Structure-dependent inhibition of the ETS-family transcription factor PU.1 by novel heterocyclic diamidines

Abstract

ETS transcription factors mediate a wide array of cellular functions and are attractive targets for pharmacological control of gene regulation. We report the inhibition of the ETS-family member PU.1 with a panel of novel heterocyclic diamidines. These diamidines are derivatives of furamidine (DB75) in which the central furan has been replaced with selenophene and/or one or both of the bridging phenyl has been replaced with benzimidazole. Like all ETS proteins, PU.1 binds sequence specifically to 10-bp sites by inserting a recognition helix into the major groove of a 5'-GGAA-3' consensus, accompanied by contacts with the flanking minor groove. We showed that diamidines target the minor groove of AT-rich sequences on one or both sides of the consensus and disrupt PU.1 binding. Although all of the diamidines bind to one or both of the expected sequences within the binding site, considerable heterogeneity exists in terms of stoichiometry, site-site interactions and induced DNA conformation. We also showed that these compounds accumulate in live cell nuclei and inhibit PU.1-dependent gene transactivation. This study demonstrates that heterocyclic diamidines are capable of inhibiting PU.1 by targeting the flanking sequences and supports future efforts to develop agents for inhibiting specific members of the ETS family.

Figure 1.

Heterocyclic diamidines and DNA target site used in this study. (A) Compounds are colored to aid visualization of the succeeding figures. (B) The high-affinity λB site hairpin used in SPR experiments. The ETS consensus sequence (5′-GGAA-3′) is in bold.

Figure 2.

SPR analysis of the dications binding to the λB site and inhibition of PU.1. (A) The binding of compounds to an immobilized DNA hairpin duplex harboring the λB site was determined by SPR. The r values (as explained in the Supplementary Methods) are plotted against compound concentration. An appropriate one or two site (DB293) model was used to fit the data. Additional points at higher concentration were used to obtain the K_D values for DB75 and DB1213. DB1977 is similar to DB1976 and is not shown. Numerical estimates of K_D are given in Table 1. (B) The protein inhibition signal in percentage is plotted against compound concentration. Numerical estimates of IC₅₀ are given in Table 1. (C) Comparison of DNA-binding affinity and PU.1-inhibitory potency of DB compounds. For DB293, the K_D value for binding the first equivalent of drug is used.

Figure 3.

DB293 induces distinct DNA conformations at the λB motif that are not shared by its homolog DB1281. A DNA fragment harboring the λB site was saturated with DB293 (0.1 mM) or DB1281 (1 µM) and probed by hydroxyl radicals (•OH). Shown here are the lane traces for the 5′-GGAA-3′ and 5′-TTCC-3′ strands; the experimental gel images are found in Supplementary Figure SD5. The pixel count is marked in the abscissa. Note the 3′→5′ direction from left to right. Although the two •OH footprints within the λB motif for DB1281 are apparent, neither strand exhibits protection by DB293 at S2. Instead, DB293 induces local hypersensitivity to •OH at both strands just 3′ to the CC. DNase I footprinting confirms the weak footprint at S1 and negligible occupancy at S2 for DB293 (Supplementary Figure SD1).

Figure 4.

The homologs DB1976 and DB1977 differentially recognize AT-rich sequences within the λB motif, a PU.1-specific binding site. A DNA fragment harboring the λB site was saturated with DB1976 (1 µM) or DB1977 (0.1 µM) and probed by DNase I (A and B) or hydroxyl radicals (•OH; C and D). Shown here are the lane traces for the 5′-GGAA-3′ and 5′-TTCC-3′ strands; the experimental gel images are found in Supplementary Figure SD6. The pixel count is marked in the abscissa. Note the 3′ → 5′ direction from left to right. Bases marked by hollow symbols (square, circle) are significantly more protected against DNase I by DB1976 than DB1977. DNase I protection by DB1976 may, therefore, be considered as a single, extended footprint. Whereas the two •OH footprints for DB1976 are apparent (S1 ad S2), neither strand exhibits protection by DB1977 at S2.

Figure 5.

Inhibition of PU.1-specific gene transactivation in live cells. (A) PU.1 activity was assayed using an EGFP reporter under the control of a minimal TATA-box promoter. An enhancer element consisting of five tandem λB sites, spaced one helical turn apart, confers specificity to PU.1. Reporter expression was measured by flow cytometry. The HEK293 cells, which do not express PU.1, do not activate the reporter except in the presence of exogenous PU.1 (as shown in fluorescence micrograph). (B) PU.1-expressing HEK293 cells were transfected with reporter plasmid with or without DB1976, DB1977 or DB1281 in the culture medium at the indicated concentrations. EGFP fluorescence was measured by flow cytometry after 24 h. The effect of the compounds on cell viability was separately determined by resazurin reduction.

Figure 6.

Localization of DB compounds in live unfixed HEK293 cells. (A) The uptake of DB1281, DB1976, and DB1977 by HEK293 cells and colocalization with DOX were monitored by fluorescence microscopy as described in ‘Materials and Methods’. Arrows indicate cells that show particularly strong DOX staining in DB1281. (B) Quantitative colocalization analysis of the dications and DOX fluorescence in terms of spatial co-distribution and intensity correlation. DB1281 correlates comparably as DB1976 and DB1977 in terms of spatial codistribution with DOX but not DOX intensity.