Synthesis and preliminary evaluation of poly(amidoamine)-melittin conjugates as endosomolytic polymers and/or potential anticancer therapeutics

The pH-responsive poly(amidoamine)s (PAAs) have been previously described. Whereas ISA23 enhances transfection in vitro and ISA1 promotes the cytosolic delivery of the non-permeant toxins this process shows poor efficiency. The aim of this study was to prepare and evaluate PAA conjugates containing the membrane disrupting peptide melittin (MLT). It was hypothesised that PAA conjugation would reduce the haemolytic activity of MLT at pH 7.4, however, upon delivery to tumours by the EPR effect, the polymer would uncoil in an acidic environment exposing MLT and allowing it to interact with membranes. PAA–MLT conjugates were prepared using MLT as a comonomer together with bis-acryloylpiperazine, 2-methylpiperazine and bis-hydroxyethylethylenediamine (ISA1-like), or bis-acrylamidoacetic acid and 2-methylpiperazine (ISA23-like). The melittin content of the conjugates was 6–19% (w/w). Although ISA1–MLT improved gelonin delivery compared to the parent polymer ISA1 ( 13-fold increase) and showed pH-dependent haemolytic activity at a polymer concentration of 0.05 mg/ml, this conjugate also displayed high haemolytic activity at pH 7.4. In contrast, ISA23–MLT like the parent compound ISA23 did not deliver gelonin. However, this conjugate could have potential as a novel polymeric anticancer conjugate due to its lack of haemolytic activity at pH 7.4 and retention of cytotoxicity.     

In vivo toxicity and immunogenicity of wheat germ agglutinin conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles for intranasal delivery to the brain

Biodegradable polymer-based nanoparticles have been widely studied to deliver therapeutic agents to the brain after intranasal administration. However, knowledge as to the side effects of nanoparticle delivery system to the brain is limited. The aim of this study was to investigate the in vivo toxicity and immunogenicity of wheat germ agglutinin (WGA) conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles (WGA-NP) after intranasal instillation. Sprague-Dawley rats were intranasally given WGA-NP for 7 continuous days. Amino acid neurotransmitters, lactate dehydrogenase (LDH) activity, reduced glutathione (GSH), acetylcholine, acetylcholinesterase activity, tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) in rat olfactory bulb (OB) and brain were measured to estimate the in vivo toxicity of WGA-NP. Balb/C mice were intranasally immunized by WGA-NP and then WGA-specific antibodies in serum and nasal wash were detected by indirect ELISA. WGA-NP showed slight toxicity to brain tissue, as evidenced by increased glutamate level in rat brain and enhanced LDH activity in rat OB. No significant changes in acetylcholine level, acetylcholinesterase activity, GSH level, TNF-α level and IL-8 level were observed in rat OB and brain for the WGA-NP group. WGA-specific antibodies in mice serum and nasal wash were not increased after two intranasal immunizations of WGA-NP. These results demonstrate that WGA-NP is a safe carrier system for intranasal delivery of therapeutic agents to the brain.     

Drug release from the enzyme-degradable and pH-sensitive hydrogel composed of glycidyl methacrylate dextran and poly(acrylic acid)

Hydrogels composed of glycidyl methacrylate dextran (GMD) and poly(acrylic acid, PAA) were prepared by UV irradiation method for colon-specific drug delivery. GMD was synthesized by coupling of glycidyl methacrylate to dextran in the presence of 4-(N,N-dimethylamino)pyridine. GMD was photo-polymerized by ammonium peroxydisulfate as initiating system in phosphate-buffered solution (0.1 M, pH 7.4). And then, acrylic acid monomer was added and subsequently heat-polymerized by 2,2'-azobisisobutyronitrile as an initiator. The hydrogels exhibited high swelling ratio (about 20) at 37 degrees C, and showed a pH-dependent swelling behavior. In addition, the swelling ratio of the hydrogel was remarkably enhanced to about 45 times in the presence of dextranase at pH 7.4. The swelling-deswelling behavior proceeded reversibly for the GMD/PAA hydrogels between pH 2 and pH 7.4. Release of 5-aminosalicylic acid from the GMD/PAA hydrogels was evaluated in simulated gastrointestinal pH fluids in the absence or presence of dextranase. We concluded that the hydrogels prepared could be used as a dual-sensitive drug carrier for sequential release in gastrointestinal tract.     

