Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex.

Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 µM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M-1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.

Effects of Doxorubicin on the Liquid-Liquid Phase Change Properties of Elastin-Like Polypeptides.

The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent ß-turn structures. Labeling with Dox further enhances ß-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Dox-labeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a -11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets.

Berenil Binds Tightly to Parallel and Mixed Parallel/Antiparallel G-Quadruplex Motifs with Varied Thermodynamic Signatures.

Diminazene, DMZ, (or berenil) has been reported as a tight binder of G-quadruplexes. G-Quadruplex structures are often located in the promotor regions of oncogenes and may play a regulatory role in gene expression based on the stability of the folding topology. In this study, attempts have been made to characterize the specificity of DMZ binding toward multiple G-quadruplex topologies or foldamers. Mutant sequences of the G-quadruplex forming promotor regions of several oncogenes were designed to exhibit restricted loop lengths and folding topologies. Circular dichroism was used to confirm the quadruplex topology of mutant BCL2, KRAS, and c-MYC sequences, human telomere (Na+ and K+) G-quadruplexes and their complexes with DMZ and analogs thereof. Isothermal titration calorimetry was used to generate a complete thermodynamic profile (ΔG, ΔH, -TΔS) for the formation of DMZ and analog complexes with the target G-quadruplexes. DMZ binds to parallel and/or mixed parallel/antiparallel quadruplex DNA motifs with stoichiometries up to 8:1 and via three binding modes with varying affinities. In the case of the parallel G-quadruplexes, with the exception of the long-looped c-MYC mutant, the highest affinity binding event (mode 1) is driven by enthalpy. DMZ binding to the long-looped c-MYC mutant exhibits a very favorable entropy change in addition to a moderately favorable enthalpy change. Mode 1 binding to the antiparallel and mixed parallel/antiparallel hTel quadruplexes is also driven by favorable enthalpy changes. In all cases, the intermediate DMZ affinity binding (mode 2) is driven almost entirely by entropy, with small or unfavorable enthalpic contributions. The weakest binding event (mode 3) is also entropically driven with small or moderate enthalpic contributions.

Alkyne-substituted diminazene as G-quadruplex binders with anticancer activities.

G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes, with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities. In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231) cancer cell lines and represent interesting new leads to develop anticancer agents.

Thermodynamic investigations of [(phen)2Ru(tatpp)Ru(phen)2](4+) interactions with B-DNA.

While the antitumor activity of P(4+) is relatively well understood, the binding mechanism and thermodynamics for formation of (P(4+)·DNA) complexes remain in question. The thermodynamic parameters (Ka, ΔG, ΔH, and -TΔS) for formation of DNA complexes of the ruthenium dimer, [(phen)2Ru(tatpp)Ru(phen)2](4+) (abbreviated as P(4+)), where phen is 1,10-phenanthroline and tatpp is 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3″,2″-1:2‴,3‴-n]-pentacene, were determined using isothermal titration calorimetry. Calorimetric and spectroscopic titration experiments were performed in which P(4+) was added to three duplex DNAs of different lengths. We determined that P(4+) binds to duplex DNA at 298 K with modest affinity (Ka ≈ 3.8 × 10(5) M(-1), ΔG ≈ -7.6 kcal/mol), that the enthalpy change is unfavorable (ΔH ≈ +2.1 kcal/mol), and that complex formation is driven by a large favorable change in entropy (-TΔS ≈ -9.7 kcal/mol). These thermodynamic values were found to be approximately independent of the length of the DNA, and the stoichiometry of the (P(4+)·DNA) complexes was determined to be 1 P(4+)/2 DNA bp, at least for the two shorter DNAs. On the basis of the thermodynamic parameters, and the binding stoichiometry (verified in ESI-MS experiments), we conclude that P(4+) is intercalating between two adjacent DNA base pairs and that the neighbor sites on either side of the bound ligand are excluded from binding additional P(4+).

Diminazene or berenil, a classic duplex minor groove binder, binds to G-quadruplexes with low nanomolar dissociation constants and the amidine groups are also critical for G-quadruplex binding.

G-quadruplexes have shown great promise as chemotherapeutic targets, probably by inhibiting telomere elongation or downregulating oncogene expression. There have been many G-quadruplex ligands developed over the years but only a few have drug-like properties. Consequently only a few G-quadruplex ligands have entered clinical trials as cancer chemotherapeutic agents. The DNA minor groove ligand, berenil (diminazene aceturate or DMZ), is used to treat animal trypanosomiasis and hence its toxicological profile is already known, making it an ideal platform to engineer into new therapeutics. Herein, using a plethora of biophysical methods including UV, NMR, MS and ITC, we show that DMZ binds to several G-quadruplexes with a Kd of ∼1 nM. This is one of the strongest G-quadruplex binding affinities reported to date and is 10(3) tighter than the berenil affinity for an AT-rich duplex DNA. Structure-activity-relationship studies demonstrate that the two amidine groups on DMZ are important for binding to both G-quadruplex and duplex DNA. This work reveals that DMZ or berenil is not as selective for AT-rich duplexes as originally thought and that some of its biological effects could be manifested through G-quadruplex binding. The DMZ scaffold represents a good starting point to develop new G-quadruplex ligands for cancer cell targeting.

