Synthesis and properties of PNA containing a dicationic nucleobase based on N4-benzoylated cytosine.

We report the synthesis of a peptide nucleic acid (PNA) monomer containing N4-bis(aminomethyl)benzoylated cytosine (BzC2+ base). The BzC2+ monomer was incorporated into PNA oligomers using Fmoc-based solid-phase synthesis. The BzC2+ base in PNA had two positive charges and exhibited greater affinity for DNA G base than the natural C base. The BzC2+ base stabilized PNA-DNA heteroduplexes through electrostatic attractions, even in high salt conditions. The two positive charges on the BzC2+ residue did not compromise the sequence specificity of PNA oligomers. These insights will aid the future design of cationic nucleobases.

Isolation and identification of the new baicalin target protein to develop flavonoid structure-based therapeutic agents.

Proteins are vital constituents of all living organisms. As many therapeutic agents alter the activity of functional proteins, identifying functional target proteins of small bioactive molecules isessential for the rational design of stronger medicines. Flavonoids with antioxidant, anti-allergy, and anti-inflammatory effects are expected to have preventive effects for several diseases closely related to oxidation and inflammation, including heart disease, cancer, neurodegenerative disorders, and eye diseases. Therefore, identifying the proteins involved in the pharmacological actions of flavonoids, and designing a flavonoid structure-based medicine that strongly and specifically inhibits flavonoid target proteins, could aid the development of more effective medicines for treating heart disease, cancer, neurodegenerative disorders, and ocular diseases with few side effects. To isolate the flavonoid target protein, we conducted a novel affinity chromatography in a column wherein baicalin, a representative flavonoid, was attached to Affi-Gel 102. Through affinity chromatography and nano LC-MS/MS, we identified GAPDH as a flavonoid target protein. Then, we performed fluorescence quenching and an enzyme inhibition assay to experimentally confirmbaicalin's binding affinity for, and inhibition of, GAPDH. We also conducted in silico docking simulations to visualize the binding modes of baicalin and the newly identified flavonoid target protein, GAPDH. From the results of this study, it was considered that one of the reasons why baicalin exhibits the effects on cancer and neurodegenerative diseases is that it inhibits the activity of GAPDH. In summary, we showed that Affi-Gel102 could quickly and accurately isolate the target protein for bioactive small molecules, without the need for isotopic labeling or a fluorescent probe. By using the method presented here, it was possible to easily isolate the target protein of a medicine containing a carboxylic acid.

Synthesis and characterization of PNA oligomers containing preQ1 as a positively charged guanine analogue.

We report the synthesis of a peptide nucleic acid (PNA) monomer containing preQ1, a positively charged guanine analogue. The new monomer was incorporated into PNA oligomers using standard Fmoc-chemistry-based solid-phase synthesis. The preQ1 unit-containing PNA oligomers exhibited improved affinity for their complementary DNA through electrostatic attraction, and their sequence specificity was not compromised. It could be beneficial to incorporate preQ1 into PNA oligomers instead of guanine when creating antisense/antigene agents or research tools.

ESI-Q-TOF-MS determination of polyamines and related enzyme activity for elucidating cellular polyamine metabolism.

We developed a new procedure for the comprehensive analysis of metabolites and enzymes involved in polyamine metabolism pathways. The procedure utilizes stable isotope-labeled polyamines and directly and precisely determines labeled products from enzymatic reactions by ESI-Q-TOF-MS. The activity of different enzymes could be determined in essentially the same manner by suitably adjusting the reaction conditions for each individual enzyme. We applied the procedure to extracts of regenerating rat liver and analyzed the changes in polyamine-metabolizing enzymes and polyamine contents during recovery from partial hepatectomy. A general outline of polyamine metabolism and information of polyamine dynamics were obtained. This kind of comprehensive information would be valuable in unifying detailed but fragmentary information obtained through conventional analyses focusing on one or a few enzymes and on a limited aspect of polyamine metabolic pathway.

Development of irreversible inactivators of spermine oxidase and N1-acetylpolyamine oxidase.