姜黄素PLGA纳米粒的制备及制剂学性质分析

目的制备姜黄素(Curcumin,Cur)聚乳酸羟基乙酸共聚物(PLGA)纳米粒(Cur-PLGA-NPs)并对其理化性质进行考察。方法采用改良的自乳化溶剂挥发法制备纳米粒,通过正交设计,以粒径、包封率和载药量为评价指标优化处方工艺。结果制备Cur-PLGA-NPs的优化条件为PLGA 100 mg,泊洛沙姆188浓度1.0%,丙酮与乙醇体积比3∶1,有机相体积15 m L。按优化条件所制备的Cur-PLGA-NPs粒径为(120.33±2.44)nm,多分散系数为0.10±0.02,包封率为84.50%±1.13%,载药量为4.75%±0.22%。结论采用改良的自乳化溶剂挥发法成功制备了Cur-PLGA-NPs,为后续"纳米粒-脂质体系统"的研究奠定了基础,有望实现药物在肝脏的浓集。     

Preparation,characterization, and in vitro/vivo evaluation of dexamethasone/poly(ε-caprolactone)-based electrode coatings for cochlear implants

With dexamethasone as the model drug and polycaprolactone (PCL) as the carrier material, a drug delivery coating for cochlear electrodes was prepared, to control cochlear fibrosis caused by cochlear implantation. A dexamethasone/poly (ε-caprolactone)-based electrode coating was prepared using the impregnation coating method. Preparation parameters were optimized, yielding 1 impregnation instance, impregnation time of 10 s, and PCL concentration of 10%. The coating was characterized in vitro using scanning electron microscopy, a universal machine, high-performance liquid chromatography, and CCK-8. The surface was porous and uniformly thick (average thickness, 48.67 µm)—with good flexibility, long-term slow drug release, and optimal drug concentration—and was biologically safe. The experimental results show that PCL is an ideal controlled-release material for dexamethasone as a drug carrier coating for cochlear implants.     

Investigation of the Occurrence of Cyanotoxins in Lake Karaoun (Lebanon) by Mass Spectrometry,Bioassays and Molecular Methods

Lake Karaoun is the largest artificial lake in Lebanon and serves multiple purposes. Recently, intensive cyanobacterial blooms have been reported in the lake, raising safety and aesthetic concerns related to the presence of cyanotoxins and cyanobacterial taste and odor (T&O) compounds, respectively. Here, we communicate for the first time results from a recent investigation by LC-MS/MS covering multiple cyanotoxins (microcystins (MCs), anatoxin-a, cylindrospermopsin, nodularin) in water and fish collected between 2019 and 2020. Eleven MCs were identified reaching concentrations of 211 and 199 μg/L for MC-LR and MC-YR, respectively. Cylindrospermopsin, anatoxin-a and nodularin were not detected. The determination of the total MCs was also carried out by ELISA and Protein Phosphatase Inhibition Assay yielding comparable results. Molecular detection of cyanobacteria (16S rRNA) and biosynthetic genes of toxins were carried out by qPCR. Untargeted screening analysis by GC-MS showed the presence of T&O compounds, such as β-cyclocitral, β-ionone, nonanal and dimethylsulfides that contribute to unpleasant odors in water. The determination of volatile organic compounds (VOCs) showed the presence of anthropogenic pollutants, mostly dichloromethane and toluene. The findings are important to develop future monitoring schemes in order to assess the risks from cyanobacterial blooms with regard to the lake’s ecosystem and its uses.     

聚(油酸/亚油酸-癸二酸)-庆大霉素在家兔体内的释药实验及相容性研究*

目的:观察聚(油酸/亚油酸-癸二酸)-庆大霉素在家兔体内释药情况及相容性。方法:将聚(油酸/亚油酸-癸二酸)-庆大霉素药丸植入家兔骨髓腔,数周后观察家兔骨、肌肉、肝、肾及血清中的庆大霉素浓度,并检测局部骨组织上清液的抑菌能力。在家兔背部脊柱旁肌肉组织和皮下植入不含庆大霉素的聚(油酸/亚油酸-癸二酸)-庆大霉素空药丸,以观察其与家兔组织的生物相容性。结果:药丸在家兔骨髓腔中释药良好,药丸释出的庆大霉素浓集在植入部位的骨组织中超过4周,在血清、肝、肾及局部肌肉中的庆大霉素含量甚微,骨组织上清液对常见的骨髓炎感染菌金葡球菌和大肠杆菌均有强大的抑菌活性。此外,聚(油酸/亚油酸-癸二酸)-庆大霉素还可在家兔体内自动降解。在观察期间没有发现明显的生物不相容性。结论:聚(油酸/亚油酸-癸二酸)-庆大霉素有望用于治疗慢性骨髓炎。     

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid–polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene—5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.     

