Thermodynamics of d(GGGGCCCC) Binding to Neomycin-Class Aminoglycosides.

DNA adopts a number of conformations that can affect its binding to other macromolecules. The conformations (A, B, Z) can be sequence- and/or solution-dependent. While AT-rich DNA sequences generally adopt a Canonical B-form structure, GC-rich sequences are more promiscuous. Recognition of GC-rich nucleic acids by small molecules has been much more challenging than the recognition of AT-rich duplexes. Spectrophotometric and calorimetric techniques were used to characterize the binding of neomycin-class aminoglycosides to a GC-rich DNA duplex, G4C4, in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of G4C4, with thermal enhancement decreasing with increasing pH and/or Na+ concentration. Although G4C4 bound to aminoglycosides demonstrated a mixed A- and B-form conformation, circular dichroism studies indicate that binding induces a conformational shift toward A-form DNA. Isothermal titration calorimetry studies reveal that aminoglycoside binding to G4C4 is linked to the uptake of protons at pH = 7.0 and that this uptake is pH-dependent. Increased pH and/or Na+ concentration results in a decrease in G4C4 affinity for the aminoglycosides. The binding affinities of the aminoglycosides follow the expected hierarchy: neomycin > paromomycin > ribostamycin. The salt dependence of DNA binding affinities of aminoglycosides is consistent with at least two drug NH3+ groups participating in electrostatic interactions with G4C4. These studies further embellish our understanding of the many factors facilitating recognition of GC-rich DNA structures as guided by their optimum charge and shape complementarity for small-molecule amino sugars.

An overview of recent advances in duplex DNA recognition by small molecules.

As the carrier of genetic information, the DNA double helix interacts with many natural ligands during the cell cycle, and is amenable to such intervention in diseases such as cancer biogenesis. Proteins bind DNA in a site-specific manner, not only distinguishing between the geometry of the major and minor grooves, but also by making close contacts with individual bases within the local helix architecture. Over the last four decades, much research has been reported on the development of small non-natural ligands as therapeutics to either block, or in some cases, mimic a DNA-protein interaction of interest. This review presents the latest findings in the pursuit of novel synthetic DNA binders. This article provides recent coverage of major strategies (such as groove recognition, intercalation and cross-linking) adopted in the duplex DNA recognition by small molecules, with an emphasis on major works of the past few years.

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.

Eukaryotic Ribosomal Expansion Segments as Antimicrobial Targets.

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10-9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 µM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for ES7CA (NeoRH, NeoFH, and NeoYH), the lowest dose needed to induce mortality in C. albicans (minimum inhibitory concentration, MIC) was determined. PAs with the lowest MIC values were tested for cytotoxicity in HEK293T cells. Molecules with high affinity for ES7CA in vitro induce mortality in C. albicans but not in HEK293T cells. The results are consistent with the hypothesis that ESs represent useful targets for chemotherapeutics directed against eukaryotic pathogens.

Probing A-form DNA: A fluorescent aminosugar probe and dual recognition by anthraquinone-neomycin conjugates.

Nucleic acids adopt a broad array of hydrogen-bonded structures that enable their diverse roles in the cell; even the familiar DNA double helix displays subtle architectural nuances that are sequence dependent. While there have been many approaches for recognition of B-form nucleic acids, A-form DNA recognition has lagged behind. Here, using a tight binding fluorescein-neomycin (F-neo) conjugate that can probe the electrostatic environment of A-form DNA major groove, we developed a fluorescent displacement assay to be used as a screen for DNA duplex-binding compounds. As opposed to intercalating dyes that can significantly perturb DNA structure, the groove binding F-neo allows the probing of native DNA conformation. In combination with the assay development and probing of DNA grooves, we also report the synthesis and binding of a series of neomycin-anthraquinone conjugates, two units with a known preference for binding GC rich DNA. The assay can be used to identify duplex DNA-binding compounds, as well as probe structural features of a target DNA duplex, and can easily be scaled up for high throughput screening of compound libraries.

Linker dependent intercalation of bisbenzimidazole-aminosugars in an RNA duplex; selectivity in RNA vs. DNA binding.

