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The four-stranded i-motif (iM) conformation of cytosine-rich DNA is important in a wide variety of biochemical systems ranging from its use in nanomaterials to a potential role in oncogene regulation. An iM is stabilized by acidic pH that allows hemiprotonated cytidines to form a C·C(+) base pair. Fundamental studies that aim to understand how the lengths of loops connecting the protonated C·C(+) pairs affect intramolecular iM physical properties are described here. We characterized both the thermal stability and the pK(a) of intramolecular iMs with differing loop lengths, in both dilute solutions and solutions containing molecular crowding agents. Our results showed that intramolecular iMs with longer central loops form at pHs and temperatures higher than those of iMs with longer outer loops. Our studies also showed that increases in thermal stability of iMs when molecular crowding agents are present are dependent on the loop that is lengthened. However, the increase in pK(a) for iMs when molecular crowding agents are present is insensitive to loop length. Importantly, we also determined the proton activity of solutions containing high concentrations of molecular crowding agents to ascertain whether the increase in pK(a) of an iM is caused by alteration of this activity in buffered solutions. We determined that crowding agents alone increase the apparent pK(a) of a number of small molecules as well as iMs but that increases to iM pK(a) were greater than that expected from a shift in proton activity.

Fisetin (3,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) are the bioactive plant flavonoids that are potentially useful therapeutic drugs for the treatment of a broad spectrum of diseases, including atherosclerosis, cardiovascular disease, obesity, hypertension, and cancer. 3-Hydroxyflavone (3HF) and 7-hydroxyflavone (7HF) are the synthetic chromophores of fisetin and quercetin. We have exploited dual luminescence properties of fisetin and quercetin along with 3-HF and 7HF to examine their efficacy of binding and compare their interactions with DNA, which is one of the macromolecular targets of flavonoids in physiological systems. Following the sequence of the human telomeric DNA 5'-d (CCCTAA-)n/(-TTAGGG)n-5', two single-stranded DNA oligonucleotides, 5'-d(C3TA2)3C3-3' and 5'-d(T2AG3)4-3', and their duplex were used as receptors to study binding by the ligands quercetin, fisetin, and their chromophores. Circular dichroism, differential absorption, UV thermal melting, and size exclusion chromatographic studies indicated the formation of unusual DNA structures (such as C4 and G4 tetraplexes) for both the C- and G-rich single-stranded DNAs. Upon binding to DNA, dramatic changes were observed in the intrinsic fluorescence behavior of the flavonoids. Molecular docking studies were performed to describe the likely binding sites for the ligands. The spectroscopic studies on flavonoid-DNA interactions described herein demonstrate a powerful approach for examining their DNA binding through exploiting the highly sensitive intrinsic fluorescence properties of the flavonoids as their own "reporter" for their interactions with macromolecular targets.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

DNA sequences with the potential to form secondary structures such as i-motifs (iMs) and G-quadruplexes (G4s) are abundant in the promoters of several oncogenes and, in some instances, are known to regulate gene expression. Recently, iM-forming DNA strands have also been employed as functional units in nanodevices, ranging from drug delivery systems to nanocircuitry. To understand both the mechanism of gene regulation by iMs and how to use them more efficiently in nanotechnological applications, it is essential to have a thorough knowledge of factors that govern their conformational states and stabilities. Most of the prior work to characterize the conformational dynamics of iMs have been done with iM-forming synthetic constructs like tandem (CCT)n repeats and in standard dilute buffer systems. Here, we present a systematic study on the consequences of epigenetic modifications, molecular crowding, and degree of hydration on the stabilities of an iM-forming sequence from the promoter of the c-myc gene. Our results indicate that 5-hydroxymethylation of cytosines destabilized the iMs against thermal and pH-dependent melting; contrarily, 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions (PEG-300, 40% w/v), the thermal stability of iMs increased by ∼10 °C, and the pKa was raised from 6.1 ± 0.1 to 7.0 ± 0.1. Lastly, the iM's stability at varying degrees of hydration in 1,2-dimethoxyethane, 2-methoxyethanol, ethylene glycol, 1,3-propanediol, and glycerol cosolvents indicated that the iMs are stabilized by dehydration because of the release of water molecules when folded. Our results highlight the importance of considering the effects of epigenetic modifications, molecular crowding, and the degree of hydration on iM structural dynamics. For example, the incorporation of 5-methylycytosines and 5-hydroxymethlycytosines in iMs could be useful for fine-tuning the pH- or temperature-dependent folding/unfolding of an iM. Variations in the degree of hydration of iMs may also provide an additional control of the folded/unfolded state of iMs without having to change the pH of the surrounding matrix.

