Design and synthesis of strong root gravitropism inhibitors with no concomitant growth inhibition.

Herein, we describe a highly potent gravitropic bending inhibitor with no concomitant growth inhibition. Previously, we reported that (2Z,4E)-5-phenylpenta-2,4-dienoic acid (ku-76) selectively inhibits root gravitropic bending of lettuce radicles at 5 µM. Based on the structure-activity relationship study of ku-76 as a lead compound, we designed and synthesized various C4-substituted analogs of ku-76. Among the analogs, 4-phenylethynyl analog exhibited the highest potency for gravitropic bending inhibition, which was effective at only 0.01 µM. Remarkably, 4-phenylethynyl analog is much more potent than the known inhibitor, NPA. Substitution in the para position on the aromatic ring of 4-phenylethynyl group was tolerated without diminished activity. In addition, evaluation using Arabidopsis indicated that 4-phenylethynyl analog inhibits gravitropism by affecting auxin distribution in the root tips. Based on the effects on Arabidopsis phenotypes, 4-phenylethynyl analog may be a novel inhibitor that differs in action from the previously reported auxin transport inhibitors.

Design and chemical synthesis of root gravitropism inhibitors: Bridged analogues of ku-76 have more potent activity.

Previously, we found (2Z,4E)-5-phenylpenta-2,4-dienoic acid (ku-76) to be a selective inhibitor of root gravitropic bending of lettuce radicles at 5 µM, with no concomitant growth inhibition, and revealed the structure-activity relationship in this inhibitory activity. The conformation of ku-76 is flexible owing to the open-chain structure of pentan-2,4-dienoic acid with freely rotating single bonds, and the (2Z)-alkene moiety may be isomerized by external factors. To develop more potent inhibitors and obtain insight into the target biomolecules, various analogues of ku-76, fixed through conformation and/or configuration, were synthesized and evaluated. Stereochemical fixation was effective in improving the potency of gravitropic bending inhibition. Finally, we found highly potent conformational and/or configurational analogues (ku-257, ku-294 and ku-308), that did not inhibit root growth. The inhibition of root curvature by these analogues was comparable to that of naptalam.

Essential structural features of (2Z,4E)-5-phenylpenta-2,4-dienoic acid for inhibition of root gravitropism.

Previously, we found (2Z,4E)-5-phenylpenta-2,4-dienoic acid (ku-76) to be a selective inhibitor of root gravitropic bending of lettuce radicles at 5 µM, with no concomitant growth inhibition. Here, we describe a structure-activity relationship study of ku-76 to determine the essential structural features for the inhibitory activity. A series of ku-76 analogues was synthesized and the key features of ku-76 that are necessary for inhibition of lettuce root gravitropic bending were determined. The (2E,4E)-, (2Z,4Z)- (2E,4Z)- analogues were inactive, and 4,5-saturated and 4,5-alkynyl analogues also did not show inhibitory activity, demonstrating the importance of the (2Z,4E) diene unit. The aromatic ring was also crucial and could not be replaced with an alkyl chain. Derivatives in which the carboxylic acid was replaced with amides, alcohols, or esters were much less potent. These results suggest that the (2Z,4E)-diene, the carboxylic acid moiety, and the aromatic ring are essential for potent inhibitory activity against gravitropic bending.

Bongkrekic acid facilitates glycolysis in cultured cells and induces cell death under low glucose conditions.

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O2 consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.

Efficient Total Synthesis of Bongkrekic Acid and Apoptosis Inhibitory Activity of Its Analogues.

