Design of anticancer drugs using modeling techniques.

As a class, minor-groove non-covalent binding small molecules generally show A/T selectivity. The lack of G/C selectivity is thought to relate to the wider minor groove in G/C regions and the presence of the exocyclic 2-amino groups of guanines which project into the minor groove and prevent multiple close van der Waals contacts with the G/C-rich minor groove. AT-selective structures such as distamycin and netropsin orientate in the minor groove in an “NH down” conformation, with their NH groups pointing into the minor groove to enhance the isohelicity of the structures and produce favourable van der Waal interactions with the groove floor below. Using a distamycin scaffold as a starting point we introduced biaryl building blocks in place of pyrroles to change the curvature and van der Waals interaction potential of this novel class of polyamides. We started by designing a library of fragments to span two DNA base pairs based on simple phenyl-substituted heterocycles of sufficient length. With each library member containing one heterocyclic and one carbocyclic component, sufficient diversification was achieved through modifications to, and substitutions within, both components. This allowed the production of libraries of building blocks possessing different curvatures and hydrogen-bonding abilities with the potential to complement functional groups and hydrogen bond donors and acceptors in the walls and floor of the minor groove. A fluorescent intercalator displacement (FID) assay was then used to select molecules with a preference for GC-rather than AT-rich sequences which identified the 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif. Molecular dynamics simulations were undertaken using the AMBER v11 software in implicit solvent over a time-scale of 20ns with the structures in both a “carbonyl-in” and “NH-in” conformation. This demonstrated that the MPB component within the biaryl polyamides undergoes a switch in orientation, with a “carbonyl down” conformation preferred which induces GC specificity. As well as occupying an idealised hydrogen-bonding distance between two side-by-side guanine residues, MPB units contained within the MPB biaryl polyamide structures showed superior hydrogen bonding interactions with GC compared to AT sequences, with each carbonyl of the MPB unit hydrogen bonding to a guanine residue. This was further supported by free-energy of binding calculations undertaken using AMBER-MMPBSA, where the MPB units showed a distinct preference for GC sequences (a difference of -13kcal/mol in favour of “carbonyl -in” over “NH-in” structures). This discovery has been applied to the design and synthesis of more-complex MPB-containing DNA minor-groove binding ligands with a high level of GC-selectivity and significant in vitro and in vivo antitumour activity. Citation Format: Paul J M Jackson, Colin M. James, Khondaker M. Rahman, David E. Thurston. Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4549. doi:10.1158/1538-7445.AM2013-4549

This research is an effort to further understand the physicochemical interaction between the novel drug molecule diethidium (2,7-diamino 9-[2,7 diamino 10-nN-phenanthridium] 10-nN-phenanthridium) and its biological receptor DNA. The ultimate goal is the elucidation of this novel class of drugs as potential pharmaceutical agents. Understanding the physicochemical properties of this drug as well as the mechanism by which it interacts with DNA should ultimately allow the rational design of novel anti-cancer or anti-viral drugs. A novel binding structure for the diethidium cation to B-form DNA is herein described. Molecular modeling on the complex formed between diethidium and a dodecamer of double-stranded B-form DNA, CGCGAATTCGCG, has shown that this complex is indeed fully capable of participating in the formation of a stable intercalation site. It was expected that diethidium would have a mechanism of intercalation significantly different from other classical intercalators because a) Its structure, that of two perpendicular planes, each known to have excellent intercalation properties, is novel b) The linker region length is zero c) The tilt between the two planes of the drug matches the geometry of the space available to this drug in the major groove. We have studied the complex formed when diethidium enters the central site of the B-DNA dodecamer through the major groove. The complex forms several classes of intercalation structures, which are all stable and vary from "partially intercalated" to "fully intercalated". Multiple minimizations show the drug to be very mobile within the intercalation site. Further, some structures show organization and concomitant stiffening of the DNA above the intercalation site, with a disorganization and disruption of the regular B-DNA structure immediately below the intercalation site. This particular phenomena may be expected to lead to significantly different physicochemical properties for the diethidium complex with respect to other known intercalators, because this sort of vectorial difference in structure above and below the site of intercalation is unknown in existing intercalators, as far as the authors are aware. In addition, we expect the mechanism of interaction between drug and DNA to be described by "direct ligand transfer", wherein the drug is transferred from duplex DNA to duplex DNA without re-entering the solvent. This work is the first instance known to the authors of a novel drug entity that was deduced solely by mathematical reasoning and described subsequently by computational methods. Evidence that diethidium should interact with its target site DNA differently from other known intercalators is strong.