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member.

Background and purpose: Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11. Experimental approach: The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined. Key results: In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results. Conclusions and implications: These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent.

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.

Background: Carboxylesterases (CEs) are ubiquitous enzymes responsible for the hydrolysis of numerous clinically useful drugs. As ester moieties are frequently included in molecules to improve their water solubility and bioavailability, de facto they become substrates for CEs. Objective: In this review, we describe the properties of human CEs with regard to their ability to activate anticancer prodrugs and demonstrate how structure-based design can be used to modulate substrate specificity and to increase efficiency of hydrolysis. Methods: A specific example using CPT-11 and a human liver CE is discussed. However, these techniques can be applied to other enzymes and their associated prodrugs. Results: Structure-guided mutagenesis of CEs can be employed to alter substrate specificity and generate novel enzymes that are efficacious at anticancer prodrug activation.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs.

We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived K(i) values with the fluorobenzoins.

A number of N,N′-disubstituted perylenetetracarboxylic diimides have been reported to bind effectively to DNA that adopts G-quadruplex motifs. In some cases, this binding may actively drive the transition from single-strand DNA to the quadruplex form. The perylenediimides in the reported cases all have amine-containing side chains, which are thought to interact with the grooves of the quadruplex and help dictate the selectivity of these compounds for quadruplex versus duplex DNA. We synthesized a polyethyleneglycol-swallowtailed (PEG-tailed) perylenediimide that is water-soluble even though it is uncharged. Binding to duplex and quadruplex DNA of this perylenediimide was studied by fluorescence quenching titrations under a variety of salt conditions, and the compound’s effect on quadruplex formation was studied by non-denaturing gel electrophoresis. Our results indicate that while the molecule binds to single-stranded DNA quite effectively and with selectivity, it does not drive the transition of the DNA to the tetrameric quadruplex structure, supporting the idea that charge neutralization is a key component of perylene compounds that stabilize tetrameric quadruplexes.

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908-0.948), with cross-correlation coefficients (q2) of approximately 0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics. Many therapeutically useful drugs are metabolized by these proteins which impacts upon the efficiency of drug treatment. In some instances, CEs convert inactive prodrugs to active metabolites, a process that is essential for biological activity. Such compounds include the anticancer agents CPT-11 (3) and capecitabine (4), the antibiotics Ceftin (9) and Vantin, as well as the illicit street drug heroin (6). However, more commonly, CEs hydrolyze many esterified drugs to inactive products that are then excreted. Agents such as flestolol (11), meperidine (5), lidocaine (8) and cocaine (7), are all hydrolyzed and inactivated by these enzymes. Therefore the efficacy of esterified drugs will be dependent upon the distribution and catalytic activity of different CEs. In this review, we examine the structural aspects of CEs and their roles in drug detoxification and propose that modulation of CE activity may allow for improvements in, and potentiation of, drug efficacy.

Benzil has been identified as a potent selective inhibitor of carboxylesterases (CEs). Essential components of the molecule required for inhibitory activity include the dione moiety and the benzene rings, and substitution within the rings affords increased selectivity toward CEs from different species. Replacement of the benzene rings with heterocyclic substituents increased the K(i) values for the compounds toward three mammalian CEs when using o-nitrophenyl acetate as a substrate. Logarithmic plots of the K(i) values versus the empirical resonance energy, the heat of union of formation energy, or the aromatic stabilization energy determined from molecular orbital calculations for the ring structures yielded linear relationships that allowed prediction of the efficacy of the diones toward CE inhibition. Using these data, we predicted that 2,2'-naphthil would be an excellent inhibitor of mammalian CEs. This was demonstrated to be correct with a K(i) value of 1 nM being observed for a rabbit liver CE. In addition, molecular simulations of the movement of the ring structures around the dione dihedral indicated that the ability of the compounds to inhibit CEs was due, in part, to rotational constraints enforced by the dione moiety. Overall, these studies identify subdomains within the aromatic ethane-1,2-diones, that are responsible for CE inhibition.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2)0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-]carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.

