Abstract 4549: Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures.

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.

Double-stranded DNA sequences have been prepared in which single atoms (the O2-carbonyls of selected thymines) have been replaced by fluorine or methyl. To maintain normal Watson-Crick hydrogen bonding with the complementary purines, these analogue derivatives have been prepared as C-nucleosides. The O2-carbonyls of interest for this study are those involved in a bifurcated (or three-centered) hydrogen bond with the minor groove binding ligand 4',6-diamidino-2-phenylindole (DAPI). TM studies of the duplexes illustrate that the DNA duplexes are destabilized when fluorine or methyl replaces one or both of the minor groove O2-carbonyls, which can in part be explained by changes in minor groove hydration. In the presence of DAPI, most of the duplexes exhibit an increased TM due to the presence of DAPI bound in the minor groove. The extent of helix overstabilization negatively correlates with the presence of one or both methyl groups in the minor groove, suggesting that ligand binding is weakened in the presence of the non-carbonyl functional groups. The presence of single fluorine appears to promote helix stabilization, and native-like stabilization occurs when both fluorines are present. KD values quantitate binding effects between DAPI and the native and analogue sequences. Sequences with one or both methyl groups exhibit very poor binding with DAPI, while those containing a single fluorine behave essentially like native carbonyl-containing sequences. With both fluorines present, KD values were observed to increase by a moderate 3-fold at 100 mM NaCl and somewhat more at 200 mM NaCl. Binding affinities with both methyl groups present were 500-1000-fold weaker than native. The results suggest that organofluorines can function as hydrogen-bond acceptors, at least in the bifurcated interaction that contributes to minor groove binding by DAPI.

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (NUm), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of NU and NUm substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either NU or NUm and of DNA:RNA hybrid duplexes containing NUm. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to NU, NUm decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the NUm containing DNA:DNA duplex structure. The conformation of NUm, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of NUm to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been standard antimalarial drugs since the early 1950s, and very recently, the possibility of their use for the treatment of COVID-19 patients has been considered. To understand the drug mode of action at the submicroscopic level (atoms and molecules), molecular modeling studies with the aid of computational chemistry methods have been of great help. A fundamental step in such theoretical investigations is the knowledge of the predominant drug molecular structure in solution, which is the real environment for the interaction with biological targets. Our strategy to access this valuable information is to perform density functional theory (DFT) calculations of 1H NMR chemical shifts for several plausible molecular conformers and then find the best match with experimental NMR profile in solution (since it is extremely sensitive to conformational changes). Through this procedure, after optimizing 30 trial distinct molecular structures (ωB97x-D/6-31G(d,p)-PCM level of calculation), which may be considered representative conformations, we concluded that the global minimum (named M24), stabilized by an intramolecular N-H hydrogen bond, is not likely to be observed in water, chloroform, and dimethyl sulfoxide (DMSO) solution. Among fully optimized conformations (named M1 to M30, and MD1 and MD2), we found M12 (having no intramolecular H-bond) as the most probable structure of CQ and HCQ in water solution, which is a good approximate starting geometry in drug-receptor interaction simulations. On the other hand, the preferred CQ and HCQ structure in chloroform (and CQ in DMSO-d6) solution was assigned as M8, showing the solvent effects on conformational preferences. We believe that the analysis of 1H NMR data in solution can establish the connection between the macro level (experimental) and the sub-micro level (theoretical), which is not so apparent to us and appears to be more appropriate than the thermodynamic stability criterion in conformational analysis studies.

Two analogue bases are described: 3-deazaadenine is a derivative of adenine from which N3 has been deleted and 3-methyl-2-pyridone is a C-nucleoside that mimics thymine but lacks the O2 carbonyl. The dc(3)A-dm(3)2P base pair is similar to dA-dT but eliminates the polar functional groups in the minor groove. The presence of this base pair in dA-dT rich sequences results in destabilized duplexes or conformational preferences for monomolecular hairpins rather than bimolecular duplexes. When present in dG-dC rich sequences, no significant differences in helix stability are observed. These differences are explained on the basis of hydration effects, most notably, the elimination of the minor groove spine of hydration normally present in dA-dT rich sequences. CD spectra suggest that sequences with a fully modified core (four analogue base pairs) are more A-like helices than B-like helices. Sequences containing two analogue base pairs can be transformed to A-like helices under conditions of high salt, or 65% trifluoroethanol. These conformational changes are also explained in terms of a loss of hydration in the minor groove that normally stabilizes the B-form conformation. In the absence of such hydration, the helices are conformationally mobile and adopt a more A-like helix form.

