Poly(triazolyl methacrylate) glycopolymers as potential targeted unimolecular nanocarriers.

Synthetic glycopolymers are increasingly investigated as multivalent ligands for a range of biological and biomedical applications. This study indicates that glycopolymers with a fine-tuned balance between hydrophilic sugar pendant units and relatively hydrophobic polymer backbones can act as single-chain targeted nanocarriers for low molecular weight hydrophobic molecules. Non-covalent complexes formed from poly(triazolyl methacrylate) glycopolymers and low molecular weight hydrophobic guest molecules were characterised through a range of analytical techniques - DLS, SLS, TDA, fluorescence spectroscopy, surface tension analysis - and molecular dynamics (MD) modelling simulations provided further information on the macromolecular characteristics of these single chain complexes. Finally, we show that these nanocarriers can be utilised to deliver a hydrophobic guest molecule, Nile red, to both soluble and surface-immobilised concanavalin A (Con A) and peanut agglutinin (PNA) model lectins with high specificity, showing the potential of non-covalent complexation with specific glycopolymers in targeted guest-molecule delivery.

The fitness burden imposed by synthesising quorum sensing signals.

It is now well established that bacterial populations utilize cell-to-cell signaling (quorum-sensing, QS) to control the production of public goods and other co-operative behaviours. Evolutionary theory predicts that both the cost of signal production and the response to signals should incur fitness costs for producing cells. Although costs imposed by the downstream consequences of QS have been shown, the cost of QS signal molecule (QSSM) production and its impact on fitness has not been examined. We measured the fitness cost to cells of synthesising QSSMs by quantifying metabolite levels in the presence of QSSM synthases. We found that: (i) bacteria making certain QSSMs have a growth defect that exerts an evolutionary cost, (ii) production of QSSMs negatively correlates with intracellular concentrations of QSSM precursors, (iii) the production of heterologous QSSMs negatively impacts the production of a native QSSM that shares common substrates, and (iv) supplementation with exogenously added metabolites partially rescued growth defects imposed by QSSM synthesis. These data identify the sources of the fitness costs incurred by QSSM producer cells, and indicate that there may be metabolic trade-offs associated with QS signaling that could exert selection on how signaling evolves.

Multiscale modeling of drug-polymer nanoparticle assembly identifies parameters influencing drug encapsulation efficiency.

Using a multiscale (dual resolution) approach combining an atomistic (GROMOS96) and coarse-grain (MARTINI) force field, we have been able to simulate the process of drug-polymer nanoparticle assembly by nanoprecipitation from mixed solvents. Here, we present the development and application of this method to the interaction of three poly(glycerol adipate) polymer variants with the anticancer drug dexamethasone phosphate. Differences in encapsulation efficiency and drug loading between the polymers are in agreement with the experimental trend. Reference atomistic simulations at key points along the predicted aggregation pathway support the accuracy of the much more computationally efficient multiscale methodology.

Atomistic simulations reveal bubbles, kinks and wrinkles in supercoiled DNA.

Although DNA is frequently bent and supercoiled in the cell, much of the available information on DNA structure at the atomistic level is restricted to short linear sequences. We report atomistic molecular dynamics (MD) simulations of a series of DNA minicircles containing between 65 and 110 bp which we compare with a recent biochemical study of structural distortions in these tight DNA loops. We have observed a wealth of non-canonical DNA structures such as kinks, denaturation bubbles and wrinkled conformations that form in response to bending and torsional stress. The simulations show that bending alone is sufficient to induce the formation of kinks in circles containing only 65 bp, but we did not observe any defects in simulations of larger torsionally relaxed circles containing 110 bp over the same MD timescales. We also observed that under-winding in minicircles ranging in size from 65 to 110 bp leads to the formation of single stranded bubbles and wrinkles. These calculations are used to assess the ability of atomistic MD simulations to determine the structure of bent and supercoiled DNA.

Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.

AIMS: Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. METHODS: An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. RESULTS: Several specific APE1 inhibitors were isolated by this approach. The IC(50) for APE1 inhibition ranged between 30 nM and 50 µM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. CONCLUSIONS: Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

The levels of telomere-binding proteins in human tumours and therapeutic implications.