Camptothecin (CPT) analogs that form more stable ternary complexes with DNA and topoisomerase I (termed cleavable complexes) show greater activity in their ability to inhibit tumor cell line growth in preclinical studies. Based on our earlier work, we hypothesized that analogs bearing hydrogen bonding moieties at the 7- through 10-position of CPT would result in more stable cleavable complexes. Consequently, we synthesized analogs with 7-mono-, 7-di-, and 7-trihydroxymethylaminomethyl groups. These analogs showed increasing cleavable complex stability as the number of hydroxyl groups was increased. The 7-trihydroxymethylaminomethyl analog of 10,11-methylenedioxycamptothecin (THMAM-MD) showed remarkable ternary complex stability with a half-life of 116 minutes. This is an order of magnitude more stable than any previously examined analog. Our in vitro analysis demonstrated that these analogs were all potent topoisomerase I poisons and could inhibit tumor cell growth in culture. We studied the effects of THMAM-MD in vivo in severe combined immunodeficient mice bearing HT-29 colon cancer and MiaPaCa-2 pancreatic cancer tumors. The THMAM-MD analog showed excellent, persisting activity in inhibiting tumor growth with both lines. Taken together, our results suggest that CPTs with hydrophilic, hydrogen-bonding groups at the 7-position hold the promise of excellent clinical activity.

Epigenetic modifications to DNA base sequences may regulate gene expression. CpG islands can contain methylated (5mC) or hydroxymethylated (5hmC) cytosine. Most CpG islands are found primarily in promoter regions that may also contain a high number of repeated cytosines and/or guanines. G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary structures that can form in repeating sequences of either guanine or cytosine, respectively. Both G4 and iM sequences may contain CpG sequences that can be methylated or hydroxymethylated. The effects of CpG islands on DNA secondary structures were determined by incorporating a single 5hmC at varying positions in the Vascular Endothelial Growth Factor (VEGF) G4 and iM sequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visible spectrometer were used to monitor the effect of 5hmC on G4 and iM thermal stability. Two of the three 5hmC-containing loops showed a notable decrease in stability for G4's and increased intermolecular structure formation. Contrastingly, the iM stability increased when 5hmC was incorporated into its sequence. Additionally, there was little change in the iM pka. In summary, our results suggest the 5hmC has little effect on iM structures, but can destabilize the G4's.

Due to the diversity of biological activities that can be found in aquatic ecosystems, marine metabolites have been an active area of drug discovery for the last 30 years. Marine metabolites have been found to inhibit a number of enzymes important in the treatment of human disease. Here, we focus on marine metabolites that inhibit the enzyme acetylcholinesterase, which is the cellular target for treatment of early-stage Alzheimer’s disease. Currently, development of anticholinesterase drugs with improved potency, and drugs that act as dual acetylcholinesterase and amyloid-β aggregation inhibitors, are being sought to treat Alzheimer’s disease. Seven classes of marine metabolites are reported to possess anti-cholinesterase activity. We compared these metabolites to clinically-used acetylcholinesterase inhibitors having known mechanisms of inhibition. We performed a docking simulation and compared them to published experimental data for each metabolite to determine the most likely mechanism of inhibition for each class of marine inhibitor. Our results indicate that several marine metabolites bind to regions of the acetylcholinesterase active site that are not bound by the clinically-used drugs rivastigmine, galanthamine, donepezil, or tacrine. We use the novel poses adopted for computational drug design of tighter binding anticholinesterase drugs likely to act as inhibitors of both acetylcholinesterase activity and amyloid-β aggregation inhibition.

The antitumor antibiotic 7-aminoactinomycin D has been previously demonstrated to bind and stabilize transient hairpin structures formed by single-stranded DNA. Those experiments suggested that DNA secondary structures are viable targets for development of novel antitumor or antiviral compounds. Interestingly, when 7-aminoactinomycin D binds to hairpins formed by selected single-stranded DNAs having a high affinity for the drug, the fluorescence quantum yield, average fluorescence lifetime, excitation wavelengths, and emission wavelengths for the drug are significantly different from those observed when it is free in solution or bound to duplex, B-form DNA. This suggests a unique physical chemical environment within certain DNA hairpins that is specifically recognized by the drug. To understand the environmental contribution to the fluorescence properties of the bound drug, we used solvatochromism techniques, whereby 7-aminoactinomycin D fluorescence properties were determined in 20 solvents with known po...

