Characterization of human DAAO variants potentially related to an increased risk of schizophrenia

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the “structurally ambivalent” peptide V47–L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d + l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.     

18F-labeled flavones for in vivo imaging of β-amyloid plaques in Alzheimer’s brains

In vivo imaging of β-amyloid (Aβ) aggregates in the brain may lead to early detection of Alzheimer’s disease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study was to develop novel ¹⁸F-labeled amyloid-imaging probes based on flavones as a core structure. Fluoropegylated (FPEG) flavone derivatives were designed and synthesized. The affinity of the derivatives for Aβ aggregates varied from 5 to 321 nM. In brain sections of AD model mice, FPEG flavones with the dimethylamino group intensely stained β-amyloid plaques. In biodistrubution experiments using normal mice, they displayed high uptake in the brain ranging from 2.9 to 4.2%ID/g at 2 min postinjection. The radioactivity washed out from the brain rapidly (1.3–2.0%ID/g at 30 min), which is highly desirable for β-amyloid imaging agents. FPEG flavones may be potential PET imaging agents for β-amyloid plaques in Alzheimer’s brains.     

Highly efficacious factor Xa inhibitors containing α-substituted phenylcycloalkyl P4 moieties

We previously disclosed a series of highly potent FXa inhibitors bearing α-substituted (CH₂NR¹R²) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the α-substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K_i), human plasma anticoagulant activity (PT EC_2×) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC₅₀s = 29–81 nM).     

Synthesis and evaluation of copper-64 labeled benzofuran derivatives targeting β-amyloid aggregates

In vivo imaging of β-amyloid (Aβ) aggregates consisting of Aβ(1–40) and Aβ(1–42) peptides by positron emission tomography (PET) contributes to the diagnosis and therapy for Alzheimer’s disease (AD). Because ⁶⁴Cu (t_1/2 = 12.7 h) is a radionuclide for PET with a longer physical half-life than ¹¹C (t_1/2 = 20 min) and ¹⁸F (t_1/2 = 110 min), it is an attractive radionuclide for the development of Aβ imaging probes that are suitable for routine use. In the present study, we designed and synthesized two novel ⁶⁴Cu labeled benzofuran derivatives and evaluated their utility as PET imaging probes for Aβ aggregates. In an in vitro binding assay, 6 and 8 showed binding affinity for Aβ(1–42) aggregates with a K_i value of 33 and 243 nM, respectively. In addition, these probes bound to Aβ plaques deposited in the brain of an AD model mouse in vitro. In a biodistribution experiment using normal mice, these probes showed low brain uptake (0.33% and 0.36% ID/g) at 2 min post-injection. Although refinement to enhance brain uptake is needed, [⁶⁴Cu]6 and [⁶⁴Cu]8 demonstrated the feasibility of developing novel PET probes for imaging Aβ aggregates.     

A 68Ga complex based on benzofuran scaffold for the detection of β-amyloid plaques

Since the imaging of β-amyloid (Aβ) plaques in the brain is believed to be a useful tool for the early diagnosis of Alzheimer’s disease (AD), a number of imaging probes to detect Aβ plaques have been developed. Because the radionuclide ⁶⁸Ga (t_1/2 = 68 min) for PET imaging could become an attractive alternative to ¹¹C and ¹⁸F, we designed and synthesized a benzofuran derivative conjugated with a ⁶⁸Ga complex (⁶⁸Ga-DOTA-C3-BF) as a novel Aβ imaging probe. In an in vitro binding assay, Ga-DOTA-C3-BF showed high affinity for Aβ(1-42) aggregates (K_i = 10.8 nM). The Ga-DOTA-C3-BF clearly stained Aβ plaques in a section of Tg2576 mouse, reflecting the affinity for Aβ(1-42) aggregates in vitro. In a biodistribution study in normal mice, ⁶⁸Ga-DOTA-C3-BF displayed low initial uptake (0.45% ID/g) in the brain at 2 min post-injection. While improvement of the brain uptake of ⁶⁸Ga complexes appears to be essential, these results suggest that novel PET imaging probes that include ⁶⁸Ga as the radionuclide for PET may be feasible.     

