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

New antimicrobials are needed to combat drug resistance and have often been equated with the identification and exploitation of novel targets. This study focused on the synthesis of new benzimidazole analogues with improved DNA minor groove-binding affinity and having lower cytotoxicity to mammalian cells as well as selective targeting of bacterial DNA over host DNA. 5-(4-Methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl]benzimidazole (DMA) cleared bacterial infections from mammalian cell culture without apparent cytotoxicity to mammalian cells. Moreover, DMA inhibited microbial topoisomerase over mammalian topoisomerase, with a 50% inhibitory concentration (IC50) value for human topoisomerase I of >54microM compared with an IC50 of <10microM for Escherichia coli topoisomerase I in vitro.

Benzimidazoles: a minor groove-binding ligand-induced stabilization of triple helix.

Nonintercalating minor groove-binding ligands netropsin, Hoechst 33258, and DAPI are reported to destabilize the triplex. Ligands with different substitutions on the phenyl ring of bis- and terbenzimidazoles were evaluated for their effect on the stability of C+.GC triplex and Hoogsteen duplex. We found that newly synthesized benzimidazoles stabilize the triplex as shown by fluorescence and melting studies. Modeling studies showed that these molecules bind in the Watson-Crick minor groove of triplex, which can exert a profound impact on the properties of the host triplex. Circular dichroism-binding studies indicate 5.77 base triplets/ligand as an apparent binding site for bis- and 8.66 for terbenzimidazoles. The stabilization of triplex can be attributed to the protonation of nitrogens and amines of benzimidazoles at pH 5.2 that compensate the negative charge of phosphate backbone to reduce the repulsion between the strands resulting in the stabilization.

Renal phosphodiesterase 4B is activated in the Dahl salt-sensitive rat.

Reduced beta-adrenoreceptor signaling is associated with increased sympathoadrenal activity in hypertensive patients and animal models of hypertension. However, the mechanism that accounts for this characteristic decline in beta-adrenergic signaling is unclear. In the present study, we investigated renal phosphodiesterase 4B, which metabolizes cAMP. Immunoblot analysis detected only the phosphodiesterase 4B4 isoform present in kidney tissue from spontaneously hypertensive rats, hypertensive Dahl salt-sensitive (SS) rats, and Dahl salt-resistant rats. The phosphorylated (activated) form of the protein was present at 2-fold greater levels in Dahl SS rats than in spontaneously hypertensive rats and Dahl salt-resistant rats, whereas the unphosphorylated form of the protein was reduced by approximately one half in SS animals. In accord with immunoblot data, rolipram-inhibitable cAMP hydrolyzing activity, a measure of PDE4 activity, was approximately 3-fold greater in kidney cytosolic extracts from SS rats than in extracts from spontaneously hypertensive rats and salt-resistant rats. Phosphodiesterase 4B expression was detected by immunohistochemistry in the renal vasculature, proximal tubules, and distal tubules. These results raise the possibility that increased PDE4 activity, specifically phosphodiesterase 4B4 activity, reduces beta-adrenergic signaling in the kidney and contributes to salt-sensitive hypertension in the Dahl SS rat.

New method for chloroamidation of olefins. Application in the synthesis of N-glycopeptides and anticancer agents.

Chloroamidation of olefins using a new reagent system (COCl)2-AgNO3-CH3CN was observed. Various glycals with this reagent system produce 2-chloro-1-acetamido sugars in good yields which, in turn, were converted to free amino derivatives and various glycopeptides. The acetamido sugar derivatives and free amines were found to be promising anticancer agents against the U-87 malignant glyoma (a brain tumor) cell line with IC-50=1 nm-22 microM, and they were found to be far less cytotoxic against a normal human embryonic kidney cell line.

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA.

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 microM concentration of DMA and TBZ and irradiated at 5 Gy; both the ligands showed nuclei condensation suggesting a probable mechanism to protect DNA from radiation damage. The bisubstituted analogs of Hoechst 33342 are found to be better free radical scavengers and protect DNA against radiation-induced damage at a lower concentration than the parent molecule. Both the ligands also quenched free radicals in isolated free radical system suggesting their dual mode of action against radiation-induced damage to DNA. Molecules binding to the chromatin alter gene expression, whereas in this study both the ligands have not shown any profound effect on the nucleosome assembly and gene expression in vitro and in vivo. Both ligands afford a 2-fold protection by altering DNA structure as well as through direct free radical quenching in bulk solution in comparison to the parent ligand, which acts only through quenching of free radicals.

Aminopeptidase N reduces basolateral Na+ -K+ -ATPase in proximal tubule cells.

Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na(+)-K(+)-ATPase, a preeminent basolateral Na(+) transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na(+)-K(+)-ATPase activity and basolateral Na(+)-K(+)-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na(+)-K(+)-ATPase activity and basolateral abundance by approximately 50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na(+)-K(+)-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na(+)-K(+)-ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.

Minor groove binding DNA ligands with expanded A/T sequence length recognition, selective binding to bent DNA regions and enhanced fluorescent properties.

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.

Influence of phenyl ring disubstitution on bisbenzimidazole and terbenzimidazole cytotoxicity: synthesis and biological evaluation as radioprotectors.

DNA minor groove binders, Hoechst 33258 and Hoechst 33342, have been reported to protect against radiation-induced DNA-strand breakage, but their mutagenicity and cytotoxicity limit their use as protectors of normal tissue during radiotherapy and as biological radioprotectors during accidental radiation exposure. On the basis of these observations, two new nontoxic disubstituted benzimidazoles were synthesized, one having two methoxy groups (5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl]benzimidazole, 5) and another having a methoxy and a hydroxyl group (5-(4-methylpiperazin-1-yl)-2-[2'[2''-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl]benzimidazole, 6) ortho to each other on the phenyl ring. The radiomodifying effects of these nontoxic ligands were investigated with a human glioma cell line exposed to low linear energy transfer radiation by determining cell survival and cell proliferation compared with effects of the parent compound, Hoechst 33342. Cytotoxicity assayed by analyzing clonogenicity, cell growth, and metabolic viability showed that both 5 and 6 were nontoxic at 100 microM after 72 h of exposure, whereas Hoechst 33342 resulted in lysis of 77% of these cells in 24 h. Macrocolony assay (clonogenicity) showed that 73%, 92%, and 10% of the cells survived when treated with 100 microM 5, 6, and Hoechst 33342, respectively. Both 5 and 6 did not affect the growth of BMG-1 cells. At 10 microM, 5 and 6 showed 82% and 37% protection against radiation-induced cell death (macrocolony assay) while 100% protection was observed against growth inhibition. Disubstitution of the phenyl ring has not only reduced cytotoxicity but also enhanced DNA-ligand stability, conferring high degree of radioprotection.