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin [CPT-11 (irinotecan)] is a water-soluble camptothecin-derived prodrug that is activated by esterases to yield the potent topoisomerase I poison SN-38. We identified a rabbit liver carboxylesterase (CE) that was very efficient at CPT-11 metabolism; however, a human homolog that was more than 81% identical to this protein activated the drug poorly. Recently, two other human CEs have been isolated that are efficient in the conversion of CPT-11 to SN-38, yet both demonstrate little homology to the rabbit protein. To understand this phenomenon, we have characterized a series of esterases from human and rabbit, including several chimeric proteins, for their ability to metabolize CPT-11. Computer predictive modeling indicated that the ability of each enzyme to activate CPT-11 was dependent on the size of the entrance to the active site. Kinetic studies with a series of nitrophenyl and naphthyl esters confirmed these predictions, indicating that activation of CPT-11 by a CE is constrained by size-limited access of the drug to the active site catalytic amino acid residues.

Our recent work has indicated that the potent antibiotic and antitumor agent actinomycin D has the ability to selectively bind and stabilize single-stranded DNA that is capable of adopting a hairpin conformation. This mechanism of DNA binding has been implicated in the drug's ability to inhibit transcription by HIV reverse transcriptase from single-stranded DNA templates. In this report, we studied the importance of the hairpin loop on the ability of the 7-amino analog of actinomycin D to selectively bind DNA hairpins. Binding dissociation constant (Kd) values were determined to be 0.22 +/- 0.11 microM for the hairpin formed from the single-stranded DNA 5'-AAAAAAATAGTTTTAAATATTTTTTT-3' (dubbed HP1). The hairpin stem without the loop resulted in binding with Kd = 2.6 +/- 0.9 microM. The drug showed low affinity for the HP1 strand fully duplexed to its complementary sequence (estimated to be at least Kd > 21 microM). Evaluation of 7-aminoactinomycin D binding to a library of thermodynamically characterized DNA hairpins revealed an affinity for the hairpin-forming sequence 5'-GGATACCCCCGTATCC-3' (dubbed ACC4) of Kd = 6.8 +/- 2.2 microM. Replacement of the terminal guanines of this sequence to generate 5'-ATATACCCCCGTATAT-3' resulted in a 10-fold increase in affinity for this hairpin compared to ACC4, to Kd = 0.74 +/- 0.06 microM. A molecular model of the ACC4actinomycin D complex reveals that significant interactions between the hairpin loop and the pentapeptide rings of the drug must occur during drug binding. Taken together, our data indicate that the composition of the stem-loop interface is critical for the selectivity of actinomycin D and its 7-amino analog for DNA hairpins and suggests that novel drugs may be designed based on selection for the desired hairpin composition.

DNA secondary structures containing regions of single-stranded DNA have now been identified in the genomic DNA of a number of prokaryotic and eukaryotic species, including humans. Many of these secondary structures are associated with regions of DNA involved in regulation of transcription: promoters or upstream elements. The secondary structures involved appear likely to be hairpin or cruciform structures that may be recognition sites for binding of transcription factors. In the case of the coliphage N4 virion RNA polymerase, a defined hairpin in the polymerase promoter necessary for binding of the polymerase and regulation of transcription has been shown to be extruded under physiological conditions in plasmid DNA. The presence of single-stranded DNA in the promoters of several species suggests that regulatory hairpins may be involved in transcription of a number of genes. In support of this, hairpin- or cruciform-binding proteins have been identified from several species. These results imply that secondary structures in regulatory regions may be targets for drugs that bind and either block or enhance binding of proteins involved in transcription. In this review, we discuss the evidence for DNA secondary structures, particularly hairpins and cruciforms, in genomic DNA and review the studies to date of development of small molecules that can selectively bind these structures.

A planar array immunosensor, equipped with a charge-coupled device (CCD) as a detector, was used to simultaneously detect 3 toxic analytes. Wells approximately 2 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Antibodies against staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis were covalently attached to the bottoms of the circular wells to form the sensing surface. Rectangular wells containing chicken immunoglobulin were used as alignment markers and to generate control signals. After removing the optical adhesive, the slides were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction lens array was used to focus the sensing surface onto the CCD. Solutions of toxins were then placed on the slide. After rinsing, Cy5-labeled antibodies were introduced. The identity and amount of toxin bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 25 ng/mL of ricin, 15 ng/mL of pestis F1 antigen, and 5 ng/mL of SEB could be routinely measured.