Bongkrekic acid (BKA), isolated from the bacterium Burkholderia cocovenenans, is an inhibitor of adenine nucleotide translocator, which inhibits apoptosis, and is thus an important tool for the mechanistic investigation of apoptosis. An efficient total synthesis of BKA has been achieved by employing a three-component convergent strategy based on Kocienski-Julia olefination and Suzuki-Miyaura coupling. It is noteworthy that segment B has been prepared as a new doubly functionalized coupling partner, which contributes to shortening of the number of steps. Torquoselective olefination with an ynolate has also been applied for the efficient construction of an unsaturated ester. Furthermore, it is revealed that 1-methyl-2-azaadamantane N-oxyl is an excellent reagent for final oxidation to afford BKA in high yield. Based on the total synthesis, several BKA analogues were prepared for structure-activity relationship studies, which indicated that the carboxylic acid moieties were essential for the apoptosis inhibitory activity of BKA. More easily available BKA analogues with potent apoptosis inhibitory activity were also developed.

Tumor cell secretion of soluble factor(s) for specific immunosuppression.

Studies of tumor models using syngeneic transplantation have advanced our understanding of tumor immunity, including both immune surveillance and evasion. Murine mammary carcinoma 4T1 cells secrete immunosuppressive soluble factors as demonstrated in splenocyte culture. Cultured primary splenocytes secrete IFN-γ, which was strikingly elevated when the cells were isolated from 4T1 tumor-bearing mice. The secretion of IFN-γ peaked a week after 4T1 inoculation and then declined. This reduction may be due to the relatively decreased lymphocytes and increased granulocytes in a spleen accompanied by splenomegaly with time after the 4T1 inoculation. IFN-γ production was further suppressed with the addition of the conditioned media from 4T1 cells to the splenocyte culture. This suppressive effect was more evident in the splenocytes isolated from mice that had 4T1 tumors for a longer period of time and was not observed in the conditioned medium either from CT26 cells or with splenocytes isolated from CT26 tumor-bearing mice. These results suggest that the IFN-γ suppression is 4T1 tumor-specific. The soluble factor(s) in the 4T1-conditioned media was a protein between 10 to 100 kDa. The cytokine tip assay demonstrated several known cytokines that negatively regulate immune responses and may be candidates for this immunosuppression activity.

Effective tumor treatment by VEGF siRNA complexed with hydrophobic poly(amino acid)-modified polyethylenimine.

Two copolymers are designed and synthesised for siRNA delivery based on polyethylenimine by grafting hydrophilic acrylamide segments and hydrophobic poly(γ-benzyl L-glutamate). The amphiphilic PEI-PBLG/siRNA complex demonstrates high gene silencing efficiency in the absence or presence of 10 vol% and 50 vol% sera in vitro. The anti-tumor effects in vivo are evaluated in luciferase-bearing mice expressing CT26 tumors. PEI-PBLG/siVEGF complex provides a higher and more sustained suppressive effect by reducing VEGF mRNA expression in the tumors, leading to higher tumor growth inhibition efficacy. Further studies on the potential use of the PEI-PBLG carrier system in mediating the silencing of genes other than VEGF or in other tumor models are recommended.

Tumor delivery of Photofrin® by PLL-g-PEG for photodynamic therapy.

Photofrin® (porfimer sodium) is a photosensitive reagent used for photodynamic therapy (PDT) of tumors and dysplasias. Because only photo-irradiated sites are damaged, PDT is less invasive than systemic treatments. However, a photosensitive reaction is a major side effect of systemically delivered Photofrin. To enhance localization of Photofrin to tumors, we have formulated Photofrin with the tumor-localizing graft copolymer poly(ethylene glycol)-grafted poly(l-lysine), PLL-g-PEG. We demonstrate that Photofrin preferentially interacts with PLL-g-PEG through both ionic and hydrophobic interactions. The serum competitive study showed that the highly PEG-grafted PLL is better for preventing serum binding to the Photofrin/PLL-g-PEG complex. In tumor-bearing mice, formulation of Photofrin with PLL-g-PEG enhanced tumor localization of Photofrin as twice as Photofrin alone and concomitantly suppressed the photosensitivity reaction drastically.

Design of UCST polymers for chilling capture of proteins.