Radiosynthesis and evaluation of IGF1R PET ligand [11C]GSK1838705A

Radiosynthesis and evaluation of [¹¹C]GSK1838705A in mice using microPET and determination of specificity in human GBM UG87MR cells are described herein. The radioligand was synthesized by reacting desmethyl-GSK1838705A with [¹¹C]CH₃I using GE FX2MeI module in ～5% yield (EOS), >95% radiochemical purity and a specific activity of 2.5 ± 0.5 Ci/μmol. MicroPET imaging in mice indicated that [¹¹C]GSK1838705A penetrated blood brain barrier (BBB) and showed retention of radiotracer in brain. The radioligand exhibited high uptake in U87MG cells with >70% specific binding to IGF1R. Our experiments suggest that [¹¹C]GSK-1838705A can be a potential PET radiotracer for the in vivo quantification of IGF1R expression in GBM and other brain tumors.     

Synthesis and evaluation of ethyleneoxylated and allyloxylated chalcone derivatives for imaging of amyloid β plaques by SPECT

We report radioiodinated chalcone derivatives as new SPECT imaging probes for amyloid β (Aβ) plaques. The monoethyleneoxy derivative 2 and allyloxy derivative 8 showed a high affinity for Aβ(1–42) aggregates with K_i values of 24 and 4.5 nM, respectively. Fluorescent imaging demonstrated that 2 and 8 clearly stained thioflavin-S positive Aβ plaques in the brain sections of Tg2576 transgenic mice. In vitro autoradiography revealed that [¹²⁵I]2 displayed no clear accumulation toward Aβ plaques in the brain sections of Tg2576 mice, whereas the accumulation pattern of [¹²⁵I]8 matched with the presence of Aβ plaques both in the brain sections of Tg2576 mice and an AD patient. In biodistribution studies using normal mice, [¹²⁵I]2 showed preferable in vivo pharmacokinetics (4.82%ID/g at 2 min and 0.45%ID/g at 60 min), while [¹²⁵I]8 showed only a modest brain uptake (1.62%ID/g at 2 min) with slow clearance (0.56%ID/g at 60 min). [¹²⁵I]8 showed prospective binding properties for Aβ plaques, although further structural modifications are needed to improve the blood brain barrier permeability and washout from brain.     

Development of dual functional SPECT/fluorescent probes for imaging cerebral β-amyloid plaques

The imaging of β-amyloid (Aβ) aggregates in the brain may lead to the early detection of Alzheimer’s disease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study was to develop dual modality SPECT and fluorescent probes based on boron dipyrromethane (BODIPY) as a core structure. We designed and synthesized an ¹²⁵I-labeled derivative of BODIPY (BODIPY7). BODIPY7 had a K_i value of 108 nM for Aβ(1–42) aggregates and exhibited peaks of absorption/emission at 606/613 nm. It detected Aβ plaques in sections of brain tissue from an animal model of AD and displayed low uptake in the brain and high uptake in the liver in normal mice. Although additional modifications of the BODIPY scaffold are necessary to improve brain uptake, these results should aid the development of dual functional SPECT/fluorescent probes for the imaging of Aβ plaques in the brain.     

A novel 18F-labeled pyridyl benzofuran derivative for imaging of β-amyloid plaques in Alzheimer’s brains

A potential probe for PET targeting β-amyloid plaques in Alzheimer’s disease (AD) brain, FPYBF-1 (5-(5-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzofuran-2-yl)-N,N-dimethylpyridin-2-amine), was synthesized and evaluated. In experiments in vitro, FPYBF-1 displayed high affinity for Aβ(1–42) aggregates (K_i = 0.9 nM), and substantial labeling of β-amyloid plaques in sections of postmortem AD brains but not control brains. In experiments in vivo, [¹⁸F]FPYBF-1 displayed good initial uptake (5.16%ID/g at 2 min postinjection) and rapid washout from the brain (2.44%ID/g at 60 min postinjection) in normal mice, and excellent binding to β-amyloid plaques in a murine model of AD. Furthermore, the specific labeling of plaques labeling was observed in autoradiographs of autopsied AD brain sections. [¹⁸F]FPYBF-1 may be a useful probe for imaging β-amyloid plaques in living brain tissue.     