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.

Eight new side-chain allylic, benzylic, and propargylic ether analogs of the natural hormone calcitriol have been rationally designed and easily synthesized. Three of these 23-oxa ether analogs lacking the typical side-chain OH group are more antiproliferative in vitro and desirably less calcemic in vivo than the natural hormone. One of these three 23-oxa analogs has transcriptional potency almost as high as that of calcitriol, even though it binds to the human vitamin D receptor only about 1% as well as calcitriol.

Extensive molecular dynamics (MD) simulations have been used to investigate the intercalative binding of 9-hydroxyellipticine to the DNA oligonucleotide d(ATATATATATAT)2. Four independent simulations differing in the initial orientation of the drug at the intercalation site were carried out, and compared both with each other and a control simulation of the free DNA sequence. The structure of the latter was compared with structures obtained from x-ray crystallography and nmr spectroscopy, as well as the theoretically derived "alternating B-DNA" model [A. Klug et al. (1979), Journal of Molecular Biology, Vol. 131, p. 669]. The alternation of twist angles observed in experimental structures was reproduced in the simulation. All four independent simulations of the drug-DNA intercalation complex converged in placing the pyridine ring of the ellipticine chromophore in the major groove; in one case this involved a 180 degrees rotation of the drug at the intercalation site. At a more detailed level, the drug is seen to be capable of adopting several distinct orientations, each stable over a period of hundreds of pico-seconds. Despite the presence of several polar groups in the drug, however, no direct hydrogen bonding to the DNA occurs; instead, interactions between the methyl groups of the drug and the thymine bases at the intercalation site appear important in determining the orientational preferences of the drug. Comparison of the intercalation complexes with the free DNA sequence shows a degree of unwinding resulting from intercalation, in good agreement with experimental results, but spread over the three central base-pair steps, not confined to the intercalation site itself. Measurements of torsional rigidity indicate only a slight stiffening of the DNA restricted to the immediate site of intercalation. The structures obtained from the MD simulations were used to calculate theoretical CD spectra, with separate simulations giving very different results. This appears to indicate that given an accurate assignment of the main electronic transition dipole moment of the ellipticine chromophore, discrimination of the more realistic binding geometries may be possible. The relative merits of the various drug orientations observed in the simulations are discussed and a perpendicular orientation of the drug at the intercalation site is considered to be the most consistent with experimental data. While the simulations themselves represent a total of over 2 ns, however, the differences apparent between independent runs indicate that longer simulation times will be required before a complete, unequivocal view of DNA intercalation is obtained.

Carbohydrates are among the most abundant molecules in living nature, with the exception of the water molecule. They are found in wide varieties as monomers, oligomers, cyclic and linear and branched polymers. They occur alone or chemically combined with purines and pyrimidines in the nucleic acids, with polypeptides in the glycoproteins and with lipids in the glycolipids. The simpler carbohydrates resemble water molecules in one important respect. Their primary hydrogen-bonding function comes from the hydroxyl group which can have both donor and acceptor properties (Box 13.1). For this reason, their hydrogen-bonding patterns have features in common with those of the ices and the high hydrates. They differ fundamentally from those of the polypeptides or proteins, in which the majority of hydrogen-bond groups are -NH groups, which are only hydrogen-bond donors, and the C = O groups, which can only be hydrogen-bond acceptors. The hydrogen bonding in the nucleosides and nucleotides, which contain functional groups which are both donor-acceptors and donors or acceptors only, has features which resemble both the carbohydrates and the peptides.

