Minor Groove Binding DNA Ligands with Expanded A/T Sequence Length Recognition, Selective Binding to Bent DNA Regions and Enhanced Fluorescent Properties

Abstract

DNA minor groove ligands provide a paradigm for double-stranded DNA recognition, where common structural motifs provide a crescent shape that matches the helix turn. Since minor groove ligands are useful in medicine, new ligands with improved binding properties based on the structural information about DNA-ligand complexes could be useful in developing new drugs. Here, two new synthetic analogues of AT specific Hoechst 33258 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole (DMA) and 5-(4-methylpiperazin-1-yl)-2-[2'[2''-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl] benzimidazole (TBZ) were evaluated for their DNA binding properties. Both analogues are bisubstituted on the phenyl ring. DMA contains two ortho positioned methoxy groups, and TBZ contains a phenolic group at C-4 and a methoxy group at C-3. Fluorescence yield upon DNA binding increased 100-fold for TBZ and 16-fold for DMA. Like the parent compound, the new ligands showed low affinity to GC-rich (K approximately 4 x 10(7) M(-1)) relative to AT-rich sequences (K approximately 5 x 10(8) M(-1)), and fluorescence lifetime and anisotropy studies suggest two distinct DNA-ligand complexes. Binding studies indicate expanded sequence recognition for TBZ (8-10 AT base pairs) and tighter binding (DeltaT(m) of 23 degrees C for d (GA(5)T(5)C). Finally, EMSA and equilibrium binding titration studies indicate that TBZ preferentially binds highly hydrated duplex domains with altered A-tract conformations d (GA(4)T(4)C)(2) (K= 3.55 x 10(9) M(-1)) and alters its structure over d (GT(4)A(4)C)(2) (K = 3.3 x 10(8) M(-1)) sequences. Altered DNA structure and higher fluorescence output for the bound fluorophore are consistent with adaptive binding and a constrained final complex. Therefore, the new ligands provide increased sequence and structure selective recognition and enhanced fluorescence upon minor groove binding, features that can be useful for further development as probes for chromatin structure stability.

Old class but new dimethoxy analogue of benzimidazole: a bacterial topoisomerase I inhibitor

“…Amongst them, ligand 5-(4-methylpiperazin-1-yl)-2-[2 -(3,4-dimethoxyphenyl)-5 -benzimidazolyl]benzimidazole (DMA) displayed higher binding affinity to DNA and stabilised the altered DNA structure [9,10]. The cytotoxicity of the ligand was investigated on a glioma cell line (U-87) by studying its effects on cell survival and cell proliferation [6,11].…”

Osmolyte Changes the Binding Affinity and Mode of Interaction of Minor Groove Binder Hoechst 33258 with Calf Thymus DNA

“…[14][15][16] New applications are emerging from studies related to radiosensitization and radioprotection in relation to modification of radiation response by Hoechst derivatives. [17][18][19][20][21] Attempts have also been made to correlate these observations with different Hoechst analogues.…”

“…55) This aspect is also demonstrated in femtosecond resolved fluorescence studies indicate the presence of weakly bound waters in the Hoechst 33258-DNA complex. 56,57) All these studies have indicated association of water in the Hoechst 33258-DNA complex.…”

“…[17][18][19][20][21] Attempts have also been made to correlate these observations with different Hoechst analogues.…”

Interaction of DNA Minor Groove Binder Hoechst 33258 with Bovine Serum Albumin

“…8,9) Our group has also studied cellular responses of a few synthesized bisbenzimidazole derivatives in in-vitro systems. 10,11) Hoechst 33258 is a bisbenzimidazole molecule that binds strongly in the minor groove region of duplex DNA with predominant specificity for AT rich sequences. [12][13][14] Bisbenzimidazole compounds have also been reported to increase radiation induced cytotoxicity in several cancerous cell lines 15) and thus have an ability to improve radiotherapy.…”