Tumours require telomeric integrity to maintain viability, conferred by adequate length of telomeric DNA replenished by telomerase, and binding of telomere-binding proteins (TBPs), thus telomeres have received attention as an anticancer target. Levels of TBPs in tumour tissue may have implications for drug development if they render some cancers relatively more sensitive or resistant to telomere targeted agents. This review gives an overview of the studies examining the levels of TBPs in tumours and discusses possible reasons for differences in the findings, given the interplay between various factors determining telomere stability. Whether cancers with lower levels of TBPs will be more susceptible to therapies targeting telomere maintenance will require clinical trials of these novel therapies.

Supercoiling and denaturation of DNA loops.

We study the statistical mechanics of small DNA loops emphasizing the competition between elasticity, supercoiling, and denaturation. Motivated by recent experiments and atomistic molecular dynamics simulation, we propose a new coarse-grained phenomenological model of DNA. We extend the classical elastic rod models to include the possibility of denaturation and nonlinear twist elasticity. Using this coarse-grained model, we obtain a phase diagram in terms of fractional overtwist and loop size that can be used to rationalize a number of experimental results which have also been confirmed by atomistic simulations.

A simple physical description of DNA dynamics: quasi-harmonic analysis as a route to the configurational entropy.

It has become increasingly apparent that the dynamic as well as the structural properties of biological macromolecules are important to their function. However, information concerning molecular flexibility can be difficult to obtain experimentally at the atomic level. Computer modelling techniques such as molecular dynamics (MD) have therefore proved invaluable in advancing our understanding of biomolecular flexibility. This paper describes how a combination of atomistic MD simulations and quasi-harmonic analysis can be used to describe the dynamics of duplex DNA, with a particular emphasis on methods for calculating differences in configurational entropies. We demonstrate that DNA possesses remarkably simple mechanical properties relative to globular proteins, making it an ideal system for exploring biomolecular flexibility in general. Our results also highlight the importance of solvent viscosity in determining the dynamic behaviour of DNA in aqueous solution.

Auger electrons--a nanoprobe for structural, molecular and cellular processes.

This paper provides a brief review of recently published work on biophysical and biological aspects of Auger processes. Three specific questions have been considered. (1) Does charge neutralisation contribute to molecular damage such as DNA strand breaks? (2) How many DNA double strand breaks are produced by a single decay of DNA bound (125)I? (3) What is the correlation between number of gammaH2AX foci and number of double strand breaks (DSB)? The paper also gives preliminary reports on two new calculations: (a) calculation of the spectrum of Auger electrons released during decay of (124)I and (b) the use of Auger electrons in the decay of (125)I as a probing agent of novel DNA structures.

The mysteries of telomere structure and recognition: could radioprobing help?

PURPOSE: Telomeres are specialized DNA-protein complexes found at the ends of eukaryotic chromosomes. In normal somatic cells these become shorter with each cell division and appear to control their replicative lifespan. However almost all tumours show activation of the enzyme telomerase, a specialised reverse transcriptase/DNA polymerase, that can add new telomeric repeats to the ends of chromosomes and this appears to be a key factor in the cell immortalization process. Consequently there is much current interest in the potential for inhibitors of telomere extension in the treatment of cancer. Several groups have found that it is possible to produce inhibitory molecules that target the telomeric repeat (substrate) DNA rather than the telomerase enzyme itself. This is thought to work because it has been found that in vitro, these DNA sequences can fold up into a four-stranded (quadruplex) structure that the drugs recognise and stabilize, but which is not recognised by the enzyme. However, while medicinal chemists continue to base rational design programs on this hypothesis, there is currently very little evidence that these structures form in vivo, and that in vivo the drugs work by binding to them. To have incontrovertible evidence of where and how these telomerase inhibitors and DNA interact is therefore a pressing concern for a basic understanding of their mechanism of action and effective drug development. MATERIALS AND METHODS: Radioprobing represents a valuable new approach to the study of DNA structures. Recently we have shown through computer simulations of radioprobing that the technique is a remarkably sensitive probe of quite fine details of DNA conformation. Here we report on our simulations of the binding of a radiolabelled telomerase inhibitor, related to a class of novel inhibitors under development at Nottingham, to a variety of possible structures for telomeric DNA. RESULTS AND CONCLUSIONS: The predicted cleavage patterns prove to be very sensitive to the DNA structure, and the mode of binding of the drug. These results suggest that radioprobing experiments should be able to provide unambiguous evidence as to the 'true' nature of the telomere-drug complexes, and so aid the rational design programme.