G-quadruplex- and i-motif-based DNA oligomers are being investigated for their integration into therapeutic and diagnostic micro-assemblies. Examples include quadruplex-forming aptamers as potential anti-HIV agents, and serum-stable quadruplexes as carriers for delivering porphyrins into cancer cells for photodynamic therapy. The i-motifs from C-rich DNA find application in pH-triggered hydrogels that can carry agents like drugs, proteins, and polymers to their targets. The pH-dependent conformational dynamics of i-motifs also make them useful as biosensors for detecting pH changes in cellular microenvironments. Due to these and many other applications, and in an effort to present a compendia of recent uses of quadruplexes and i-motifs, this chapter will concern itself with formation of G-quadruplexes and i-motifs; their physical and chemical properties; the effects of molecular crowding and hydration on their structure and stability; and their application in therapeutics as drug targets, drug-delivery vehicles, and diagnostic tools.

The sequence specificity and structural aspects of the mode of interaction of the antitumor drug actinomycin D (AMD) with single-stranded DNA were studied by fluorescence, absorption and NMR spectroscopy, calorimetry, ultrasonic velocity and density measurements, and molecular modeling. The binding is length and sequence dependent, with the tetranucleotide motif TAGT showing the highest affinity. A “hemi-intercalation” model for the interaction is proposed.

We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by fluorescence and NMR spectroscopy and by molecular modeling. The significant absorption and emission changes accompanying the interaction of the fluorescent derivative 7-amino-AMD with DNAs varying in length and base composition were used to calculate affinity constants for the drug-DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted favorable binding sites. In contrast, the sequence TGA did not bind the drug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TAGT. The binding was length dependent, an oligonucleotide of at least 14 bases being required for effective complex formation (Ka > 10(4) M1=). AMD also bound to poly(d(AGT)). Gel electrophoresis confirmed that the complex was formed between the drug and individual unstructured DNA strands. The 1H NMR spectra of oligonucleotides containing the TAGT site and their complexes with AMD provided further insight into the mode(s) of interaction. A comparison of the measured chemical shifts with those estimated from ring-current calculations provided strong evidence for a hemi-intercalation of AMD between the A and G purine bases with a preference for one of two possible relative orientations. The latter were modeled as complexes with the sequence T3AGT3 and refined by force field calculations with the AMBER program. The biological implications for this novel form of interaction of AMD with single-stranded DNA are discussed.

Topological variants of single-strand DNA (ssDNA) structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC) and 1,3-diaza-2-oxophenoxazine (tC∘), can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA) to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD) spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

Of the compounds that have now been shown to circumvent acquired cellular multidrug resistance, little or no structure-activity relationship has been found, although their proposed mechanism of action is through modulation of function of p-glycoprotein. While it has been suggested that this inhibition is a direct binding to p-glycoprotein, we show here that such a model seriously neglects the effects many of these compounds have on lipid physical properties. We have characterized the interactions between 16 structurally diverse pharmacological agents (nine of which are known to reverse multidrug resistance) and a variety of lipids. Potent modulators inhibit the membrane binding of rhodamine 6G, and we have observed a correlation of the measured K i values with the effectiveness of the compounds in situ. We have determined the effects of the compounds on detergent micellization, and have shown substantial changes on the critical micelle concentration of detergents in the presence of modulators. Finally, we have examined the changes in model membrane ‘viscosity’ induced by the compounds. These results indicate that both direct p-glycoprotein and indirect lipid interactions of modulators should be considered in the mechanism by which these compounds reverse multidrug resistance.

We present here the initial characterization of the mechanism of reversal of cellular resistance to Vinca alkaloids by phenoxazine (PZ). Changes in fluorescence upon cellular accumulation of PZ allowed measurement of the membrane transport kinetics in a sensitive KB-3-1 cell line and two multi-drug resistant (MDR) counterparts. The accumulation of PZ is characterized by two uptake routes, with pseudo-first order rate constants of 0.3 s−1 and 0.07 s−1, while efflux of PZ from cells revealed rate constants of 0.2 s−1. PZ rapidly reaches steady-state concentrations within cells, which may make it more clinically useful than modulators that accumulate more slowly (e.g. verapamil).

