normalized to the BrdU incorporation of untreated cells as a function of rhodium concentration. Standard error bars for five trials are shown. MMR-proficient HCT116N cells (green) and MMR-deficient HCT116O cells (red) were plated and allowed 24 h to adhere before incubation with 0–25 μM Rh(NH₃)₄chrysi³⁺ for 12, 24, 48, or 72 h. At the end of the 12, 24, and 48 h incubations, the medium containing Rh was replaced with fresh medium for the remainder of the 72 h, followed by ELISA analysis. BrdU was added to the medium 24 h prior to analysis.

Figure 4

Inhibitory effects of rhodium metalloinsertors as a function of incubation time. Shown are plots of BrdU incorporation normalized to the BrdU incorporation of untreated cells as a function of rhodium concentration. The inhibition differential is the difference of the normalized percentages of cellular proliferation for each cell line, with standard error bars (S_N–O = √(S_N + S_O2)). ELISA analyses were performed as in Figure 3. Cells were incubated with no rhodium, 2 μM rac-Rh(DIP)2chrysi³⁺, 10 μM Rh(NH₃)₄chrysi³⁺, or 20 μM rac-Rh(L)₂chrysi³⁺ (L = HDPA, bpy, or phen).

Figure 5

Inhibitory effects of rac-Rh(DIP)₂chrysi³⁺. Shown are plots of BrdU incorporation normalized to the BrdU incorporation of untreated cells as a function of rhodium concentration. Standard error bars for five trials are shown. MMR-proficient HCT116N cells (green) and MMR-deficient HCT116O cells (red) were plated and allowed 24 h to adhere before incubation with 0–5 μM rac-Rh(DIP)₂chrysi³⁺ for 12 h. At the end of the incubation, the medium containing Rh was replaced with fresh medium, and cells were grown for an additional 60 h before ELISA analysis. BrdU was added to the medium 24 h prior to analysis.

Figure 6

Inhibitory effects of rhodium metalloinsertors as a function of metal complex identity. Shown are bar graphs of BrdU incorporation normalized to the BrdU incorporation of untreated cells as a function of rhodium concentration. The inhibition differential is the difference of the normalized percentages of cellular proliferation for the two cell lines, HCT116O versus HCT116N. ELISA analyses were performed as in Figure 3. Cells were incubated with no rhodium, 2 μM rac-Rh(DIP)₂chrysi³⁺, 10 μM Rh(NH₃)₄chrysi³⁺, or 20 μM rac-Rh(L)₂chrysi³⁺ (L = HDPA, bpy, or phen). A correlation between mismatch binding affinity and differential inhibition of MMR-deficient cells is evident.

Figure 7

Crystal and model structures of rhodium metalloinsertors bound to the mismatch site. Rhodium insertors (red) are shown bound to the DNA (gray) from the minor groove at the mismatch site with the bases (adenine in blue, cytosine in yellow) ejected and the chrysi ligand stacked fully with the adjacent base pairs. The crystal structure of Δ-Rh(bpy)₂chrysi³⁺ bound to the CA mismatch is shown in panel (A), along with structural models of Δ-Rh(DIP)₂chrysi³⁺ (B) and Δ-Rh(NH₃)₄chrysi³⁺ (C) binding based on the crystal structure. Superposition of the DIP complex upon the rhodium center of the bpy complex leads to steric clashes with the sugar–phosphate backbone (possible atoms involved in green), whereas the tetraammine complex is easily accommodated.