Synthesis of redox derivatives of lysine and related peptides containing phenothiazine or tris(2,2′-bipyridine)ruthenium(II)

Boc-l -Lysine derivatives and lysine-containing peptides bearing the electron donor 10H-phenothiazine (PTZ) or the redox chromophore tris(2,2′-bipyridine)ruthenium(II) dication ([Rub₃,]^2H, where b is 2,2-bipyridine) have been synthesized and characterized. SeO₂ oxidation (53% yield) of 4,4′-dimethyl-2,2′-bipyridine, Ag_2:O oxidation (85% yield) of the monoaldehyde, complexation (96% yield) of 4′-methyl-2,2′-bipyridine-4-carboxylic acid (m-OH) with Rub₂Cl_2:. activation (81% yield) of the acid [Rub₂m-OH]²⁺ (PF₆^?)₂, and condensation (83% yield) of the succinimido ester [Rub₂m-OSu]²⁺ (PF₆^?)₂ with Boc-Lys furnished the protected redox-chromophore module [Boc-Lys(Rub₂m)-OH]²⁺(PF₆^?)₂ in 29% overall yield over five steps. The first two steps constitute the first practical synthesis of the monocarboxylic acid m-OH (45% overall yield). Also prepared were m-OSu, Boc-Lys(m)-OH, Boc-Lys(m)-OCH₃, and [Rub₂m-NHCH₃]²⁺ (PF₆^?)_2:. Activation (91% yield) of 3-(10H-phenothiazine-10)propanoic acid (PTZpn-OH) and condensation (92% yield) of the succinimido ester PTZpn-OSu with Boc-Lys furnished the protected electron-donor module Boc-Lys(PTZpn)-OH (84% overall yield). The latter was used in solid-phase syntheses of two redox tripeptides. CH₃CO-Ala-Lys(PTZpn)-Ala-OH and [Rub₂m-Ala-Lys(PTZpn)-Ala-OH]² (PF₆^?)₂. The electrochemical properties of these redox amino acids and peptides were similar to those of PTZpn-OH, [Rub₂ m-OH]2⁺(PF₆)₂. or [Rub₂ m-NHCH₃]²⁺ (PF₆^?)₂. Lys(PTZpn). [Lys(Rub₂m)]²⁺ (PF₆)_2:. and other redox modules may be useful for engineering light-harvesting proteins, photovoltaic cells, and other molecular electronic devices.     

Isolation and structure characterization of related impurities in 10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H) bulk drug and quantitation by a validated RP-LC

10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H), a novel active derivative of ginkgolide B, is a platelet-activating factor antagonist which is being under clinical trial. Two unknown related impurities were observed in analysis of XQ-1H bulk drug. A scaling up preparative liquid chromatography (Prep LC) was used for isolation of the two impurities. Based on LC–MS/MS and nuclear magnetic resonance (NMR) spectra, they were characterized as 10-O-(N,N-dimethylaminoethyl)-11,12-seco-ginkgolide B (imp-1) and 10-O-(N,N-dimethylaminoethyl)-11,12,2,15-diseco-3,14-dehydroginkgolide B (imp-2), respectively. A reversed-phase liquid chromatography (RP-LC) was developed for simultaneous determination of XQ-1H as well as imp-1 and imp-2. Main variables that significantly influence the chromatographic procedure were optimized and efficient chromatographic separation was achieved on a CN column with mobile phase consisting of 5 mM dipotassium hydrogen phosphate (pH 7.5) and methanol delivered in a gradient mode at the flow rate of 1.0 mL min⁻¹. The method was validated and found to be suitable to check the quality of bulk samples of XQ-1H at test concentration of 5.0 mg mL⁻¹ for a 20 μL injection volume.     

Synthesis and cytotoxicity of a novel 1-alkylaminomethyl-2,4-diaryl-butadiene-1,3 fragment integrated within cyclohex(pent)enes

A group of 1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes 3a-n with a 1-alkylaminomethyl-2,4-diaryl-1,3-butadiene fragment and a group of their congeners 3-alkylaminomethyl-1,2-diarylcyclohexene 7a-f have been synthesised for the first time. The conjugated system in 1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes 3a-n was unambiguously confirmed by X-ray crystallography. Cytotoxicity tests revealed that 3a-n possess inconsistent cytotoxicity against cancer cells, not their congeners 7a-f.     

