Novel approach to determine ghrelin analogs by liquid chromatography with mass spectrometry using a monolithic column.

In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 µg/mL.

Enantiospecific tritium labeling of 28-homocastasterone.

A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2 /Pd[0]/Et3 N system, providing 28-[3ß-3 H]homocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).

Synthesis of the derivatives of 6-amino-uracil labelled with 14 C.

The radioactively labelled 6-amino-5-nitroso-uracil (1) and 5-acetyl-6-amino-1,3-dimethyl-uracil (2) were required for metabolic studies to assess their suitability as drug candidates. A common precursor for both compounds was [cyano-14 C]cyanoacetic acid (6), readily prepared from potassium [14 C]cyanide. ACS reagents, namely, diethyl ether, acetic acid and acetic anhydride, had to be rigorously repurified to achieve a successful synthesis of 14 C-labelled compounds on a tenth-of-a-milligramme scale. 6-Amino-5-nitroso-[6-14 C]uracil (1-14 C) (0.55 mCi) was prepared with radiochemical purity > 98% and specific activity (SA) = 55.6 mCi/mmol. 5-Acetyl-6-amino-1,3-dimethyl-[6-14 C]uracil (2-14 C) (8 mCi) was prepared with radiochemical purity > 97% and SA = 55.6 mCi/mmol. It has been shown that a SA assay can be made from standard 13 C NMR spectra, thus avoiding the need to perform lengthier inverse-gated 13 C NMR experiments.

The labeling of unsaturated γ-hydroxybutyric acid by heavy isotopes of hydrogen: iridium complex-mediated H/D exchange by C─H bond activation vs reduction by boro-deuterides/tritides.

3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. [3 H]-HOCPCA selectively labeled on the position C-3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.

Transport mechanisms of a novel antileukemic and antiviral compound 9-norbornyl-6-chloropurine.

6-Chloropurines substituted at the position 9 with variously modified bicyclic skeletons represent promising antiviral and anticancer agents. This work aimed to investigate the transport mechanisms of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP) and their relationship to the metabolism and biological activity of the compound. Transport experiments were conducted in CCRF-CEM cells using radiolabeled compound ([(3)H]NCP). The pattern of the intracellular uptake of [(3)H]NCP in CCRF-CEM cells pointed to a combination of passive and facilitated diffusion as prevailing transport mechanisms. NCP intracellular metabolism was found to enhance its uptake by modifying NCP concentration gradient. The transport kinetics reached steady state under the conditions of MRP and MDR proteins blockade, indicating that NCP is a substrate for these efflux pumps. Their inhibition also increased the cytotoxicity of NCP. Our findings suggest that the novel nucleoside analog NCP has potential to become a new orally available antileukemic agent due to its rapid membrane permeation.

Characterization of new stable ghrelin analogs with prolonged orexigenic potency.

Ghrelin, the only known peripherally produced and centrally acting peptide that stimulates food intake, is synthesized primarily in the stomach and acts through the growth hormone secretagogue receptor (GHS-R1a). In addition to its orexigenic effect, ghrelin stimulates the release of growth hormone (GH). In this study, we investigated the biological properties of full-length and shortened ghrelin analogs in which octanoylated Ser(3) is replaced with an octanoic acid moiety coupled to diaminopropionic acid (Dpr). Ghrelin analogs stabilized with Dpr(N-octanoyl) in position 3 and noncoded amino acids in position 1 (sarcosine) and/or position 4 (naphthylalanine or cyclohexylalanine) were found to possess affinities similar to those of ghrelin for cell membranes with transfected GHS-R1a. In vivo, the prolonged orexigenic effects of analogs containing Dpr(N-octanoyl)(3) compared with that of ghrelin in adult mice and a similar impact on GH secretion in young mice were found. Full-length [Dpr(N-octanoyl)(3)]ghrelin and its analogs with a noncoded amino acid in position 1 and/or 4 showed significantly prolonged stability in blood plasma compared with that of ghrelin. Ghrelin analogs with a prolonged orexigenic effect are potential treatments for GH deficiency or cachexia that accompanies chronic diseases. Desoctanoylated ghrelin analogs and N-terminal penta- and octapeptides of ghrelin did not show any biological activity.

Structure-activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

Cocaine- and amphetamine-regulated transcript (CART) peptide specific binding in pheochromocytoma cells PC12.

CART (cocaine- and amphetamine-regulated transcript) peptides have been studied for ten years. We report specific binding of 125I-CART(61-102) to the rat adrenal pheochromocytoma PC12 cell line, both intact cells and cell membranes. Saturation binding to intact plated cells resulted in Kd of 0.48+/-0.16 nM and Bmax of 2228+/-529 binding sites/cell. 125I-CART(61-102) was also bound to PC12 cells differentiated using nerve growth factor to the neuronal phenotype with non-specific binding below 20%, and Kd of 1.90+/-0.27 nM and Bmax of 11,194+/-261 binding sites/cell. In competitive binding experiments, CART(61-102), CART(55-102) and di-iodinated CART(61-102) were bound to PC12 cell membranes with Ki in low nM range; their affinity to intact non-differentiated and differentiated cells was in low 10(-8) M range. In order to prove that iodination did not eliminate the pharmacological properties of CART, we tested the biological activity of di-iodinated CART(61-102). It decreased food intake in in vivo feeding experiment on fasted mice in a dose of 1 microg/mouse to the same extent as CART(61-102) in a dose of 0.5 microg/mouse.

Immunoaffinity chromatography combined with tandem mass spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones.