Neomycin and Hoechst 33258 are two well-known nucleic acid binders that interact with RNA and DNA duplexes with high affinities respectively. In this manuscript, we report that covalent attachment of bisbenzimidazole unit derived from Hoechst 33258 to neomycin leads to intercalative binding of the bisbenzimidazole unit (oriented at 64-74° with respected to the RNA helical axis) in a linker length dependent manner. The dual binding and intercalation of conjugates were supported by thermal denaturation, CD, LD and UV-Vis absorption experiments. These studies highlight the importance of linker length in dual recognition by conjugates, for effective RNA recognition, which can lead to novel ways of recognizing RNA structures. Additionally, the ligand library screens also identify DNA and RNA selective compounds, with compound 9, containing a long linker, showing a 20.3°C change in RNA duplex Tm with only a 13.0°C change in Tm for the corresponding DNA duplex. Significantly, the shorter linker in compound 3 shows almost the reverse trend, a 23.8°C change in DNA Tm, with only a 9.1°C change in Tm for the corresponding RNA duplex.

Multivalency in the recognition and antagonism of a HIV TAR RNA-TAT assembly using an aminoglycoside benzimidazole scaffold.

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA with a low nanomolar affinity. 5 displayed enhanced binding compared to its individual building blocks including the neomycin dimer azide and benzimidazole alkyne. In essence, a high affinity multivalent ligand was designed and synthesized to target HIV TAR RNA.

Influence of linker length and composition on enzymatic activity and ribosomal binding of neomycin dimers.

The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.

Influence of linker length in shape recognition of B* DNA by dimeric aminoglycosides.

DNA-protein recognition has shown us the importance of DNA shapes in the recognition process. Specific high-affinity targeting of DNA shapes by small molecules is desirable for many biological applications that involve regulation of DNA based processes. Here, the effect of linker length and rigidity on the affinity of a conjugated neomycin dimer for a specific DNA shape (B* form) AT-rich DNA was explored. Binding constants approximating 10(8)M(-1) for optimal linker lengths of 18-19 atoms are reported herein.

Shape readout of AT-rich DNA by carbohydrates.

Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove-binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA "shape readout" properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT-rich) DNA. Isothermal titration calorimetry (ITC), circular dichroism (CD) titration, ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT-rich DNA duplex d[5'-G2 A6 T6 C2 -3']. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two-binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy-driven with a binding constant of approximately 10(8) M(-1) . ITC-derived binding enthalpies were used to obtain the binding-induced change in heat capacity (ΔCp ) of -225 ± 19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT-tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT-rich DNA (d[5'-G2 A6 T6 C2 -3']) >B form alternate AT-rich DNA (d[5'-G2 (AT)6 C2- 3']) > A form GC-rich DNA (d[5'-A2 G6 C6 T2 -3']), demonstrating the preference of ligand 1 for B* form DNA.

Recognition of RNA duplex by a neomycin-Hoechst 33258 conjugate.

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin-Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.

Analysis of diazofluorene DNA binding and damaging activity: DNA cleavage by a synthetic monomeric diazofluorene.

The lomaiviticins and kinamycins are complex DNA damaging natural products that contain a diazofluorene functional group. Herein, we elucidate the influence of skeleton structure, ring and chain isomerization, D-ring oxidation state, and naphthoquinone substitution on DNA binding and damaging activity. We show that the electrophilicity of the diazofluorene appears to be a significant determinant of DNA damaging activity. These studies identify the monomeric diazofluorene 11 as a potent DNA cleavage agent in tissue culture. The simpler structure of 11 relative to the natural products establishes it as a useful lead for translational studies.

A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient.

With the recent success of lipid nanoparticle (LNP) based SARS-CoV-2 mRNA vaccines, the potential for RNA therapeutics has gained widespread attention. LNPs are promising non-viral delivery vectors to protect and deliver delicate RNA therapeutics, which are ineffective and susceptible to degradation alone. While food and drug administration (FDA) approved formulations have shown significant promise, benchmark lipid formulations still require optimization and improvement. In addition, the translatability of these formulations for several different RNA cargo sizes has not been compared under the same conditions. Herein we analyze "gold standard" lipid formulations for encapsulation efficiency of various non-specific RNA cargo lengths representing antisense oligonucleotides (ASO), small interfering RNA (siRNA), RNA aptamers, and messenger RNA (mRNA), with lengths of 10 bases, 21 base pairs, 96 bases, 996 bases, and 1929 bases, respectively. We evaluate encapsulation efficiency as the percentage of input RNA encapsulated in the final LNP product (EEinput%), which shows discrepancy with the traditional calculation of encapsulation efficiency (EE%). EEinput% is shown to be < 50% for all formulations tested, when EE% is consistently > 85%. We also compared formulations for LNP size (Z-average) and polydispersity index (PDI). LNP size does not appear to be strongly influenced by cargo size, which is a counterintuitive finding. Thoughtful characterization of LNPs, in parallel with consideration of in vitro or in vivo behavior, will guide design and optimization for better understanding and improvement of future RNA therapeutics.

