Pharmacologic efficacy of PU.1 inhibition by heterocyclic dications: a mechanistic analysis

Abstract

Heterocyclic dications are receiving increasing attention as targeted inhibitors of transcription factors. While many dications act as purely competitive inhibitors, some fail to displace protein efficiently at drug concentrations expected to saturate their DNA target. To achieve a mechanistic understanding of these non-competitive effects, we used a combination of dications, which are intrinsically fluorescent and spectrally-separated fluorescently labeled DNA to dissect complex interactions in multi-component drug/DNA/protein systems. Specifically, we interrogated site-specific binding by the transcription factor PU.1 and its perturbation by DB270, a furan-bisbenzimidazole-diamidine that strongly targets PU.1 binding sites yet poorly inhibits PU.1/DNA complexes. By titrating DB270 and/or cyanine-labeled DNA with protein or unlabeled DNA, and following the changes in their fluorescence polarization, we found direct evidence that DB270 bound protein independently of their mutual affinities for sequence-specific DNA. Each of the three species competed for the other two, and this interplay of mutually dependent equilibria abrogated DB270's inhibitory activity, which was substantively restored under conditions that attenuated DB270/PU.1 binding. PU.1 binding was consistent with DB270's poor inhibitory efficacy of PU.1 in vivo, while its isosteric selenophene analog (DB1976), which did not bind PU.1 and strongly inhibited the PU.1/DNA complex in vitro, fully antagonized PU.1-dependent transactivation in vivo.

Figure 1.

DB270 and DB1976: two isosteres with contrasting inhibitory efficacy on the transcription factor PU.1. (A) DB270 and DB1976 are classic heterocyclic dications with strong affinity and selectivity for AT-rich sequences commonly found in cognate DNA binding sites for PU.1. (B) Despite similar DNA-binding properties on their own, the two isosteres differ markedly in their ability to inhibit PU.1. When inhibition of PU.1 binding to the λB motif (5′-AATAAAAGGAAGTG-3′), a natural high-affinity DNA binding site, was measured by biosensor-SPR, only DB1976 effectively displaced with DNA-bound PU.1 (5). The poor inhibitory efficacy seen with DB270 indicated additional non-competitive interactions at work.

Figure 2.

Sequence-dependent binding of DB270 with duplex DNA sites as measured by fluorescence polarization (FP). (A) Emission spectra of DB270 at 10 nM in the absence (dashed) or 1 μM of duplex DNA harboring the [5′]AGC sequence (solid). (B) Representative titrations of DB270 (10 nM) with unlabeled duplex DNA harboring the λB (circles), [5′]AGC (squares) or SC1 site (diamonds) in the presence of 300 mM Na+. DNA sequences of the three sites are given in Table 1. Curves represent a global fit of the three datasets with a 1:1 binding model in which the anisotropies of the free and bound states are shared parameters. (C) Log–log plot of the dissociation constants for DB270/DNA binding at various NaCl concentrations. Lines represent a global linear fit of all the data points with a shared, fitted slope of 2.1 ± 0.2. The extrapolated affinities of DB270 at 150 mM Na+ for the λB and [5′]AGC sequences are 0.19 ± 0.04 and 1.3 ± 0.3 nM, respectively (Table 2).

Figure 3.

Characterization of PU.1/DNA interactions using internally cyanine-labeled DNA probes. (A) Structure of the Cy-labeled DNA probes harboring the [5′]AGC sequence. The labels (in highlight) were incorporated on the strand harboring the 5′-GGAA-3′ consensus. For Cy3, n = 1; for Cy5, n = 2. An unpaired adenine in the complementary strand provides spacing for the dye. (B) Representative direct titrations of Cy3- (1 nM) and Cy5-labeled [5′]AGC probes (3 nM) by PU.1 ETS domain. Curves represent a global fit of the data to a sequential 2:1 binding model as represented by Scheme 1. For the Cy5-[5′]AGC, the dissociation constant for the second equivalent of PU.1 was fixed at log K₀₁₂ = −3. (C) Representative competitive titrations of 1 nM of Cy3-[5′]AGC by unlabeled [5′]AGC (solid symbols) and a non-specific site (NS, open symbols) in the presence of 2 nM of PU.1. Curves represent a global fit of the data to a one-site competitive model with the dissociation constant of the PU.1/Cy3-[5′]AGC complex fixed at the value determined by direct titration of the probe in Panel B. The dissociation constant for the NS site was set at log K₀₁₁ = −3. Parametric values from replicate experiments are given in Table 2.