Ureido-derivatized polymers, such as poly(allylurea) (PU) and poly(L-citrulline) derivatives, exhibited upper critical solution temperature (UCST) behavior under physiological buffer conditions as we previously reported. The PU derivatives having amino groups (PU-Am) also showed UCST behavior. In this study, we modified the amino groups of the polymer with succinyl anhydride (PU-Su) or acetyl anhydride (PU-Ac) to determine the effects of these ionic groups on the UCST behavior and to control interactions between the PU derivatives and biocomponents such as proteins and cells. Succinylation of PU-Am resulted in a significant decrease in phase separation temperature (Tp), whereas acetylation of PU-Am resulted in an increase in Tp. As expected, the Tp of PU-Am and PU-Su changed when the pH of the solution was changed. The Tp of PU-Am increased at higher pH, whereas that of PU-Su increased at lower pH, indicating that ionic charge decreases Tp of PU derivatives by increasing osmotic pressure and by increasing hydrophilicity of the polymer chains. Interestingly, these groups did not significantly change UCST when these groups were nonionic. We then examined capture and separation of particular proteins from a protein mixture by cooling-induced phase separation. Selective and rapid capture of particular proteins from protein mixture by PU derivatives was shown, indicating that the ureido-derivatized polymers are potential media for bioseparation under biofriendly conditions.

Polyelectrolyte-assisted transconformation of a stem-loop DNA.

A cationic copolymer triggered dimerization of a self-complementary stem-loop DNA. The dimerization was faster than spontaneous dissociation of the dimer. Reversible transformation between stem-loop and dimer structures was driven by switching on/off copolymer activity.

Dual crosslinked hydrogel nanoparticles by nanogel bottom-up method for sustained-release delivery.

Polysaccharide-PEG hybrid nanogels (CHPOA-PEGSH) crosslinked by both covalent ester bonds and physical interactions were prepared by the reaction of a thiol-modified poly(ethylene glycol) (PEGSH) with acryloyl-modified cholesterol-bearing pullulan (CHPOA). Experimental parameters, including CHPOA concentration, the degree of acryloyl substitution of CHPOA, and the initial amounts of CHPOA and PEGSH, were modified in order to assess their effect on the size of the nanogels (50-150 nm) and on their degradation kinetics, monitored by dynamic light scattering (DLS) and asymmetrical flow field-flow fractionation (AF4) chromatography. Rhodamine-labeled nanogels were injected intravenously into mice and their concentration in blood was determined by a fluorescence assay as a function of post-injection time. The elimination half-life (t(1/2)) of CHPOA-PEGSH nanoparticles was about 15-fold longer (18 h) than that of CHP nanogels (1.2 h). The half-life enhancement of CHPOA-PEGSH was attributed to the presence of the crosslinker PEG chains, which prevent non-specific protein adsorption, and to the slow hydrolysis kinetics of the crosslinking esters in the biological milieu. The hybrid CHPOA-PEGSH nanogels are expected to be useful as injectable nanocarriers for drugs and proteins, in view of their low surface fouling and slow hydrolysis rate.

Ureido-derivatized polymers based on both poly(allylurea) and poly(L-citrulline) exhibit UCST-type phase transition behavior under physiologically relevant conditions.

There are few examples of polymers that exhibit upper critical solution temperature (UCST) behavior under physiological conditions of temperature, pH, and ionic strength. In this study, we demonstrated that polymers with ureido groups undergo UCST-type phase transitions under physiologically relevant conditions. Poly(allylurea) copolymers showed UCST behavior at pH 7.5 in 150 mM NaCl even at the low polymer concentration of 0.13 mg/mL. Their phase separation temperatures (T(p)) could be controlled up to 65 °C. Similar thermosensitivity was observed with copolypeptides consisting of L-citrulline having an ureido group. This is the first demonstration of a non-vinyl polymer that shows UCST behavior under physiologically relevant conditions. We suggest that the ureido modification will be useful for production of polymer materials with UCST behavior in aqueous media.

Enhanced cell uptake via non-covalent decollation of a single-walled carbon nanotube-DNA hybrid with polyethylene glycol-grafted poly(l-lysine) labeled with an Alexa-dye and its efficient uptake in a cancer cell.