The influence of surfactant type on migration behavior and chemical selectivity in micellar electrokinetic chromatography (MEKC) is investigated through linear solvation energy relationships (LSER) and functional group selectivities. In LSER modeling, solutes' capacity factors are correlated with their structural descriptors such as size, dipolarity, and hydrogen-bonding abilities. Using the LSER methodology, useful information about the nature of solute interactions with different types of surfactant aggregates can be obtained since capacity factor in MEKC is directly related to solute distribution between the bulk aqueous solvent and micelles. High correlations were observed for different LSER models of migration behavior in MEKC for a group of 60 uncharged aromatic compounds of non-hydrogen bonding (NHB), hydrogen-bonding acceptor (HBA) bases, and hydrogen-bonding donor (HBD) acids. In two anionic, hydrocarbon micellar systems of sodium dodecyl sulfate (SDS) and sodium cholate (SC), retention is primarily influenced by the size of molecules and their hydrogen bond accepting basicity. Their dipolarity/polarizability and hydrogen bond donating acidity play minor roles. Capacity factors of solutes in SDS and SC systems increase with their size and decrease for stronger hydrogen bond acceptor bases. These results are similar to those observed for other systems where hydrophobic interactions play a major role, e.g., solute distribution in the 1-octanol-water solvent system or retention in reversed phase LC. In MEKC with an anionic fluorocarbon surfactant, lithium perfluorooctanesulfonate (LiPFOS), however, size and solute HBD acidity are the two predominant factors. The LSER results indicate that compounds find the SDS micellar environments slightly less cohesive (i.e., more apolar) than the SC micelles, while the LiPFOS micelles are the most cohesive among the three surfactant aggregates and 1-octanol provides the least cohesive environment. The fluorocarbon micelles of LiPFOS, on the other hand, are the strongest hydrogen bond donor acids, followed by SDS, SC, and 1-octanol, respectively. The SC micelles have the most hydrogen bond acceptor basic characteristics, followed by 1-octanol, SDS, and LiPFOS micelles. It can be concluded that selectivity differences between these surfactant types in MEKC is primarily due to hydrogen-bonding interactions rather than the dipolar interactions. Comparing the perfluorinated and the hydrocarbon surfactants, even solute size can play a role in selective migration patterns. In addition, information from polar and hydrophobic group selectivities confirm the LSER conclusions about the underlying interactions that control migration behavior and chemical selectivity in MEKC.(ABSTRACT TRUNCATED AT 400 WORDS)

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 103/1.44 × 103 L mol−1 at 291 K. The values of ΔHθ, ΔSθ, and ΔGθ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern–Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA–AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS–DNA–AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A–T rich region of DNA.

Homeodomains are helix-turn-helix type DNA-binding domains that exhibit sequence-specific DNA binding by insertion of their "recognition" alpha helices into the major groove and a short N-terminal arm into the adjacent minor groove without inducing substantial distortion of the DNA. The stability and DNA binding of four representatives of this family, MATalpha2, engrailed, Antennapedia, and NK-2, and truncated forms of the last two lacking their N-terminal arms have been studied by a combination of optical and microcalorimetric methods at different temperatures and salt concentrations. It was found that the stability of the free homeodomains in solution is rather low and, surprisingly, is reduced by the presence of the N-terminal arm for the Antennapedia and NK-2 domains. Their stabilities depend significantly upon the presence of salt: strongly for NaCl but less so for NaF, demonstrating specific interactions with chloride ions. The enthalpies of association of the homeodomains with their cognate DNAs are negative, at 20 degrees C varying only between -12 and -26 kJ/mol for the intact homeodomains, and the entropies of association are positive; i.e., DNA binding is both enthalpy- and entropy-driven. Analysis of the salt dependence of the association constants showed that the electrostatic component of the Gibbs energy of association resulting from the entropy of mixing of released ions dominates the binding, being about twice the magnitude of the nonelectrostatic component that results from dehydration of the protein/DNA interface, van der Waals interactions, and hydrogen bonding. A comparison of the effects of NaCl/KCl with NaF showed that homeodomain binding results in a release not only of cations from the DNA phosphates but also of chloride ions specifically associated with the proteins. The binding of the basic N-terminal arms in the minor groove is entirely enthalpic with a negative heat capacity effect, i.e., is due to sequence-specific formation of hydrogen bonds and hydrophobic interactions rather than electrostatic contacts with the DNA phosphates.

Synthesis, characterization and biological applications of a new nano-sized binuclear copper(II) complex containing bathophenanthroline and ellagic acid as ligands

The conformational variability of RNA duplexes with internal loop 5′GAGU/3′UGAG was investigated by nuclear magnetic resonance (NMR) spectroscopy and all-atom molecular dynamics (MD) simulations. It was previously found that the CG-flanked internal loop in 5′GACGAGUGUCA/3′ACUGUGAGCAG existed in a major conformation (I) characterized by U7 and U7∗ bulging into solution, A5 and A5∗ stacking, and G4-G6∗ and G6-G4∗ basepairs closing the loop. A minor conformation also existed with G-U pairs with a bifurcated hydrogen bond and A-G pairs with a single hydrogen bond (II). A maximum hydrogen bonding structure with wobble G-U pairs and two-hydrogen-bond A-G pairs (III) was not observed by NMR or MD. Here, flanking pairs A-U, U-A, and G-C were substituted for the C-G basepair adjacent to the GAGU internal loop. NMR demonstrated changes in conformational preference depending on the identity of the flanking pair. Clustering of MD structures for C-G- and U-A-flanked duplexes showed transitions between conformations II and III with a greater fraction of the structures in conformation II, although the simulations started in conformation III. Additionally, U-A-flanked simulations contained 25% of structures in a state between conformations II and III. A-U- and G-C-flanked structures were in a single cluster whose centroid was conformation II. MD showed a dominance of structure II over III, in agreement with NMR for C-G closure, but in disagreement with other closures. MD starting in conformation I did not transition to II or III, with structures in a single cluster for all flanking basepairs except for G-C where 22% of the structures were defined by a C3′-endo sugar pucker for G4 and G4∗. Bases G4 and G4∗ were also syn around the glycosidic bond for all flanking pairs, whereas G6 and G6∗ transitioned between syn and anti for C-G- and A-U-flanked simulations, consistent with NMR.