DNA secondary structures containing regions of single-stranded DNA have now been identified in the genomic DNA of a number of prokaryotic and eukaryotic species, including humans. Many of these secondary structures are associated with regions of DNA involved in regulation of transcription: promoters or upstream elements. The secondary structures involved appear likely to be hairpin or cruciform structures that may be recognition sites for binding of transcription factors. In the case of the coliphage N4 virion RNA polymerase, a defined hairpin in the polymerase promoter necessary for binding of the polymerase and regulation of transcription has been shown to be extruded under physiological conditions in plasmid DNA. The presence of single-stranded DNA in the promoters of several species suggests that regulatory hairpins may be involved in transcription of a number of genes. In support of this, hairpin- or cruciform-binding proteins have been identified from several species. These results imply that secondary structures in regulatory regions may be targets for drugs that bind and either block or enhance binding of proteins involved in transcription. In this review, we discuss the evidence for DNA secondary structures, particularly hairpins and cruciforms, in genomic DNA and review the studies to date of development of small molecules that can selectively bind these structures.

CPT-11 is an antitumor prodrug that is hydrolyzed by carboxylesterases (CE) to yield SN-38, a potent topoisomerase I poison. However, the dose limiting toxicity is delayed diarrhea that is thought to arise, in part, from activation of the prodrug by a human intestinal CE (hiCE). Therefore, we have sought to identify selective inhibitors of hiCE that may have utility in modulating drug toxicity. We have evaluated one such class of molecules (benzene sulfonamides), and developed QSAR models for inhibition of this protein. Using these predictive models, we have synthesized a panel of fluorene analogues that are selective for hiCE, demonstrating no cross reactivity to the human liver CE, hCE1, or towards human cholinesterases, and have Ki values as low as 14nM. These compounds prevented hiCE-mediated hydrolysis of the drug and the potency of enzyme inhibition correlated with the clogP of the molecules. These studies will allow the development and application of hiCE-specific inhibitors designed to selectively modulate drug hydrolysis in vivo.

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.

Carboxylesterases are enzymes that hydrolyze a broad suite of endogenous and exogenous ester-containing compounds to the corresponding alcohol and carboxylic acid. These enzymes metabolize a number of therapeutics including the anti-tumor agent CPT-11, the anti-viral drug oseltamivir, and the anti-thrombogenic agent clopidogrel as well as many agrochemicals. In addition, carboxylesterases are involved in lipid homeostasis, including cholesterol metabolism and transport with a proposed role in the development of atherosclerosis. Several different scaffolds capable of inhibiting carboxylesterases have been reported, including organophosphates, carbamates, trifluoromethyl ketone-containing structures (TFKs), and aromatic ethane-1,2-diones. Of these varied groups, only the 1,2-diones evidence carboxylesterase isoform-selectivity, which is an important characteristic for therapeutic application and probing biological mechanisms. This study constructed a series of classical and 3D-QSAR models to examine the physiochemical parameters involved in the observed selectivity of three mammalian carboxylesterases: human intestinal carboxylesterase (hiCE), human carboxylesterase 1 (hCE1), and rabbit carboxylesterase (rCE). CoMFA-based models for the benzil-analogs described 88%, 95% and 76% of observed activity for hiCE, hCE1 and rCE, respectively. For TFK-containing compounds, two distinct models were constructed using either the ketone or gem-diol form of the inhibitor. For all three enzymes, the CoMFA ketone models comprised more biological activity than the corresponding gem-diol models; however the differences were small with described activity for all models ranging from 85–98%. A comprehensive model incorporating both benzil and TFK structures described 92%, 85% and 87% of observed activity for hiCE, hCE1 and rCE, respectively. Both classical and 3D-QSAR analysis showed that the observed isoform-selectivity with the benzil-analogs could be described by the volume parameter. This finding was successfully applied to examine substrate selectivity, demonstrating that the relative volumes of the alcohol and acid moieties of ester-containing substrates were predictive for whether hydrolysis was preferred by hiCE or hCE1. Based upon the integrated benzil and TFK model, the next generation inhibitors should combine the A-ring and the 1,2-dione of the benzil inhibitor with the long alkyl chain of the TFK-inhibitor in order to optimize selectivity and potency. These new inhibitors could be useful for elucidating the role of carboxylesterase activity in fatty acid homeostasis and the development of atherosclerosis as well as effecting the controlled activation of carboxylesterase-based prodrugs in situ.

Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values5 routinely yielded Ki values in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo.

CpG islands are prone to epigenetic cytosine modification via methylation (5mC) or 5-hydroxymethylation (5hmC). Although their potential sequences and regions for occurrence in G/C-rich DNA overlap, throughout the genome 5mC and 5hmC, and 5mC and G-quadruplex (G4) and i-motif (iM) structures, are found to not be co-existent. Moreover, in some regions of DNA, 5hmCs and G4/iM forming regions have high potential to form in concurrence. However, minimal research has been done examining the effect of 5hmC incorporation into G4 and iMs in order to determine the effect of cytosine modification on non-B-DNA structures. These higher order secondary DNA formations have been described within the VEGF promoter and have great potential to silence its transcription and prevent subsequent angiogenesis. In addition, there are three cytosines in the promoter on both the guanine- and the cytosine-rich strands found in CpG sequences that have the potential for 5mhC modification. Therefore, the current study was undertaken to examine the whether there is an effect of 5hmC incorporation into G4 loops and iM stems on structure formation and stabilization. Significant changes in structure or stability in buffers with varying pH’s, cationic strength, and molecular crowding conditions were examined by circular dichroism and confirmed by UV-monitored melting. Electromobility shift assays differentiated inter- and intra-molecular structures, further identifying the number of isoforms. Notable changes in G4 dynamics were seen with two of the three loop modifications, which decreased overall stability and increased intermolecular structures. Much less marked effects were discovered for the iM structures. Ongoing studies examining the role of G4 and iM 5hmC incorporation in regulation by endogenous proteins are part of a global effort to best understand the physiological conditions regulating G4 and iM stability and function. These VEGF promoter higher order structures are promising therapeutic targets for anti-angiogenic therapy, and a comprehensive understanding of the physiological principles governing their formation will best inform future drug discovery efforts. Brooks Lab startup funds, R15, University of Mississippi School of Pharmacy Citation Format: Rhianna Morgan, Randy Wadkins, Tracy Brooks. Effects of 5-hydroxymethylcytosine epigenetic modification on G-quadruplex and i-motif structure and stability within the VEGF promoter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1050. doi:10.1158/1538-7445.AM2015-1050

The anticancer prodrug CPT-11 is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. The 2.6 angstroms resolution crystal structure of its complex with Torpedo californica acetylcholinesterase (TcAChE) demonstrates that CPT-11 binds to TcAChE and spans its gorge similarly to the Alzheimer drug, Aricept. The crystal structure clearly reveals the interactions, which contribute to the inhibitory action of CPT-11. Modeling of the complexes of CPT-11 with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug, shows how binding to either of the two enzymes yields a productive substrate-enzyme complex.

CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is an anticancer prodrug that has been approved for the treatment of colon cancer. It is a member of the camptothecin class of drugs and activation to the active metabolite SN-38, is mediated by carboxylesterases (CE). SN-38 is a potent topoisomerase I poison and is highly effective at killing human tumor cells, with IC50 values in the low nM range. However, upon high dose administration of CPT-11 to cancer patients, a cholinergic syndrome is observed, that can be rapidly ameliorated by atropine. This suggests a direct interaction of the drug or its metabolites with acetylcholinesterase (AChE). Kinetic studies indicated that CPT-11 was primarily responsible for AChE inhibition with the 4-piperidinopiperidine moiety, the major determinant in the loss of enzyme activity. Structural analogs of 4-piperidinopiperidine however, did not inhibit AChE, including a benzyl piperazine derivate of CPT-11. These results suggest that novel anticancer drugs could be synthesized that do not inhibit AChE, or alternatively, that novel AChE inhibitors could be designed based around the camptothecin scaffold.