Cooperativity in drug-DNA recognition: a molecular dynamics study.

NMR studies have shown that the minor groove-binding ligand Hoechst 33258 binds to the two T4/A4 tracts within the duplex d(CTTTTCGAAAAG)2 in a highly cooperative manner, such that in titration experiments no intermediate 1:1 complex can be detected. The NMR-derived structures of the free DNA and the 2:1 complex have been obtained, but can shed little light on what the origins of this cooperativity may be. Here we present the results of a series of molecular dynamics simulations on the free DNA, the 1:1 complex, and the 2:1 complex, which have been designed to enable us to calculate thermodynamic parameters associated with the molecular recognition events. The results of the molecular dynamics studies confirm that structural factors alone cannot explain the cooperativity observed, indeed when enthalpic and hydration factors are looked at in isolation, the recognition process is predicted to be slightly anticooperative. However, when changes in configurational entropy are taken into account as well, the overall free energy differences are such that the calculated cooperativity is in good agreement with that observed experimentally. The results indicate the power of molecular dynamics methods to provide reasonable explanations for phenomena that are difficult to explain on the basis of static models alone, and provide a nice example of the concept of "allostery without conformational change".

Structural studies on bioactive compounds. 34. Design, synthesis, and biological evaluation of triazenyl-substituted pyrimethamine inhibitors of Pneumocystis carinii dihydrofolate reductase.

The triazenyl-pyrimethamine derivative 3a (TAB), a potent and selective inhibitor of Pneumocystis carinii DHFR, was selected as the starting point for a lead optimization study. Molecular modeling studies, corroborated by a recent crystal structure determination of the ternary complex of P. carinii DHFR--NADPH bound to TAB, predicted that modifications to the acetoxy residue of the lead inhibitor could exploit binding opportunities in the vicinity of an active site pocket bounded by residues Ile33, Lys37, and Leu72. Substitutions in the benzyl moiety with electron-donating and electron-withdrawing groups were predicted to probe face-edge interactions with amino acid Phe69 unique to the P. carinii enzyme. New triazenes 10a--v and 12a--f were prepared by coupling the diazonium tetrafluoroborate salt 6b of aminopyrimethamine with substituted benzylamines or phenethylamines. The most potent of the new inhibitors against P. carinii DHFR was the naphthylmethyl-substituted triazene 10t (IC(50): 0.053 microM), but a more substantial increase in potency against the rat liver DHFR led to a reduction in selectivity (ratio rat liver DHFR IC(50)/P. carinii DHFR IC(50): 5.36) compared to the original lead structure 3a (ratio rat liver DHFR IC(50)/P. carinii DHFR IC(50): 114).

Dynamic model of base pair breathing in a DNA chain with a defect.

We model and analyze a short section of a DNA chain with a defect, with the aim of understanding how the frequency, amplitude, and localization of breathing events depend on the strength of the bonds between base pairs, both along the chain and between the chains. Our results show that the presence of a defect in the chain permits the existence of a localized breather mode. The models we analyze are linear and hence solvable, with solvability extending to the statistical mechanics formulation of the problem. Parameter values for the interaction energy of a base with its nearest neighbors are obtained from AMBER. The results indicate good agreement with both the amplitude and the number of base pairs affected by defect-induced breathing motion.

Force-induced melting of a short DNA double helix.

The dynamic behaviour of DNA is of fundamental importance to many cellular processes. One principal characteristic, central to transcription and replication, is the ability of the duplex to "melt". It has recently been shown that dynamic force spectroscopy provides information about the energetics of biomolecular dissociation. We have employed this technique to investigate the unbinding of single dodecanucleotide molecules. To separate the duplex to single-stranded DNA, forces ranging from 17 to 40 pN were required over a range of loading rates. Interpretation of the dependence of melting force on loading rate revealed that the energy barrier to rupture is between 9 and 13 kcal mol-1 in height and situated 0.58 nm from an intermediate structural state. Thermal melting studies show that, prior to dissociation, the oligonucleotide underwent a transition which required between 7 and 11 kcal mol-1 in energy. Through combined dynamic force spectroscopy and thermal melting studies we show the derivation of an energy landscape to dissociate a 12-mer duplex. Until very recently, this type of information was only accessible by computational analysis. Additionally, the force spectroscopy data allow an estimation of the kinetics of duplex formation and melting.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations.