Scheme 1.

Figure 4.

DB270/DNA titrations in the presence of PU.1 reveal multiple interactions. (A) Representative direct titrations of DB270 with (unlabeled) duplex DNA harboring the [5′]AGC site at fixed concentrations of PU.1 ETS domain as indicated along the diagonal abscissa. Curves represent empirical fits of the data to the Hill equation. (B) The fitted capacities of the binding curves in Panel A as a function of the PU.1 concentration present.

Figure 5.

DB270 binds PU.1 in the absence of DNA. Representative direct titrations of 10 nM of DB270 with PU.1 ETS domain at 150 mM (A), 300 mM (B) and 600 mM NaCl (C). Curves represent an empirical fit of the data to the Hill equation (dashed) or a mechanistic fit (solid) to a model in which DB270 binds n independent sites on PU.1 with a microscopic dissociation constant k₁₀₁, as defined by Scheme 2. Parametric values are given in Table 2. (D) Emission spectra of 10 nM of DB270 in the free state (dashed) and when saturated with 1 μM of PU.1Δ167 (solid). The sample was excited at 358 nm. (E) Electrophoretic resoloution of the PU.1/DB270 complex. Mixtures of 1 μg of PU.1 (∼70 pmol) were titrated with graded amounts of DB270 before separation on a 12% non-denaturing polyacrylamide gel. Note the anode at the bottom of the gel.

Scheme 2.

Figure 6.

Two-dimensional FP analysis of DB270/DNA/PU.1 interactions. DB270 at 1 or 10 nM, plus 1 nM of Cy3-labeled DNA probe harboring the [5′]AGC sequence, were co-titrated with PU.1 ETS domain. At each step the anisotropies of Cy3-[5′]AGC (A and B) and DB270 (C and D) were measured. The anisotropy of the Cy3-[5′]AGC probe alone is shown as open symbols. Curves represent a direct computation of the mutually competitive model as represented by Scheme 3 using only relevant parametric values from Table 2 (solid), or the same set of parameters with exception of the DB270/DNA dissociation constant set at log K₁₁₀ = −10. N.B.: The curves are not optimized fits to the data; rather they are predicted anisotropies computed from Table 2, without adjustment or any additional floating parameters.

Scheme 3.

Figure 7.

DNA-independent binding to PU.1 is the mechanism of impaired PU.1 inhibition. (A) Abundance of the transcriptionally active 1:1 PU.1/DNA complex and unbound DB270 as computed from a mutually competitive model (Scheme 3) for 10 nM of DB270 and 1 nM of [5′]AGC site using the parametric values in Table 2 (solid curve). The case of no DB270 (dashed curve) and if DB270/PU.1 interactions were absent (log k₁₀₁ = −3; dotted curve) are also shown for comparison. Note that excess PU.1 drives the formation of the 2:1 protein/DNA complex, and dissociation of free DNA-bound DB270 in the absence of other interactions. (B) Inhibition of the PU.1/DNA complex by DB270 at 600 mM Na⁺, an ionic condition that abolished DB270/PU.1 binding (c.f. Figure 5C) and restored inhibition of the 1:1 PU.1/DNA complex (bottom Panel, square symbols). PU.1/DNA binding in the absence of DB270 is represented by squares. Curves represent fits of the data to the sequential 2:1 model as represented by Scheme 1.

Figure 8.

The lack of PU.1 binding by DB1976 under physiological conditions is correlated with PU.1 inhibitory efficacy in vivo. (A) Representative direct titration of 10 nM of DB1976 with PU.1 ETS domain at 150 mM Na⁺, an ionic condition under which DB270 avidly binds protein (c.f. Figure 5A). (B) Dose-dependent inhibition of PU.1 by DB1976 (triangles) and DB1076 (squares) in cultured HEK293 cells. PU.1-dependent transactivation at an engineered λB-based promoter was reported by the expression of enhanced GFP, and quantitatively measured by flow cytometry. No detectable EGFP fluorescence was observed in the absence of induced ectopic expression of PU.1 (×).