Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment.

Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common platform fragment-based drug discovery, two fragments that bind to adjacent sites on a target are identified and are then linked together using different linkers to identify the linkage for optimum activity. What are not known from these studies are the effects these linkers, which typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, and photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258-modeled bisbenzimidazoles (1-9) that contain linkers of varying lengths (3-21 atoms) display length- and composition-dependent variation in B-DNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3 to 9.0 °C as the linker length increased from 3 to 21 atoms, respectively. Compounds with linker lengths of ≤11 atoms (such as compounds 1 and 5) are localized in the nucleus, while compounds with long linkers (such as compounds 8 and 9) are distributed in the extranuclear space, as well, with possible interactions with extranuclear targets. These findings provide insights into future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.

Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase I by Hoechst 33258 Derived Mono- and Bisbenzimidazoles.

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC50 values <5.0 µM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 µg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 µg/mL, Klebsiella pneumoniae > 32 µg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2-23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.

Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase.

The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1.

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.

Rapid synthesis, RNA binding, and antibacterial screening of a peptidic-aminosugar (PA) library.

A 215-member mono- and diamino acid peptidic-aminosugar (PA) library, with neomycin as the model aminosugar, was systematically and rapidly synthesized via solid phase synthesis. Antibacterial activities of the PA library, on 13 bacterial strains (seven Gram-positive and six Gram-negative bacterial strains), and binding affinities of the PA library for a 27-base model of the bacterial 16S ribosomal A-site RNA were evaluated using high-throughput screening. The results of the two assays were correlated using Ribosomal Binding-Bacterial Inhibition Plot (RB-BIP) analysis to provide structure-activity relationship (SAR) information. From this work, we have identified PAs that can discriminate the E. coli A-site from the human A-site by up to a 28-fold difference in binding affinity. Aminoglycoside-modifying enzyme activity studies indicate that APH(2″)-Ia showed nearly complete removal of activity with a number of PAs. The synthesis of the compound library and screening can both be performed rapidly, allowing for an iterative process of aminoglycoside synthesis and screening of PA libraries for optimal binding and antibacterial activity for lead identification.

Selective Inhibition of Bacterial Topoisomerase I by alkynyl-bisbenzimidazoles.

Hoechst dyes are well known DNA binders that non-selectively inhibit the function of mammalian topoisomerase I and II. Herein, we show that Hoechst 33258 based bisbenzimidazoles (DPA 151-154), containing a terminal alkyne, are effective and selective inhibitors of E. coli. topoisomerase I. These bisbenzimidazoles displayed topoisomerase I inhibition much better than Hoechst 33342 or Hoechst 33258 with IC50 values in the range of 2.47-6.63 µM. Bisbenzimidazoles DPA 151-154 also display selective inhibition of E. coli. topoisomerase I over DNA gyrase and Human topoisomerases I and II, and effectively inhibit bacterial growth.

Characterization of ribosomal binding and antibacterial activities using two orthogonal high-throughput screens.

We report here the affinity and antibacterial activity of a structurally similar class of neomycin dimers. The affinity of the dimer library for rRNA was established by using a screen that measures the displacement of fluorescein-neomycin (F-neo) probe from RNA. A rapid growth inhibition assay using a single drug concentration was used to examine the antibacterial activity. The structure-activity relationship data were then rapidly analyzed using a two-dimensional ribosomal binding-bacterial inhibition plot analysis.

A genome-wide over-expression screen identifies genes involved in phagocytosis in the human protozoan parasite, Entamoeba histolytica.

Functional genomics and forward genetics seek to assign function to all known genes in a genome. Entamoeba histolytica is a protozoan parasite for which forward genetics approaches have not been extensively applied. It is the causative agent of amoebic dysentery and liver abscess, and infection is prevalent in developing countries that cannot prevent its fecal-oral spread. It is responsible for considerable global morbidity and mortality. Given that the E. histolytica genome has been sequenced, it should be possible to apply genomic approaches to discover gene function. We used a genome-wide over-expression screen to uncover genes regulating an important virulence function of E. histolytica, namely phagocytosis. We developed an episomal E. histolytica cDNA over-expression library, transfected the collection of plasmids into trophozoites, and applied a high-throughput screen to identify phagocytosis mutants in the population of over-expressing cells. The screen was based on the phagocytic uptake of human red blood cells loaded with the metabolic toxin, tubercidin. Expression plasmids were isolated from trophozoites that survived exposure to tubercidin-charged erythrocytes (phagocytosis mutants), and the cDNAs were sequenced. We isolated the gene encoding profilin, a well-characterized cytoskeleton-regulating protein with a known role in phagocytosis. This supports the validity of our approach. Furthermore, we assigned a phagocytic role to several genes not previously known to function in this manner. To our knowledge, this is the first genome-wide forward genetics screen to be applied to this pathogen. The study demonstrates the power of forward genetics in revealing genes regulating virulence in E. histolytica. In addition, the study validates an E. histolytica cDNA over-expression library as a valuable tool for functional genomics.

Whole tumor cell vaccine with irradiated S180 cells as adjuvant.

Whole tumor cell vaccines have been widely studied and remain promising cancer immunotherapies. In the present study, we discovered that vaccination with irradiated mouse sarcoma S180 tumor cells stimulated robust antitumor immunity to autologous tumor cells in both syngenic and allogenic mice. The antitumor activity requires both T and B cells, but not NK cells. When a mouse lung carcinoma (TC-1) whole tumor cell vaccine was combined with the S180 vaccine, the antitumor immunity against live TC-1 tumor cells is significantly enhanced compared to a TC-1 whole cell vaccine alone. This antitumor immunity not only prevents live tumor challenge but also eradicates existing tumor cells. A similar phenomenon was also observed when S180 vaccine was combined with LL2 Lewis lung carcinoma tumor cells. Therefore, S180 vaccine may serve as an adjuvant for other whole tumor cell vaccines.