This book provides an update on heterocyclic compounds that serve as key components of anti-cancer agents administered in pre-clinical settings. Many of the compounds highlighted in the book are being actively investigated for the bioactive properties against a range of cancer cell lines. There is potential for heterocyclic compounds to design agents that can target specific molecules to treat different types of cancers. Chapters are contributed by experts in pharmaceutical chemistry and are written to give a general overview of the topic to readers involved in all levels of research and decision-making in pharmaceutical chemistry and anti-cancer drug design. Part 1 of the book set covers these topics: - Heterocyclic anticancer compounds derived from natural sources with their mechanism of action - The role of terpenoids as anticancer compounds: an insight into prevention and treatment - Recent advances in synthesis and anticancer activity of benzothiazole hybrids as anticancer agents - Structure-activity relationship studies of novel hybrid quinoline and quinolone derivatives as anticancer agents - Tetrazoles: structure and activity relationship as anticancer agents - Progress in nitrogen and oxygen-based heterocyclic compounds for their anticancer activity: an update (2017-2020)

Proline-rich peptides are known to adopt preferentially the extended polyproline II (PPII) helical conformation, which is involved in several protein-protein recognition events. By resorting to molecular modelling techniques, we wished to investigate the extent to which PPII helices could be used for the formation of isochelical peptide-DNA complexes leading to the selective recognition of the major groove of B-DNA. For that purpose, we have grafted to a cationic intercalator, 9-amino-acridine, an oligopeptide having the sequence: Pro- Arg-Pro-Pro-Arg-Pro-Pro-Arg-Pro-Pro-Asp-Pro-Pro. Each residue in the sequence was set in the D configuration, to prevent enzymatic hydrolysis, and each Arg residue was designed to target O6/N7 of a guanine base following the intercalation site. The Asp residue was designed to target a cytosine base, whilst simultaneously forming a bidentate complex with the Arg three residues upstream. Energy-minimization, using the JUMNA procedure, led to the following conclusions : 1) major groove binding is favoured over minor groove or exclusive binding to the phosphates by large energy differences, of over 50 and 90 kcal/mole, respectively: 2) the two best bound sequences are those having three successive guanine bases on the same DNA strand, immediately adjacent to the intercalation site. Sequence d(CGGGC G), encountered in the Primer Binding Site of the HIV retrovirus, thus ranks amongst the best-bound sequences; 3) replacement of an individual guanine amongst the three ones upstream of the intercalation site, by an adenine base, weakens by > 6 kcal/mole the binding energetics; 4) the conformational rigidity of the DNA-bound PPII helix should enable for a modulation of the base sequence selectivity, by appropriate replacements of the Arg and Asp residues. Thus sequence CGGCAAG, also encountered in the HIV genome, could be targeted by an oligopeptide having the sequence Pro-Arg-Pro-Pro-Asp-Pro-Pro-Asn-Pro-Pro-Asn-Pro-Pro-Arg-Ala.

Two series of dendritically modified tryptophan derivatives have been synthesised and their emission spectra measured in a range of different solvents. This paper presents the syntheses of these novel dendritic structures and discusses their emission spectra in terms of both solvent and dendritic effects. In the first series of dendrimers, the NH group of the indole ring is available for hydrogen bonding, whilst in the second series, the indole NH group has been converted to NMe. Direct comparison of the emission wavelengths of analogous NH and NMe derivatives indicates the importance of the Kamlet-Taft solvent beta3 parameter, which reflects the ability of the solvent to accept a hydrogen bond from the NH group, an effect not possible for the NMe series of dendrimers. For the NH dendrimers, the attachment of a dendritic shell to the tryptophan subunit leads to a red shift in emission wavelength. This dendritic effect only operates in non-hydrogen-bonding solvents. For the NMe dendrimers, however, the attachment of a dendritic shell has no effect on the emission spectra of the indole ring. This proves the importance of hydrogen bonding between the branched shell and the indole NH group in causing the dendritic effect. This is the first time a dendritic effect has been unambiguously assigned to individual hydrogen-bonding interactions and indicates that such intramolecular interactions are important in dendrimers, just as they are in proteins. Furthermore, this paper sheds light on the use of tryptophan residues as a probe of the microenvironment within proteins--in particular, it stresses the importance of hydrogen bonds formed by the indole NH group.