Effect of basic cell-penetrating peptides on the structural, thermodynamic, and hydrodynamic properties of a novel drug delivery vector, ELP[V5G3A2-150].

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II ß-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

The effect of molecular crowding on the stability of human c-MYC promoter sequence I-motif at neutral pH.

We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5-5.5). In terms of drug targeting, the question is whether c-MYC promoter sequence i-motifs will exist in the nucleus at neutral pH. In this work, we have investigated the stability of a mutant c-MYC i-motif in solutions containing a molecular crowding agent. The crowded nuclear environment was modeled by the addition of up to 40% w/w polyethylene glycols having molecular weights up to 12,000 g/mol. CD and DSC were used to establish the presence and stability of c-MYC i-motifs in buffer solutions over the pH range 4 to 7. We have shown that the c-MYC i-motif can exist as a stable structure at pH values as high as 6.7 in crowded solutions. Generic dielectric constant effects, e.g., a shift in the pKa of cytosine by more than 2 units (e.g., 4.8 to 7.0), or the formation of non-specific PEG/DNA complexes appear to contribute insignificantly to i-motif stabilization. Molecular crowding, largely an excluded volume effect of added PEG, having a molecular weight in excess of 1,000 g/mol, appears to be responsible for stabilizing the more compact i-motif over the random coil at higher pH values.

Bcl-2 promoter sequence G-quadruplex interactions with three planar and non-planar cationic porphyrins: TMPyP4, TMPyP3, and TMPyP2.

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)1/DNA complex (K1 = 3.7 × 10(6)) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)1/DNA complex, (K1 = 7.0 × 10(4)). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)3/DNA and (TMPyP4)4/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.

Structural and hydrodynamic analysis of a novel drug delivery vector: ELP[V5G3A2-150].

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II ß-turn structure, forming a ß-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous ß structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 µM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

DSC deconvolution of the structural complexity of c-MYC P1 promoter G-quadruplexes.

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P(1) promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K(+)] and 130 mM [K(+)]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).

Effect of Ionic Strength on Porphyrin Drugs Interaction with Quadruplex DNA Formed by the Promoter Region of C-myc and Bcl2 Oncogenes.

C-myc and Bcl2 are well characterized oncogenes that are capable of forming G-quadruplex structures. Promoter regions of C-myc and Bcl2 forming G-quadruplex structures are chemically synthesized and G-quadruplex structure is formed in presence of 100 mM potassium ion. Three different porphyrin drugs, namely TMPyP2, TMPyP3, and TMPyP4 are allowed to interact with quadruplex DNA complex and the site and nature of interaction are studied. Drug interactions with quadruplex DNA were carried out in different potassium ionic strengths using fluorescence spectroscopy. It is found that fluorescence hypochromicity decreases with an increase in ionic strength in the case of TMPyP4, TMPyP3, and TMPyP2. Fluorescence titration studies and Job plots indicate that four molecules of TMPyP4, two molecules of TMPyP3 and TMPyP2 are interacting with one molecule of quadruplex DNA.

Biophysical characterization of an ensemble of intramolecular i-motifs formed by the human c-MYC NHE III1 P1 promoter mutant sequence.

i-Motif-forming sequences are present in or near the regulatory regions of >40% of all genes, including known oncogenes. We report here the results of a biophysical characterization and computational study of an ensemble of intramolecular i-motifs that model the polypyrimidine sequence in the human c-MYC P1 promoter. Circular dichroism results demonstrate that the mutant sequence (5'-CTT TCC TAC CCTCCC TAC CCT AA-3') can adopt multiple "i-motif-like," classical i-motif, and single-stranded structures as a function of pH. The classical i-motif structures are predominant in the pH range 4.2-5.2. The "i-motif-like" and single-stranded structures are the most significant species in solution at pH higher and lower, respectively, than that range. Differential scanning calorimetry results demonstrate an equilibrium mixture of at least three i-motif folded conformations with Tm values of 38.1, 46.6, and 49.5 degrees C at pH 5.0. The proposed ensemble of three folded conformations includes the three lowest-energy conformations obtained by computational modeling and two folded conformers that were proposed in a previous NMR study. The NMR study did not report the most stable conformer found in this study.

Studies on the site and mode of TMPyP4 interactions with Bcl-2 promoter sequence G-Quadruplexes.