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel,high affinity ligands for CB1 and CB2 cannabinoid receptors

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R² = H, R¹ = F) and 13 (R = COOCH₃, R¹ = R² = H) exhibited high affinity for CB2 receptors with K_i values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (K_i values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.     

Anti-inflammatory and anti-apoptotic effects of (RS)-glucoraphanin bioactivated with myrosinase in murine sub-acute and acute MPTP-induced Parkinson’s disease

This study was focused on the possible neuroprotective role of (R_S)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive R_S-GRA), in an experimental mouse model of Parkinson’s disease (PD). R_S-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale seeds.R_S-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration and analyzed by HPLC. Following, pure R_S-GRA was characterized by ¹H and ¹³C NMR spectrometry and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method. The obtained purity has been of 99%.To evaluate the possible pharmacological efficacy of bioactive R_S-GRA (administrated at the dose of 10 mg/kg, ip +5 μl/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experiment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an acute (2 injections · 40 mg/kg MPTP) and a sub-acute (5 injections · 20 mg/kg MPTP) model of PD.Behavioural test, body weight changes measures and immunohistochemical localization of the main PD markers were performed and post-hoc analysis has shown as bioactive R_S-GRA is able to reduce dopamine transporter degradation, tyrosine hydroxylase expression, IL-1β release, as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP immunolocalization). These effects have been correlated with the release of neurotrophic factors, such as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioactive R_S-GRA. Moreover, after PD-induction mice treated with bioactive R_S-GRA are appeared more in health than animals that did not received the treatment both for phenotypic behaviour and for general condition (movement coordination, presence of tremors, nutrition).Overall, our results suggest that bioactive R_S-GRA can protect neurons against the neurotoxicity involved in PD via an anti-apoptotic/anti-inflammatory action.     

Preparation of classical Re/99mTc(CO)3+ and novel 99mTc(CO)2(NO)2+ cores complexed with flavonol derivatives and their binding characteristics for Aβ(1–40) aggregates

Classical ^99mTc(CO)₃⁺ and novel ^99mTc(CO)₂(NO)²⁺ cores complexed with flavonol derivatives were prepared. Autoradiography of postmortem AD transgenic mice (Tg C57, APP, PS1 12-month-old) brain section confirmed the binding property of [^99mTc(CO)₃⁺-3-OH-flavone]⁰ to Aβ_(1–40) aggregates, while the novel ^99mTc(CO)₂(NO)²⁺ labeled compounds showed no binding sites in AD transgenic mice sections. Intravenous administration of [^99mTc(CO)₃⁺-3-OH-flavone]⁰ resulted in moderate brain uptake (0.48 ± 0.05%ID/g) at 5 min post-injection and slow clearance from the brain issues in 2 h post-injection (120 min: 0.39 ± 0.08%ID/g). Then an Aβ_(1–40)-receptor-targeted Re(CO)₃⁺-3-OH-flavone, was prepared to identify the structure of the technetium complex. UV–vis absorption and fluorescence emission properties have been studied at room temperature in order to determine the natures of the lowest electronically excited states of Re(CO)₃⁺-3-OH-flavone and the ligand. The fluorescent rhenium complex Re(CO)₃⁺-3-OH-flavone showed high affinity for Aβ_(1–40) aggregates in vitro by fluorescence spectra (dissociation constant (K_d) = 11.16 nM). In conclusion, the results suggested that ^99mTc(CO)₃⁺-3-OH-flavone should be a suitable candidate as Aβ plaque SPECT imaging agent for AD.     