Luminol: Extended hydrogen bond network in water solution

Quinacridones are industrially important hydrogen-bonded pigments. The color in the solid state is vivid red, while it is pale yellow in solution, indicating evidently the involvement of intermolecular interactions in the solid state. The electronic structure has therefore been investigated with special attention to the role of intermolecular NH...O hydrogen bonds for three representative quinacridone compounds with different hydrogen-bond forming characteristics: unsubstituted gamma-quinacridone (gamma-QA) with two NH groups, mono-N-methylquinacridone (MMQA) with one NH and one CH(3), and N,N'-dimethyl-quinacridone (DMQA) with two CH(3) groups. The number of the NH...O hydrogen bonds per molecule is four, two, and zero for gamma-QA, MMQA, and DMQA, respectively. In solution, no significant difference in absorption maximum is recognized between gamma-QA, MMQA, and DMQA. However, in the solid state, the absorption maximum of gamma-QA appears at the longest wavelength, followed by MMQA and then DMQA, depending on the number of NH...O intermolecular hydrogen bonds. The role of the hydrogen bond is found to align transition dipoles in a "head-to-tail" fashion and to displace the absorption band toward longer wavelengths due to excitonic interactions. The extent of the spectral shift increases with increasing number of hydrogen bonds per molecule.

As part of an effort to expand the genetic alphabet, we examined the synthesis of DNA with six different unnatural nucleotides bearing methoxy-derivatized nucleobase analogues. Different nucleobase substitution patterns were used to systematically alter the nucleobase electronics, sterics, and hydrogen-bonding potential. We determined the ability of the Klenow fragment of E. coli DNA polymerase I to synthesize and extend the different unnatural base pairs and mispairs under steady-state conditions. Unlike other hydrogen-bond acceptors examined in the past, the methoxy groups do not facilitate mispairing, implying that they are not recognized by any of the hydrogen-bond donors of the natural nucleobases; however, they do facilitate replication. The more efficient replication results largely from an increase in the rate of extension of primers terminating at the unnatural base pair and, interestingly, requires that the methoxy group be at the ortho position where it is positioned in the developing minor groove and can form a functionally important hydrogen bond with the polymerase. Thus, ortho methoxy groups should be generally useful for the effort to expand the genetic alphabet.

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT]2, d[GCGATATCGC]2 and d[ATAICICTAT]2, were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT]2 complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.

The X-ray structures of two crystal forms of the RNA duplex [CGCG(5-FC)G]2 are the first crystal structures for an RNA duplex containing alternating CG base pairs with unmodified 2′-hydroxyl groups. The triclinic and the rhombohedral crystal forms (PDB ids 3jxq, 3jxr) differ in the molecular packing, the amount of ordered structure and in molecular details, especially the hydration. The P1 data extend to 1.45 Å resolution, while the R32 data reach 1.25 Å. In both structures the RNA oligomers take the form of a right-handed half-turn with conformational parameters close to the canonical A-RNA. The duplexes stack coaxially in the crystal lattice. The geometry of C:G vs. 5-FC:G pairing is similar in terms of H-bond geometry. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. Both structures contain ordered water molecules, forming characteristic hydration network in the major and the minor grooves. In addition to water, the triclinic structure contains four magnesium cations, one of them forming an inner complex with a phosphate. Two types of unusual interactions are observed. In the major groove, the 5-fluorocytidine residues form C–F⋯H–O–H hydrogen bonds with water molecules. In the minor groove, intermolecular contacts include C–H hydrogen bonds (C1′–H1⋯O2′ and C4′–H4′⋯O4′) between two ribose rings.

The crystal structure of the RNA duplex [r(CCCCGGGG)]2 has been refined to 1.46 A resolution with room temperature synchrotron diffraction data. This represents the highest resolution reported to date for an all-RNA oligonucleotide and is well beyond the best resolution ever achieved with an A-form DNA duplex. The analysis of the ordered hydration around the octamer duplex reveals conserved regular arrangements of water molecules in both grooves. In the major groove, all located first shell water molecules can be fitted into a pattern that is repeated through all eight base pairs, involves half the phosphate oxygens, and joins the two strands. In the minor groove, roughly across its narrowest dimension, tandem water molecules link the 2'-hydroxyl groups of adjacent nucleotides in base-pair steps in a similarly regular fashion. The structure provides evidence for an important role of the 2'-hydroxyl groups in the thermodynamic stabilization of RNA, beyond their known functions of locking the sugar pucker and mediating 3' --> 5' intrastrand O2'...O4' hydrogen bonds. The ribose 2'-hydroxyls lay the foundation for the enthalpic stability of the RNA relative to the DNA duplex, both as a scaffold for the water network in the minor groove and through their extensive individual hydration.