TMPyP4 (Mesotetra(N-methyl-4-pyridyl)porphine) is known to have a high affinity for G-quadruplex DNA. However, there is still some controversy over the exact site(s) and mode(s) of TMPyP4 binding to G-quadruplex DNA. We examined TMPyP4 interactions with seven G-quadruplex forming oligonucleotides. The parent oligonucleotide is a 27-mer with a wild-type (WT) G-rich sequence of the Bcl-2 P1 promoter mid-region (5'-d(CGG GCG CGG GAG GAA GGG GGC GGG AGC-3')). This sequence folds into at least three unique loop isomer quadruplexes. The two mutant oligonucleotides used in this study are shorter (23-mer) sequences in which nonquadruplex core bases were eliminated and two different (-G-G-) --> (-T-T-) substitutions were made to restrict the folding complexity. The four additional mutant oligonucleotides were labeled by substituting a 2-aminopurine (2-AP) base for an A or G in either the first three-base lateral loop or the second five- or seven-base lateral loop (depending on the G-->T mutation positions). Spectroscopic and microcalorimetric studies indicate that four molecules of TMPyP4 can be bound to a single G-quadruplex. Binding of the first two moles of TMPyP4 appears to occur by an end or exterior mode (K approximately 1 x 10(7) M(-1)), whereas binding of the third and fourth moles of TMPyP4 appears to occur by a weaker, intercalative binding mode (K approximately 1 x 10(5) M(-1)). As the mid-loop size decreases from seven to five bases, end binding occurs with significantly increased affinity. 2-AP-labeled Bcl-2 promoter region quadruplexes show increased fluorescence of the 2-AP base on addition of TMPyP4. The change in fluorescence for 2-AP bases in the second half of the TMPyP4 titration lends support to our previous speculation regarding the intercalative nature of the weaker binding mode.

Molecular modeling and biophysical analysis of the c-MYC NHE-III1 silencer element.

G-Quadruplex and i-Motif-forming sequences in the promoter regions of several oncogenes show promise as targets for the regulation of oncogenes. In this study, molecular models were created for the c-MYC NHE-III(1) (nuclease hypersensitivity element III(1)) from two 39-base complementary sequences. The NHE modeled here consists of single folded conformers of the polypurine intramolecular G-Quadruplex and the polypyrimidine intramolecular i-Motif structures, flanked by short duplex DNA sequences. The G-Quadruplex was based on published NMR structural data for the c-MYC 1:2:1 loop isomer. The i-Motif structure is theoretical (with five cytosine-cytosine pairs), where the central intercalated cytosine core interactions are based on NMR structural data obtained for a tetramolecular [d(A(2)C(4))(4)] model i-Motif. The loop structures are in silico predictions of the c-MYC i-motif loops. The porphyrin meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4), as well as the ortho and meta analogs TMPyP2 and TMPyP3, were docked to six different locations in the complete c-MYC NHE. Comparisons are made for drug binding to the NHE and the isolated G-Quadruplex and i-Motif structures. NHE models both with and without bound cationic porphyrin were simulated for 100 ps using molecular dynamics techniques, and the non-bonded interaction energies between the DNA and porphyrins calculated for all of the docking interactions.

Biophysical studies of the c-MYC NHE III1 promoter: model quadruplex interactions with a cationic porphyrin.

Regulation of the structural equilibrium of G-quadruplex-forming sequences located in the promoter regions of oncogenes by the binding of small molecules has shown potential as a new avenue for cancer chemotherapy. In this study, microcalorimetry (isothermal titration calorimetry and differential scanning calorimetry), electronic spectroscopy (ultraviolet-visible and circular dichroism), and molecular modeling were used to probe the complex interactions between a cationic porphryin mesotetra (N-methyl-4-pyridyl) porphine (TMPyP4) and the c-MYC PU 27-mer quadruplex. The stoichiometry at saturation is 4:1 mol of TMPyP4/c-MYC PU 27-mer G-quadruplex as determined by isothermal titration calorimetry, circular dichroism, and ultraviolet-visible spectroscopy. The four independent TMPyP4 binding sites fall into one of two modes. The two binding modes are different with respect to affinity, enthalpy change, and entropy change for formation of the 1:1 and 2:1, or 3:1 and 4:1 complexes. Binding of TMPyP4, at or near physiologic ionic strength ([K(+)] = 0.13 M), is described by a "two-independent-sites model." The two highest-affinity sites exhibit a K(1) of 1.6 x 10(7) M(-1) and the two lowest-affinity sites exhibit a K(2) of 4.2 x 10(5) M(-1). Dissection of the free-energy change into the enthalpy- and entropy-change contributions for the two modes is consistent with both "intercalative" and "exterior" binding mechanisms. An additional complexity is that there may be as many as six possible conformational quadruplex isomers based on the sequence. Differential scanning calorimetry experiments demonstrated two distinct melting events (T(m)1 = 74.7 degrees C and T(m)2 = 91.2 degrees C) resulting from a mixture of at least two conformers for the c-MYC PU 27-mer in solution.