With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.

Sixteen 2-cyclohexenone and 6-(ethoxycarbonyl)-2-cyclohexenone analogs of combretastatin-A4 (CA-4, 1) were synthesized, and their ability to inhibit the growth of two murine cancer cell lines (B16 melanoma and L1210 leukemia) was determined using an MTT assay. One of the cyclohexenone analogs, 8, which contains the same substituents as CA-4 (1) is the most potent (IC 50 = 0.91 µM, L1210). Exposure of A-10 aortic cells to cyclohexenone 8 and its ester congener 7 produced significant reduction in cellular microtubules, with EC 50 values of 27 and 37 µM, respectively. Molecular modeling studies indicate that analog 8 adopts a twisted conformation, similar to CA-4, suggesting that conformation and structure are crucial for activity. These compounds are worthy of further investigation as potential tubulin inhibitors in the quest for novel anti-cancer agents.

Pyrrole (P)-Imidazole (I) containing polyamides are small synthetic molecules which can target predetermined DNA sequences with high affinity and modulate gene expression by interfering with the binding of transcription factors to DNA. As a model system we have previously used the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα promoter and shown that targeted polyamides inhibit the binding of the transcription factor NF-Y and induce expression of topo IIα in confluent cancer cells. Critical to this approach is the need for low molecular weight polyamides that readily enter in the nucleus. In this study, the fluorophore p-anisylbenzimidazolecarboxamido (Hx) moiety was rationally designed to mimic the recognition of A/T base pairs by two consecutive pyrrole units. The hybrid polyamide HxIP is the first example of a small fluorescent molecule that fluoresces significantly more intensely upon binding to its target sequence, 5′-TACGAT-3′ of the 5′-flank of ICB2. Sequence specificity was confirmed by DNAse I footprinting, with HxIP binding with increased affinity compared to the corresponding triamides/tetraamides (f-PIP/PPIP). Thermal denaturation, circular dichroism, and Surface Plasmon Resonance studies confirmed the sequence specificity of HxIP. A dose-dependent inhibition of protein binding to ICB2 by HxIP with complete abolition at concentrations >3 μM was seen in an Electrophoretic Mobility Shift Assay (EMSA). A supershift assay confirmed NF-Y binding to the target sequence. HxIP was also able to displace bound protein factors from DNA at the same concentrations. The incorporation of Hx in the hybrid molecule provides an intrinsic probe to directly monitor cellular uptake and migration into the nucleus. Confocal microscopy in both fixed and live NIH3T3 and A549 cells showed that HxIP is readily taken up by cells and quickly localises in the cell nucleus within 5 minutes. Exposure of confluent cells to 20-40 μM HxIP resulted in time-dependent upregulation of topo IIα expression, reaching levels comparable to those of proliferating cells within 24h as shown by immunoblotting analysis. Confluence-associated repression of topo IIα expression contributes to cellular resistance to agents such as etoposide. De-repression of topo IIα by pre-incubation with HxIP was shown to resensitise NIH3T3 cells to the cytotoxic effect of etoposide (MTT assay) and enhance its DNA damaging effects by increasing levels of etoposide-induced DNA strand breaks as assessed by the single cell gel electrophoresis (comet) assay and the phosphorylation of H2AX. Subcellular localisation of HxIP and potential synergies with etoposide treatment is currently being investigated in vivo against human tumour xenografts. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3521.