Early detection is critical in the administration of definitive and curative therapy of cancer. However, current detection methods are ineffective at identifying the presence of circulating metastatic cancer cells in the blood because they typically sample only a relatively small volume of blood. One strategy for sampling larger blood volumes would be to capture circulating cells in vivo over an extended period of time. The development of such a method would be substantially facilitated by the identification of peptide ligands that bind selectively to metastatic cancer cells in the blood with high affinity. To identify such ligands a combinatorial peptide library was synthesized on polyethylene acrylamide (PEGA) resin and screened for binding to malignant epithelial cells. Using Biacore, cell binding assays were performed to demonstrate that peptides selected from PEGA bead screen can bind selectively to malignant epithelial cancer cells and not to circulating leukocytes under physiologic shear stress conditions. One peptide, with the sequence QMARIPKRLARH, was used to demonstrate selective labeling of malignant epithelial cells spiked in whole blood. When immobilized on appropriate surfaces, these peptides could be used in both in vivo and ex vivo cell separation devices to efficiently and selectively capture metastatic epithelial cancer cells from flowing blood.

The purpose of this proposal was to investigate the response of prostate cancer to a series of camptothecin analogs, which are drugs that target DNA topoisomerase I. This final report summarizes the accomplishments toward the tasks outlined in the original proposal for this grant. The goal of the project was to try to understand why, despite the availability of topoisomerase I in prostate cancer, chemotherapeutic agents that target this enzyme are ineffective in treating prostate cancer. Members of the camptothecin family that specifically target topo I were studied in prostate cancer cell lines grown in vitro. The project was meant to attack this problem from three separate directions using three assays. The first assay was that of initial topo I distribution in normal and cancer cells and its response to camptothecin analog treatment. The second assay was for selection of camptothecin analogs that form slowly reversing topo I-DNA complexes. The third was measurement of the onset of apoptosis in prostate cells treated with the different analogs. This final report documents the work performed throughout the lifetime of the funded project. The authors note that while some experimental problems were encountered that limited the completion of all tasks, good progress was made overall on the proposed research.

The mechanism of cytotoxicity of the camptothecin family of antitumor drugs is thought to be the consequence of a collision between moving replication forks and camptothecin-stabilized cleavable DNA-topoisomerase I complexes. One property of camptothecin analogs relevant to their potent antitumor activity is the slow reversal of the cleavable complexes formed with these drugs. The persistence of cleavable complexes with time may be an essential property for increasing the likelihood of a collision between the replication fork and a cleavable complex, giving rise to lethal DNA lesions. In this paper, we examined a number of camptothecin analogs forming cleavable complexes with distinctly different stabilities. Absolute reaction rate analysis was carried out for each derivative. Our results indicate that the stability of the cleavable complex is dominated by the activation entropy (DeltaS++) of the reversal process. We measured the relative lipophilicity of the CPT analogs by reverse-phase HPLC, but the DeltaS++ of complex reversal is not directly related to the lipophilicity of the CPT analog being used. We suggest that solvent ordering around the 7- through 10-position of the CPT ring may be responsible for reversal rate's dependence on DeltaS++. We demonstrate that the cleavable complex stability conferred by each camptothecin analog is directly correlated with the induction of apoptosis and cytotoxicity to tumor cells.

The dose-limiting toxicity of the highly effective anticancer agent 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (irinotecan; CPT-11) is delayed diarrhea. This is thought to be caused by either bacteria-mediated hydrolysis of the glucuronide conjugate of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) or direct conversion of CPT-11 to SN-38 by carboxylesterases (CE) in the small intestine. After drug administration, a very high level of CPT-11 is present in the bile; this is deposited into the duodenum, the region of the gut with the highest levels of CE activity. Hence, it is likely that direct conversion of the drug to SN-38 is partially responsible for the diarrhea associated with this agent. In an attempt to ameliorate this toxicity, we have applied Target-Related Affinity Profiling to identify novel CE inhibitors that are selective inhibitors of the human intestinal enzyme (hiCE). Seven inhibitors, all sulfonamide derivatives, demonstrated greater than 200-fold selectivity for hiCE compared with the human liver CE hCE1, and none was an inhibitor of human acetylcholinesterase or butyrylcholinesterase. Quantitative structure-activity relationship (QSAR) analysis demonstrated excellent correlations with the predicted versus experimental Ki values (r2 = 0.944) for hiCE. Additionally, design and synthesis of a tetrafluorine-substituted sulfonamide analog, which QSAR indicated would demonstrate improved inhibition of hiCE, validated the computer predictive analyses. These and other phenyl-substituted sulfonamides compounds are regarded as lead compounds for the development of effective, selective CE inhibitors for clinical applications.