Enhanced Sequence-Specific DNA Recognition Using Oligodeoxynucleotide-Benzimidazole Conjugates.

Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties. In the present study, five Hoechst 33258 derived benzimidazoles were conjugated to T rich ODNs and their hybridization effectiveness was tested. Thermal denaturation studies revealed significant stabilization of complementary duplexes by ODN-benzimidazole conjugates, with the extent of stabilization being highly dependent on the length of the linker between DNA and benzimidazole. The increases in thermal stability were determined to be due to the binding of the benzimidazole moiety to the duplex. Circular dichroism and molecular modeling studies provided insights toward the influence of conjugation on duplex structure and how linker length impacts placement of the benzimidazole moiety in the minor groove. Furthermore, thermal denaturation studies with the complementary strand containing a single base mismatch or being RNA revealed that covalent conjugation of benzimidazoles to an ODN also enhances the sequence specificity. The fundamental studies reported herein provide a strategy to improve the stability and specificity properties of the ODN probes, which can be of use for targeting and diagnostics applications.

Understanding Antisense Oligonucleotide Efficiency in Inhibiting Prokaryotic Gene Expression.

Oligonucleotides offer a unique opportunity for sequence specific regulation of gene expression in bacteria. A fundamental question to address is the choice of oligonucleotide, given the large number of options available. Different modifications varying in RNA binding affinities and cellular uptake are available but no comprehensive comparisons have been performed. Herein, the efficiency of blocking expression of ß-galactosidase (ß-Gal) in E. coli was evaluated utilizing different antisense oligomers (ASOs). Fluorescein (FAM)-labeled oligomers were used to understand their differences in bacterial uptake. Flow cytometry analysis revealed significant differences in uptake, with high fluorescence seen in cells treated with FAM-labeled peptidic nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO) and phosphorothioate (PS) oligomers, and low fluorescence observed in cells treated with phosphodiester (PO) oligomers. Thermal denaturation (Tm) of oligomer:RNA duplexes and isothermal titration calorimetry (ITC) studies reveal that ASO binding to target RNA demonstrates a good correlation between Tm and Kd values. There was no correlation between Kd values and reduction of ß-Gal activity in bacterial cells. However, cell-free translation assays demonstrated a direct relationship between Kd values and inhibition of gene expression by antisense oligomers, with tight binding oligomers such as LNA being the most efficient. Membrane active compounds such as polymyxin B and A22 further improved the cellular uptake of FAM-PNA and FAM-PS oligomers in wild-type E. coli cells. PNA and PMO were most effective in cellular uptake and reducing ß-Gal activity as compared to oligomers with PS or those with PO linkages. Overall, cell uptake of the oligomers is shown as the key determinant in predicting their differences in bacterial antisense inhibition, and the RNA affinity is the key determinant in inhibition of gene expression in cell free systems.

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 fluorescence-based screen for ribosome binding antibiotics.

The development of new antibacterial agents has become necessary to treat the large number of emerging bacterial strains resistant to current antibiotics. Despite the different methods of resistance developed by these new strains, the A-site of the bacterial ribosome remains an attractive target for new antibiotics. To develop new drugs that target the ribosomal A-site, a high-throughput screen is necessary to identify compounds that bind to the target with high affinity. To this end, we present an assay that uses a novel fluorescein-conjugated neomycin (F-neo) molecule as a binding probe to determine the relative binding affinity of a drug library. We show here that the binding of F-neo to a model Escherichia coli ribosomal A-site results in a large decrease in the fluorescence of the molecule. Furthermore, we have determined that the change in fluorescence is due to the relative change in the pK(a) of the probe resulting from the change in the electrostatic environment that occurs when the probe is taken from the solvent and localized into the negative potential of the A-site major groove. Finally, we demonstrate that F-neo can be used in a robust, highly reproducible assay, determined by a Z'-factor greater than 0.80 for 3 consecutive days. The assay is capable of rapidly determining the relative binding affinity of a compound library in a 96-well plate format using a single channel electronic pipette. The current assay format will be easily adaptable to a high-throughput format with the use of a liquid handling robot for large drug libraries currently available and under development.

An assay for human telomeric G-quadruplex DNA binding drugs.

Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K(+) or Na(+).

Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

Targeting C-myc G-quadruplex: dual recognition by aminosugar-bisbenzimidazoles with varying linker lengths.

G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.