Involvement of potassium channels in the antidepressant-like effect of venlafaxine in mice

AimsStudies have shown that the acute administration of venlafaxine elicits an antidepressant-like effect in the mouse forced swim test (FST) by a mechanism dependent on the l-arginine–nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway. Because it has been reported that NO activates different types of potassium (K⁺) channels in the brain, this study investigated the involvement of K⁺ channels in the antidepressant-like effect of venlafaxine in the mouse FST.Main methodsMale adult Swiss mice were pretreated with different K⁺ channel inhibitors or openers 15 min before venlafaxine administration. After 30 min, the open-field test (OFT) and FST were carried out.Key findingsIntracerebroventricular (i.c.v.) pretreatment of mice with subeffective doses of tetraethylammonium (TEA, a non-specific inhibitor of K⁺ channels, 25 pg/site), glibenclamide (an ATP-sensitive K⁺ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K⁺ channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K⁺ channel inhibitor, 10 pg/site) was able to potentiate the action of a subeffective dose of venlafaxine (2 mg/kg, i.p.). Moreover, the reduction in the immobility time elicited by an effective dose of venlafaxine (8 mg/kg, i.p.) in the FST was prevented by the pretreatment of mice with the K⁺ channel openers cromakalim (10 µg/site, i.c.v.) and minoxidil (10 µg/site, i.c.v.). The drugs used in this study did not produce any change in locomotor activity.SignificanceThe results demonstrate that the neuromodulatory effects of venlafaxine, via the inhibition of K⁺ channels, are possibly involved in its anti-immobility activity in the mouse FST.     

Discovery and optimization of N-(3-(1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yloxy)phenyl)benzenesulfonamides as novel GPR119 agonists

The discovery and optimization of novel N-(3-(1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yloxy)phenyl)benzenesulfonamide GPR119 agonists is described. Modification of the pyridylphthalimide motif of the molecule with R¹ = –Me and R² = –ⁱPr substituents, incorporated with a 6-fluoro substitution on the central phenyl ring offered a potent and metabolically stable tool compound 22.     

Methylnickel compounds containing 2-phosphinylethanolato ligands – Syntheses,properties, and ethene coupling reactions

Combining dimethylphosphinylethanols HO(R¹R²)CCH₂PMe₂ (1: R¹ = R² = C₆H₅; 2: R¹ = R² = 4-OMe–C₆H₄; 5: R¹ = R² = 4-NMe₂–C₆H₄) with methyl(methoxo)(trimethylphosphine)nickel gave mononuclear methyl(trimethylphosphine)nickel(chelate) compounds 7–9. Ligand 6 (R¹ = Me, R² = 4-OMe–C₆H₅) afforded a dinuclear methylnickel compound 14. By reacting (TMEDA)lithium-dimethylphosphinylmethanide with ketones OC(R¹R²), the dimethylphosphinylethanols HO(R¹R²)CCH₂PMe₂ (3: R¹R² = 9-fluorenyl; 4: R¹ = H, R² = C₆H₅) were synthesized as prechelate ligands. Under otherwise similar conditions, the fluorenyl substituted anion in 3 gave rise to a mononuclear complex 10 which was found to act as a source of trimethylphosphine forming dinuclear 11 and at the same time to act as an acceptor of trimethylphosphine forming pentacoordinate 10 · PMe₃. Ni(COD)(PMe₃)₂ was used as a scavenger of PMe₃ in converting 8 or 9 to the dinuclear methylnickel compounds 12 and 13, respectively. Modifying the P,O chelating unit of a methyl nickel compound by introducing 2-phosphinylethanolato ligands leads to novel single-component catalysts for ethene oligomerization showing moderate reactivity and thermal stability.     

Synthesis and biological evaluation of 18F-labled 2-phenylindole derivatives as PET imaging probes for β-amyloid plaques

A novel series of fluorinated 2-phenylindole derivatives were synthesized and evaluated as β-amyloid imaging probes for PET. The in vitro inhibition assay demonstrated that their binding affinities for Aβ_1–42 aggregates ranged from 28.4 to 1097.8 nM. One ligand was labeled with ¹⁸F ([¹⁸F]1a) for its high affinity (K_i = 28.4 nM), which was also confirmed by in vitro autoradiography experiments on brain sections of transgenic mouse (C57BL6, APPswe/PSEN1, 11 months old, male). In vivo biodistribution experiments in normal mice showed that this radiotracer displayed high initial uptake (5.82 ± 0.51% ID/g at 2 min) into and moderate washout (2.77 ± 0.31% ID/g at 60 min) from the brain. [¹⁸F]1a could be developed as a promising new PET imaging probe for Aβ plaques although necessary modifications are still needed.     

Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production

We examined glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation, using a recombinant strain of Saccharomyces cerevisiae W303-181 overexpressing this enzyme. The cultivations were carried out in a 3 L fermenter at pH 5.7, 30 °C, 2.0 vvm aeration, 200 rpm agitation and an inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process varied from 1.333 to 2.0 L, due to the addition of 15.0 g/L glucose solution during 5 h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time constant), namely: 0.2, 0.5 and 0.8 h⁻¹. The best enzyme production (847 U/L) was obtained with an exponentially increasing feeding rate and K = 0.2 h⁻¹. The results attained also showed that this process is promising for G6PD production.     

Activity of resveratrol triesters against primary acute lymphoblastic leukemia cells

Resveratrol is a common polyphenol of plant origin known for its cancer prevention and other properties. Its wider application is limited due to poor water solubility, low stability, and weak bioavailability. To overcome these limitations, a series of 13 novel resveratrol triesters were synthesized previously. In this paper, we describe the synthesis of 3 additional derivatives and the activity of all 16 against primary acute lymphoblastic leukemia cells. Of these, 3 compounds were more potent than resveratrol (IC₅₀ = 10.5 µM) namely: resveratryl triacetate (IC₅₀ = 3.4 µM), resveratryl triisobutyrate (IC₅₀ = 5.1 µM), and resveratryl triisovalerate (IC₅₀ = 4.9 µM); all other derivatives had IC₅₀ values of >10 µM. Further studies indicated that the active compounds caused G1 phase arrest, increased expression of p53, and induced characteristics of apoptotic cell death. Moreover, the compounds were only effective in cycling cells, with cells arrested in G1 phase being refractory.     

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for β-amyloid plaques in Alzheimer’s disease

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential β-amyloid probes. In vitro binding studies using Aβ aggregates showed that all of them demonstrated high binding affinities with K_i values ranged from 0.11 to 4.64 nM. In vitro fluorescent staining results showed that these compounds can intensely stained Aβ plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [¹²⁵I]-2-(5′-iodo-2,2′-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [¹²⁵I]10 and [¹²⁵I]-2-(2,2′-bithiophen-5-yl)-6-iodobenzo[d]thiazole [¹²⁵I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [¹²⁵I]10 and [¹²⁵I]13 labeled the Aβ plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [¹²⁵I]13 exhibited high brain uptake (3.42% ID/g at 2 min) and rapid clearance from the brain (0.53% ID/g at 60 min), while [¹²⁵I]10 showed lower brain uptake (0.87% ID/g at 2 min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [¹²⁵I]13 may be a candidate as an in vivo imaging agent for detecting β-amyloid plaques in the brain of AD patients.     

Discovery of isatin and 1H-indazol-3-ol derivatives as d-amino acid oxidase (DAAO) inhibitors

d-Amino acid oxidase (DAAO) is a potential target in the treatment of schizophrenia as its inhibition increases brain d-serine level and thus contributes to NMDA receptor activation. Inhibitors of DAAO were sought testing [6+5] type heterocycles and identified isatin derivatives as micromolar DAAO inhibitors. A pharmacophore and structure-activity relationship analysis of isatins and reported DAAO inhibitors led us to investigate 1H-indazol-3-ol derivatives and nanomolar inhibitors were identified. The series was further characterized by pK_a and isothermal titration calorimetry measurements. Representative compounds exhibited beneficial properties in in vitro metabolic stability and PAMPA assays. 6-fluoro-1H-indazol-3-ol (37) significantly increased plasma d-serine level in an in vivo study on mice. These results show that the 1H-indazol-3-ol series represents a novel class of DAAO inhibitors with the potential to develop drug candidates.