Three functional groups (2-propenyl, 2-propynyl, and 2,3-butadienyl) were introduced onto one of the terminal amino groups of spermidine. Of the six compounds synthesized, N-(3-aminopropyl)-N'-2,3-butadienyl-1,4-butanediamine (N(8)-butadienyl Spd) and N-[3-(2,3-butadienylamino)propyl]-1,4-butanediamine (N(1)-butadienyl Spd) irreversibly inactivated human spermine oxidase (SMO) and N(1)-acetylpolyamine oxidase (APAO). Interestingly, N(8)-butadienyl Spd inactivated SMO far more potently than N,N'-di-2,3-butadienyl-1,4-butanediamine (MDL 72527).

Construction of an immunochromatographic determination system for N¹,N¹²-diacetylspermine.

BACKGROUND: N(1),N(12)-diacetylspermine (DiAcSpm) is a recently identified tumor marker. Its concentration increases in the urine of cancer patients at early clinical stages. To utilize this characteristic feature and thus contribute to the early detection of cancer, we developed an immunochromatographic determination system for DiAcSpm. METHODS: We examined the factors that affect the performance and stability of our determination system, including antibody selection and the conditions for the formation of stably dispersed antibody-coated gold nanoparticles. We then tested the performance of the system by determining the DiAcSpm concentration in human urine samples. RESULTS: We constructed an immunochromatographic strip using anti-DiAcSpm antibody-coated gold nanoparticles in the conjugate pad and an acetylspermine-protein conjugate (a DiAcSpm mimic) immobilized on the analyzing membrane. The use of the immunochromatographic strip and an immunochromato-reader allowed for the quantitative determination of DiAcSpm in the range of 20 to 700 nM. The analytical values obtained by this method were well correlated with those determined by a colloidal gold aggregation procedure using an automatic biochemical analyzer. The immunochromatographic strip was stable for at least 8 weeks at 50°C. CONCLUSIONS: A competitive immunochromatographic device for DiAcSpm determination was developed in this study. This simple device will contribute to increasing the opportunities for early cancer detection and timely care.

Synthesis of 1-(4-aminobutyl)-2-iminoimidazolidin-4-one aimed at preparation of a creatinine-specific antibody.

For the purpose of obtaining a creatinine-specific antibody, a creatinine derivative with 4-aminobutyl, which was served as a linker for preparing the creatinine-bovine serum albumin (BSA) conjugate, was synthesized from 4-benzylaminobutan-1-ol in 8 steps. Production of anti-creatinine antibodies was observed in two rabbits using the creatinine-BSA conjugate, although their titer was rather low.

Differentiation of Positional Isomers of Halogenated Benzoylindole Synthetic Cannabinoid Derivatives in Serum by Hybrid Quadrupole/Orbitrap Mass Spectrometry.

Legally regulated synthetic cannabinoids (SCs) are continuously being created by making minor positional modifications to pre-existing analogs; thus, compounds with minor structural differences must be isolated and identified accurately. For iodo-benzoylindole derivatives of SCs, only specific isomers are currently the target of legal control, and it is necessary to establish an analytical method for accurately identifying positional isomers. In this study, we synthesized a series of 57 designer drugs and developed a screening method for identifying halogen positional isomers on the phenyl ring of benzoylindole derivative SCs in serum. Analytical methods using the Discovery F5 pentafluorophenyl column gave the best selectivity and retention of the positional isomer analytes. Some of the meta and para iodo-substituted SCs were eluted at similar retention times and were difficult to separate by liquid chromatography (LC). However, they were identified via the relative abundance of the two product ions in the collision-induced dissociation reaction using LC-hybrid quadrupole/orbitrap high-resolution mass spectrometry. Our synthesized halogen-substituted positional isomer SC library and method for differentiating positional isomers of halogenated benzoylindole SC derivatives could provide an indispensable analysis tool for identifying illegal drugs in serum of drug users.