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)(2) and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar-phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving 'lock and key' recognition.

Evaluation of the mechanism of aromatase cytochrome P450. A site-directed mutagenesis study.

Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the Ki value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the Ki value of 7alpha-APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The Ki value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.

Enantioselectivity of some 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as aromatase (P450AROM) inhibitors.

The enantioselectivity ratio ((+)-:(-)-forms) of three substituted 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as inhibitors of aromatase (P450AROM) was 2.16, 12.3 and 1.0 for the 4-methyl-, 4-fluoro- and 4-chloro-substituted compounds, respectively. The (+/-)-compounds were all >1000 times more potent than (+/-)-aminoglutethimide (IC50 = 12 x 10(3) nM). High potency (5.3-65.0 nM) for all the enantiomers studied is unusual since activity usually resides in one form for chiral inhibitors of P450AROM. The 4-methyl derivative was fitted into the model [Furet, P., Batzl, C., Bhatnager, A.S., Francotte, E., Rihs, G. and Lang, M. (1993) J. Med. Chem. 36, pp. 1393-1400] for binding of S-(-)-fadrazole to the active site and the (R)- and (S)- forms both gave a good fitting pattern with (S)-(-)-fadrazole so accounting for their close activity. Docking of both forms into the active site model for P450AROM [Laughton, C.A., Zvelebil, M.J.J.M. and Neidel, S. (1993) J. Steroid Biochem. Mol. Biol. 44, pp. 399-407], using the orientation of (S)-(-)-fadrazole, gave similar strong binding along the position of the C and D rings of the steroid substrate and in the hydrophobic cavity below the A/B rings. The site was probed for group size accommodation using the less potent 4-phenyl analogue (IC50(+/-) = 242 nM): the (S)-form showed restricted access to the region under the A ring due to the extended bulk of the biphenyl group.

Distribution of strand breaks produced by Auger electrons in decay of 125I in triplex DNA.

In this study we investigate the possibility of using Auger electrons as a probing agent for the study of structures of nucleic acids. To this end, we present the distribution of breaks produced in strands of a DNA duplex and a triplex-forming oligonucleotide (TFO) carrying Auger emitting radionuclide 125I. The method of calculation includes use of a molecular model of plasmid DNA duplex with bound TFO carrying a labelled 125I at position C5 of a single deoxycytosine residue, a source of Auger spectra, Monte Carlo electron track structure and the ensuing chemistry codes, to simulate the distribution of breaks produced in both strands of a plasmid DNA. Frequencies of fragment length distributions were obtained for the TFO, the purine and the pyrimidine strands. The frequency of breaks in the purine strand showed good correlation with the published experimental results, while that for the pyrimidine strand is lower by a factor of 3. It is concluded that the true structure of triplex DNA may not be purely of B-form.

Atomic force microscopy studies of intercalation-induced changes in plasmid DNA tertiary structure.

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide:base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic:toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.

Structural studies on bioactive compounds. Part 29: palladium catalysed arylations and alkynylations of sterically hindered immunomodulatory 2-amino-5-halo-4,6-(disubstituted)pyrimidines.

The immunological agent bropirimine 5 is a tetra-substituted pyrimidine with anticancer and interferon-inducing properties. Synthetic routes to novel 5-aryl analogues of bropirimine have been developed and their potential molecular recognition properties analysed by molecular modelling methods. Sterically challenged 2-amino-5-halo-6-phenylpyrimidin-4-ones (halo = Br or I) are poor substrates for palladium catalysed Suzuki cross-coupling reactions with benzeneboronic acid because the basic conditions of the reaction converts the amphoteric pyrimidinones to their unreactive enolic forms. Palladium-mediated reductive dehalogenation of the pyrimidinone substrates effectively competes with cross-coupling. 2-Amino-5-halo-4-methoxy-6-phenylpyrimidines can be converted to a range of 5-aryl derivatives with the 5-iodopyrimidines being the most efficient substrates. Hydrolysis of the 2-amino-5-aryl-4-methoxy-6-phenylpyrimidines affords the required pyrimidin-4-ones in high yields. Semi-empirical quantum mechanical calculations show how the nature of the 5-substituent influences the equilibrium between the 1H- and 3H-tautomeric forms, and the rotational freedom about the bond connecting the 6-phenyl group and the pyrimidine ring. Both of these factors may influence the biological properties of these compounds.