Studies on the inhibition of poly(A) polymerase from rat liver and hepatoma 3924A by rifamycin SV derivatives

The inhibition of poly (A) polymerase activity from liver and hepatoma 3924A by several O-n-alky derivatives of rifamycin SV:3-formyloxime was studied. Only the O-n-pentyl (AF/012) and the O-n-octyl (AF/013) analogs were active at the maximum tested concentration of 1 × 10^?4 M. Equivalent concentrations of liver and hepatoma enzymes were inhibited to the same degree by both compounds. The 50 per cent inhibitory concentration (IC₅₀) for AF/013 and AF/012 was 1.2 × 10^?5M and 7.5 × 10^?5M respectively. Poly (A) polymerase activity was more sensitive to AF/013 if the enzyme was preincubated with the drug before the initiation of the reaction with poly (A) than if the reaction was initiated by the addition of the enzyme. Addition of AF/013 after initiation of the assay led to a rapid inhibition of poly (A) synthesis. The mechanism of inhibition by AF/012 and AF/013 of poly(A) polymerase was of the non-competitive type with respect to ATP.     

The effect of DDT and its metabolite (DDE) on prostaglandin secretion from epithelial cells and on contractions of the smooth muscle of the bovine oviduct in vitro

The insecticide DDT and its metabolite (DDE), due to their lipolytic nature and resistance to biodegradation, are accumulated in the living tissues. In cows, DDT and DDE were found to affect prostaglandin (PG) secretion from the endometrium and contractions of the myometrium.In this study, the impact of both xenobiotics (0.1, 1, 10 or 100 ng/ml) on the function of epithelial cells and muscle strips of bovine oviducts from 1 to 5 day of the oestrous cycle was examined. Therefore the concentration of PGE2 and PGFM (a metabolite of PGF2α) in culture media, mRNA expression of genes involved in PGs synthesis in epithelial cells and the force and amplitude of strips contractions were measured after 2 and 24 or 48 h of incubation.Neither DDT nor DDE affected the viability of cells after 48 h (P > 0.05). Both DDT and DDE increased the concentrations of PGFM in culture medium and secretion of PGE2 after only 2 h of cell culture (P < 0.05). Similar effects were seen for the influence of DDE on amount of PGFM after 48 h, while DDT decreased secretion of PGE2 (P < 0.05). DDT after 2 h increased (P < 0.05) mRNA expression of PGF2α synthase (PGFS), while both xenobiotics decreased (P < 0.05) mRNA expression of cyclooxygenase-2 (COX-2) after 24 h. DTT also increased the force of isthmus contractions after 2 h, as did both xenobiotics after 48 h (P < 0.05). Moreover, after 2 and 48 h, DDE stimulated the amplitude of contractions of the isthmus as well as the ampulla, (P < 0.05). The effect of both compounds on oviduct contractions was diminished by indomethacin, which blocks PG synthesis.We conclude that oviductal secretion of prostaglandins is affected, by DDT and DDE. The influence of these xenobiotics on PGF2α and PGE2 secretion and ratio may be part of the mechanism by which both DDT and its metabolite disturb the contractions of oviductal muscle.     

High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro

[(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand - adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC(50) and IC(90) concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC(50) or IC(90)) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G(2)/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC(50) or IC(90) of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.     

血吸虫病化学治疗的研究——ⅩⅩⅩⅦ.β-(5-硝基-4-溴-2-呋喃)-及β-(4,5-二溴-2-呋喃)丙烯酰胺及其酯类的合成

本文报道β-(4,5-二溴-2-呋喃)-及β-(5-硝基-4-溴-2-呋喃)丙烯酰胺及其酯类衍生物26个的合成。动物筛选结果表明;化合物Ⅲ₆,Ⅲ₈和Ⅲ₁₃对感染日本血吸虫小白鼠有明显的治疗作用。化合物Ⅱ₆有较明显的预防作用。     

Genotoxicity studies of glycidol fatty acid ester (glycidol linoleate) and glycidol

Glycidol fatty acid esters (GEs) are found in refined edible oils. Safety concerns have been alleged due to the possible release of glycidol (G), an animal carcinogen.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.     

血吸虫病化学治疗的研究——ⅩⅪⅤ.5-[2-(5-硝基呋喃基-2)-1-取代乙烯基]-1,3,4-噁二唑酮及其1,3,4-噁二唑衍生物的合成

本文报道硝基呋喃乙烯基-1,3,4-噁二唑及其酮类衍生物44个的合成。关键中间体α-取代β-(5-硝基呋喃基-2)丙烯酰彤魄仿照文献已知方法制备的,然后分别与光气或溴化腈作用生成相应的1,3,4-噁二唑衍生物Ⅰ,Ⅱ。经感染日本血吸虫病小白鼠的预防和治疗试验,发现化合物Ⅰ₁具有显著的抗血吸虫作用,并试用于家犬的血吸虫病,证明有一定的疗效。但效果不及呋喃丙胺,故未作临床观察。     

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.