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB. METHODS AND FINDINGS: Twenty-one patients participated in a phase I study of daily oral DFMO alone for three weeks, followed by additional three-week cycles of DFMO plus daily oral etoposide. No dose limiting toxicities (DLTs) were identified in patients taking doses of DFMO between 500-1500 mg/m2 orally twice a day. DFMO pharmacokinetics, single nucleotide polymorphisms (SNPs) in the ODC gene and urinary levels of substrates for the tissue polyamine exporter were measured. Urinary polyamine levels varied among patients at baseline. Patients with the minor T-allele at rs2302616 of the ODC gene had higher baseline levels (p=0.02) of, and larger decreases in, total urinary polyamines during the first cycle of DFMO therapy (p=0.003) and had median progression free survival (PFS) that was over three times longer, compared to patients with the major G allele at this locus although this last result was not statistically significant (p=0.07). Six of 18 evaluable patients were progression free during the trial period with three patients continuing progression free at 663, 1559 and 1573 days after initiating treatment. Median progression-free survival was less among patients having increased urinary polyamines, especially diacetylspermine, although this result was not statistically significant (p=0.056). CONCLUSIONS: DFMO doses of 500-1500 mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway. TRIAL REGISTRATION: Clinicaltrials.gov NCT#01059071.

Excretion of N(1), N(12)-diacetylspermine in the urine of healthy individuals.

BACKGROUND: Urinary N(1),N(12)-diacetylspermine (DiAcSpm) is a novel tumour marker that can be used to detect early cancers. In this study, we examined whether spot urine samples could represent the daily excretion of DiAcSpm after creatinine normalization and which factors should be taken into account in determining reference values for this biomarker. METHODS: We collected the following urine samples: (1) samples from seven healthy volunteers collected on each day of two 2-day sessions to examine the circadian variation of DiAcSpm excretion; (2) samples from 3952 male and 1782 female volunteers to estimate the DiAcSpm concentrations in apparently healthy adults and (3) samples from 16 female volunteers collected every morning over a 3-month period to examine the menstruation-related variation in DiAcSpm excretion. The DiAcSpm concentrations were determined by enzyme-linked immunosorbent assay or a colloidal gold aggregation procedure using DiAcSpm-specific antibodies. RESULTS: (1) The circadian variation of DiAcSpm in the urine was greatly diminished after creatinine normalization. (2) DiAcSpm was higher in females than in males, and the creatinine-normalized medians (95th percentile) of the urinary DiAcSpm concentrations were 149 (305) and 100 (192) nmol/g creatinine for females and males, respectively. (3) The mean concentrations of urinary DiAcSpm were lower after menstruation than before menstruation by approximately 30 nmol/g creatinine. CONCLUSION: Spot urine samples obtained at any time of a day may be used to estimate the daily excretion of DiAcSpm in nmol DiAcSpm per gram creatinine. Sex, age and menstrual condition should be considered when determining the reference values for urinary DiAcSpm.

Increase of N1, N12-diacetylspermine in tissues from colorectal cancer and its liver metastasis.

PURPOSE: N (1),N (12)-Diacetylspermine (DiAcSpm) is a tumor marker featured by increase in the urine of patients with cancers, including early colorectal cancer, but where and how DiAcSpm is made remains unclear. We aimed to clarify whether colorectal cancer tissues produce increased amounts of DiAcSpm, and if they do, to examine whether tissue DiAcSpm level may serve as a criterion of tissue malignancy. METHODS: Tissue samples were obtained from 140 patients (13 low-grade intraepithelial neoplasia, 98 high-grade intraepithelial neoplasia and 29 colorectal cancer) treated for colorectal cancer and intraepithelial neoplasia at Tokyo Metropolitan Komagome Hospital between November 2007 and April 2011. The DiAcSpm level in cancer and adjacent normal tissue extracts was compared, and its relationship with clinical stages of the diseases was analyzed. RESULTS: DiAcSpm levels were higher in colorectal cancer tissue (p < 0.01, n = 12) and its liver metastasis (p < 0.05, n = 5) than in adjacent normal tissues. The tumor/normal ratio of tissue DiAcSpm content was examined for endoscopically obtained tumor and adjacent normal tissues from patients with intraepithelial neoplasia. The ratio was greater than 1.5 in 38 % (5/13) and 78 % (84/108) of low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia, respectively. CONCLUSIONS: Tissue DiAcSpm levels increase in the tissue of colorectal cancer and also in precancerous lesion, such as high-grade intraepithelial neoplasia. The increase is considered a sign that a tissue is acquiring malignant characteristics. It is likely that the DiAcSpm produced by cancer cells is responsible for the frequent increase in urinary DiAcSpm in early cancer patients.