(A) Chemical structures of the heterocyclic diamidines. (B) Model of DB2313 docked to the track (5′-AAATAAAA-3′) upstream of the 5′-GGAA-3′ ETS core consensus in the λB motif. (C) Representative SPR sensorgrams for the interaction of DB2313 with the 5′ AT-rich binding site of the λB promoter DNA sequence. Note the lack of binding by DB2313 to an alternative site specific to the ETS homolog ETS1 (5′-GCCGGAAGTG-3′), even at high concentrations (100 nM, asterisk). (D) Comparison of the binding affinities for the λB promoter DNA sequence with different compounds. RU values from the SPR sensorgrams, as in B, were converted to r (r = RU/RUmax, moles of compound bound/mole of promoter DNA) and are plotted against the unbound compound concentration. (E) Specificity of the λB motif for PU.1. Under identical solution conditions, ETS1 bound negligibly at concentrations that saturated the target in the case of PU.1. (F) Normalized PU.1 inhibition resulted from biosensor SPR experiments. The plots represent the amount of PU.1-DNA complex inhibition as a function of the added compound concentration. (G) Perturbations of DNA minor groove width and depth by bound DB2313 or PU.1. The base steps marked “X_i” denote the bases 5′ to the ETS consensus (G₀G₁AA). Dashed lines indicate the expected values of B-form DNA. Aligned structures of the DB2313-bound (gray) and PU.1-bound (orange) DNA, rendered as van der Waals surfaces, show the mutually incompatible minor groove conformations induced by the diamidine and protein. (H) DNase I footprints of compound binding to the λB motif. The subsite at which the compounds bind is marked by a bracket. Arrows indicate distinct perturbations to the drug-induced DNA structure among the compounds as detected by DNase I. As a reference, the PU.1-bound footprint is also shown (red); note the DNase I–hypersensitive band (asterisk) in the reverse strand that is diagnostic of site-specific ETS-DNA complexes.