The use of single-walled carbon nanotubes (SWNTs) for biomedical applications is a promising approach due to their unique outer optical stimuli response properties, such as a photothermal response triggered by near-IR laser irradiation. The challenging task in order to realize such applications is to render the SWNTs biocompatible. For this purpose, the stable and homogeneous functionalization of the SWNTs with a molecule carrying a biocompatible group is very important. Here, we describe the design and synthesis of a polyanionic SWNT/DNA hybrid combined with a cationic poly(l-lysine) grafted by polyethylene glycol (PLL-g-PEG) to provide a supramolecular SWNT assembly. A titration experiment revealed that the assembly undergoes an approximately 1 : 1 reaction of the SWNT/DNA with PLL-g-PEG. We also found that SWNT/DNA is coated with PLL-g-PEG very homogeneously that avoids the non-specific binding of proteins on the SWNT surface. The experiment using the obtained supramolecular hybrid was carried out in vitro and a dramatic enhancement in the cell uptake efficiency compared to that of the SWNT/DNA hybrid without PLL-g-PEG was found.

The role of cationic comb-type copolymers in chaperoning DNA annealing.

G-rich oligonucleotides tend to fall into kinetically trapped unstable structures because of their conformational polymorphism. Nucleic acid chaperones accelerate association of nucleic acids assemblies into the thermodynamically most stable conformations by decreasing the energy barrier for breakage or re-assembly of base pairings. Here, we report that an artificial nucleic acid chaperone, a cationic comb-type copolymer, promotes tetramolecular quadruplex assembly from mixtures of two different G-rich sequences, 5'-TGGGGT-3' (TG(4)T) and 5'-TGGGGGT-3' (TG(5)T). A 1:1 mixture of TG(4)T and TG(5)T mainly gave [TG(4)T▪(TG(5)T)(3)], [(TG(4)T)(2)▪(TG(5)T)(2)] and [(TG(4)T)(3)▪TG(5)T] heteroquadruplexes when the mixture was annealed by cooling from 90 °C to 4 °C at 1.0 °C/min. At a cooling rate of 0.01 °C/min the mixture mostly assembled into [TG(4)T](4) and [TG(5)T](4) homoquadruplexes, indicating that homoquadruplexes were thermodynamically more stable than heteroquadruplexes. In the presence of the copolymer, mainly homoquadruplexes were obtained at cooling rate of 1 °C/min, suggesting that the copolymer promoted formation of the thermodynamically most stable structures. We also showed that the copolymer facilitated the recombination of heteroquadruplexes to homoquadruplexes even at 20-30 °C, implying that the copolymer can promote thermodynamically preferred quadruplex assembly from oligonucleotides trapped in metastable structures. These results show that the copolymer works as a DNA annealer that induces proper assembly of stable DNA structures from heterogeneous kinetically trapped mixtures of structures.

Nuclear localization and antisense effect of PNA internalized by ASGP-R-mediated endocytosis with protein/DNA conjugates.

In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37°C for at least 3h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs.

Grafting of poly(ethylene glycol) to poly-lysine augments its lifetime in blood circulation and accumulation in tumors without loss of the ability to associate with siRNA.

Poly-lysine has been studied as a carrier for the delivery of drugs and nucleic acids for at least a decade. It is an especially attractive carrier for DNA and RNA, because of its condensed cationic charges. In our previous study, we showed that poly(ethylene glycol) (PEG) grafted to poly-L-lysine (PLL) remarkably increased the life time of a small interfering RNA (siRNA) in blood circulation. In this study, we prepared a new series of PEG-grafted PLL (PLL-g-PEG) with various lengths (PEG 2kDa, 5kDa, and 10kDa and PLL 28kDa and 40kDa), to evaluate masking effects of PEG on cationic charges of PLL in vivo and the structural implications for biodistribution and tumoral accumulation. The best in the series, 40K10P37 (40kDa of PLL, 10kDa of PEG, 37mol% grafting) with molecular weight of 10(6) as determined by Multi-Angle Laser Light Scattering (MALLS), accumulated in tumors at about 8% of the injected dose per gram of tissue. Interestingly, a PLL-g-PEG conjugate pre-mixed with murine sera prevented degradation of siRNA, suggesting that PLL-g-PEG preferentially associates with siRNA in sera. Our results indicate grafting of PEG to the side chains of PLL augments its lifetime in blood circulation and tumoral accumulation without loss of the ability to associate with siRNA and support further evaluation of these cationic delivery carriers.