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

Development of a human vasopressin V1a-receptor antagonist from an evolutionary-related insect neuropeptide.

Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action.

7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.

Neuropeptide FF analog RF9 is not an antagonist of NPFF receptor and decreases food intake in mice after its central and peripheral administration.

Neuropeptide FF (NPFF) belongs to the RF-amide family of peptides bearing the identical C-terminal amino acid sequence (R-F-NH2). In addition to NPFF, prolactin-releasing peptide (PrRP), another RF-amide, binds to NPFF receptors with high affinity. A selective antagonist of PrRP has not yet been identified, but a selective antagonist of NPFF, 1-adamantanecarbonyl-RF-NH2 (RF9), was recently reported to antagonize the hyperalgesic effect of NPFF after central administration to mice. In the present study, RF9 competed with NPFF analog D-Y-L-(N-Me)-F-Q-P-Q-R-F-NH2 (1DMe) in binding to CHO-K1 cell membranes transfected with the human NPFF2 receptor. In rat pituitary RC-4B/C cells, where the expression of the NPFF2 receptor was proved by immunodetection, RF9 did not reverse the phosphorylation of MAPK/ERK1/2 induced by [Tyr(1)]NPFF. In vivo experiments with fasted mice confirmed that centrally injected [Tyr(1)]NPFF significantly lowered food intake. However, RF9, a putative NPFF2 antagonist, did not reverse the anorectic effect of [Tyr(1)]NPFF. Paradoxically, RF9 itself exhibited an anorectic effect in fasted mice not only after intracerebroventricular but also after subcutaneous administration. This finding casts doubt on claims that RF9 is an NPFF antagonist.

9-Norbornyl-6-chloropurine is a novel antileukemic compound interacting with cellular GSH.

AIM: 6-Chloropurines substituted at position 9 with bicyclic skeletons represent promising chemotherapeutic agents. We explored the metabolism and membrane transport of 9-norbornyl-6-chloropurine (NCP) aiming to understand its mechanism of action. MATERIALS AND METHODS: The metabolism of NCP was studied in vitro in whole cells (CCRF-CEM), cellular extracts, subcellular fractions and purified enzymes. Transport experiments were conducted in Caco-2 cell monolayers. RESULTS: Three metabolites were identified, a glutathione conjugate (NCP-GS), NCP-cysteinylglycine and NCP-cysteine. Both glutathione-S-transferase inhibition and glutathione (GSH) depletion prevented metabolite formation and increased the cytotoxicity of NCP. Transepithelial transport (Caco-2) indicated good permeability, with Papp (12.6±0.3) ×10(-5) cm/s. Importantly, the drug induced glutathione depletion in treated cells and affected the activity of several GSH-dependent enzymes. CONCLUSION: The novel nucleoside analog NCP represents a promising orally available antileukemic agent, acting through lowering of GSH levels in tumor cells.

Double-headed sulfur-linked amino acids as first inhibitors for betaine-homocysteine S-methyltransferase 2.

Betaine-homocysteine S-methyltransferase 2 (BHMT-2) catalyzes the transfer of a methyl group from S-methylmethionine to l-homocysteine, yielding two molecules of l-methionine. It is one of three homocysteine methyltransferases in mammals, but its overall contribution to homocysteine remethylation and sulfur amino acid homeostasis is not known. Moreover, recombinant BHMT-2 is highly unstable, which has slowed research on its structural and catalytic properties. In this study, we have prepared the first series of BHMT-2 inhibitors to be described, and we have tested them with human recombinant BHMT-2 that has been stabilized by copurification with human recombinant BHMT. Among the compounds synthesized, (2S,8RS,11RS)-5-thia-2,11-diamino-8-methyldodecanedioic acid (11) was the most potent (K(i)(app) ∼77 nM) and selective inhibitor of BHMT-2. Compound 11 only weakly inhibited human BHMT (IC(50) about 77 µM). This compound (11) may be useful in future in vivo studies to probe the physiological significance of BHMT-2 in sulfur amino acid metabolism.

Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor.

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K(d) in the 10(-9)M range and a B(max) in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with (125)I-PrRP31 with a similar K(i). The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.

In vitro binding and in vivo biodistribution studies of the neuroprotective peptide humanin using [125I]humanin derivatives.

Humanin (HN) and HN-derivatives are a family of peptides first reported in the last decade with potent in vitro and in vivo neuroprotective activity, which is mediated through a not completely elucidated mechanism. Recently, our group has evaluated the effect of various HN-derivatives on the 3-quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation and memory in rats, by employing the T-maze test. In the present work four new, tyrosine containing HN-derivatives were synthesized (Y-PAGASRLLLTGEIDLP, peptide I; Y-PAGASRLLLLTGEIDLP, peptide II; Y-SALLRSIPAPAGASRLLLTGEIDLP, peptide III; Y-SALLRSIPAPAGASRLLLLTGEIDLP, peptide IV). The neuroprotective action of these peptides was evaluated in the T-maze test and the most active among them (peptides I and III) was radiolabeled with (125)I. The pure monoradioiodinated peptides were used in: (i) in vitro binding studies with various neuronal cell lines and with brain and stomach membranes from rats and mice and (ii) in vivo biodistribution studies in rats and mice. Moreover, the metabolic stability of the above radiolabeled peptides was studied. Under the experimental conditions used, our data do not confirm the existence of specific binding sites for HN on the neuronal tissue. Nevertheless, they are setting the basis for further relevant studies aiming at the clarification of the mode of the neuroprotective action of HN-peptides.