We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)2-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isoprop yl)2 where (SMe)Pen(O)2 stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the psi of Phe(cep) is maintained about 130 degrees and the phi angle of (cep)Gly prefers +/- 150 degrees [where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule ('flap water'), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.

Recently, there have been numerous reports on the use of different fluorescent DNA stains for specific minor groove binding. The exploration of biological markers increases the safety of their use as diagnostic criteria. Single crystal analysis of DNA–ligand binding interactions is of essential importance to obtain the requirements for their usage in the pharmaceutical and medical industries. Dyes that bind to DNA, such as Hoechst 33342 or 4′,6-diamidino-2-phenylindole (DAPI), can be used not only for analytical use, but for medical purposes. DAPI and Hoechst 33342 are fluorescent dyes that bind to the minor groove of DNA, fluorescing brightly in the blue region with an emission maximum at approximately 461 nm when excited by ultraviolet light (~350 nm). This work focuses on the binding interactions of Hoechst 33342 with the specific DNA sequence d(CGTGAATTCACG)2. The structure of the complex was determined using single-crystal X-ray diffraction at a resolution of 1.9 Å in the space group P212121. The coordinates and structure factors are deposited in the RCSB Protein Data Bank (PDB) under entry 9FT8. The structure is nearly isomorphous with that of previously reported crystal structures of the oligonucleotide d(CGTGAATTCACG)2 alone (PDB ID: 5JU4) and with that in complexes with DAPI (5T4W). The adjustments in crystal interactions between the native DNA molecule and the DNA–DAPI complex are described. Hoechst 33342 selectively binded to the tight minor groove close to the midpoint of the B-DNA segment, adjacent to the A–T base pairs. It interacted with DNA through hydrogen bonding and van der Waals forces. The structural comparison revealed that Hoechst 33342 inserts itself in the minor groove in a strongly specific manner, displacing the ordered spine waters.

A computational insight on the noncovalent interactions of aminothiazole-based palladium(II) complexes with DNA as a potent anticancer agent

Trifluridine (FTD), known as main component of TAS-102, is beginning to be applied as anti-tumor agent due to the highly efficacious antitumor potency although there is little to distinguish FTD structure from thymidine structure. TAS-102 has first been used in Japan in clinical therapy as an oral agent. Some experimental results hypothesized that FTD pharmacological antitumor effect can be arisen by inhibition of thymidylate synthase and incorporation of FTD itself into DNA. One of them is the thermal denaturation experiments for DNA duplexes of containing some FTD or not, performed by J. C. Markley et al. Their experiments have shown that the DNA duplexes containing FTD are slightly less stable, with a melting temperature about three degrees lower than not containing duplexes. However, the change of thermodynamic structure and inter-nucleobase interaction changes are not yet known. In this presentation, we have performed molecular dynamics (MD) simulations and ab initio molecular orbital (MO) calculations to analyze the differences of thermodynamics structure and inter-nucleobase interaction between containing and not containing FTD. From these results, we sought to qualitatively understand the effects of FTD. At the first step, we have estimated the stabilization structures of same two DNA duplexes (containing FTD and not) used by J. C. Markley, with energy minimization. Then, a large differences were shown between these structures. The hydrogen bond breaking is observed between FTD and complementary Adenine base while the not containing DNA duplex keeps having a stabilized structure. The behavior means that the essential difference of intra-DNA interactions was arisen. At the second step, MD simulations were performed to analyze the distribution of distances between FTD and complementary base (i.e. thermally available conformations). With some conformations from MD sampling, the MO calculations were performed to discuss the difference of intra-base interactions. From our results, we found that thermodynamic conformational change of DNA duplex containing FTD occurs even if it is in internal body temperature, and the break hydrogen bonding between FTD and paired base was observed more frequently. In addition, strong interactions between FTD and nearby π-conjugate base were observed. These behaviors might be one of the factor of inducing unstabilization in DNA duplexes. Citation Format: Jun Koseki, Kenta Tsunekuni, Masamitsu Konno, Naohiro Nishida, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Thermodynamic and molecular orbital analysis of the effects caused by incorporation of novel anti-tumor agent Trifluridine to DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2706.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes. We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate. Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.