DNA assembly and re-assembly activated by cationic comb-type copolymer.

Guanine-rich oligonucleotides, such as TG(4)T and TG(5)T, assemble into a tetramolecular quadruplexes with layers of G-quartets stabilized by coordination to monovalent cations. Association rates of the quadruplexes are extremely slow, likely owing to electrostatic repulsion among the four strands. We have shown that comb-type copolymers with a polycation backbone and abundant hydrophilic graft chains form water-soluble polyelectrolyte complexes with DNA and promote DNA hybridization. Here, we report the effect of cationic comb-type copolymers on the kinetics of tetramolecular quadruplex formation. The copolymer significantly increased the association rate of tetramolecular quadruplexes without altering kinetic effects of metal cations in quadruplex formation. Dissociation rates of the quadruplexes were also accelerated by the copolymer suggesting that the copolymer has chaperone-like activity that reduces the energy barriers associated with dissociation and re-assembly of base pairs. This hypothesis was further supported by the observation that the copolymer activated the strand exchange reaction between the quadruplex and a constituting single-stranded.

Evaluation of polyanion-coated biodegradable polymeric micelles as drug delivery vehicles.

Polymeric micelles, as drug delivery vehicles, must achieve specific targeting and high stability in the body for efficient drug delivery. We recently reported the preparation of polyanion-coated biodegradable polymeric micelles by coating positively charged polymeric micelles consisting of poly(L-lysine)-block-poly(L-lactide) (PLys-b-PLLA) AB diblock copolymers with anionic hyaluronic acid (HA) by polyion complex (PIC) formation. The obtained HA-coated micelles showed significantly higher stability in aqueous solution. In this study, to evaluate the HA-coated polymeric micelles as a drug carrier, model drug release from the micelles and cytotoxicity of the micelles were investigated. The HA-coated micelles showed sustained release of model drugs and low cytotoxicity. It is known that there are receptors for HA on liver sinusoidal endothelial cells (LSEC). Specific interactions of HA-coated micelles with LSECs and Kupffer cells were investigated and compared with polymeric micelles coated with other polyanionic polysaccharides, i.e., heparin (Hep) and carboxymethyl-dextran (CMDex). Although Hep-coated micelles and CMDex-coated micelles were incorporated into both Kupffer cells and LSECs, HA-coated micelles were taken up only into LSECs. These results suggest HA-coated micelles have potential utility as drug delivery vehicles exhibiting specific accumulation into LSECs.

Cationic graft copolymer as a DNA B-z transition inducer: effect of copolymer structure.

The biological importance of left-handed Z-DNA was supported by the identification of Z-DNA binding proteins. We previously reported that poly(l-lysine)-graft-dextran copolymers composed of a poly(l-lysine) main chain and dextran graft chains induced the B-Z transition under low salt conditions. Not only the cationic main chain but also the dextran grafts play an important role in induction of the B-Z transition. In this study, we prepared a series of poly(l-lysine) graft copolymers with different graft chains (dextran, poly(ethylene glycol) (PEG), or both) and graft contents to further elucidate the roles of copolymer structures and properties. The copolymers with higher graft contents more effectively stabilized the Z form of DNA than those having lower graft contents. An increase in the B-Z transition rate was observed as graft content increased. We propose that the side chains of the copolymers change microenvironmental factors, such as water activity and dielectric constant, around the DNA and stabilize the Z conformer. Interestingly, hetero graft copolymers with both dextran and PEG side chains stabilized Z-DNA more effectively than the homo graft copolymers with either dextran or PEG side chains.