Highly Specific and Sensitive Target Binding by the Humanized pS396-Tau Antibody hC10.2 Across a Wide Spectrum of Alzheimer's Disease and Primary Tauopathy Postmortem Brains.

BACKGROUND: Deposits of hyperphosphorylated tau fibrils are hallmarks of a broad spectrum of tauopathies, including Alzheimer's disease (AD). OBJECTIVE: To investigate heterogeneity of tau pathology across brain extracts from a broad selection of different tauopathies and examine the binding properties of the humanized pS396-tau antibody hC10.2 and six other anti-tau antibodies. METHODS: 76 individual tauopathy tissue samples were analyzed in a battery of assays: immunohistochemistry, ELISA, tau aggregation assay, western blot, [3H]PI-2620 and [3H]MK-6240 tau tracer binding, and aggregated seeding activity in RD_P301S HEK293T Biosensor cells. The efficiency of seven anti-tau antibodies to engage with pathological tau species was directly compared. RESULTS: Our data indicate that a strong correlation existed between the tau tracer binding, amount of tau aggregates, pS396-tau phosphorylation, and seeding activity. The hC10.2 antibody, which has entered clinical development, effectively engaged with its epitope across all individual cases of mid-stage and late AD, and primary tauopathies. hC10.2 was superior compared to other phospho- and total tau antibodies to prevent seeded tau aggregation in the biosensor cells. hC10.2 effectively depleted hyperphosphorylated and aggregated tau species across all tauopathy samples proportionally to the amount of tau aggregates. In AD samples, hC10.2 bound to ghost tangles which represent extracellular pathological tau species. CONCLUSION: S396 hyperphosphorylation is a feature of the formation of seeding-competent tau across different tauopathies and it is present both in intra- and extracellular pathological tau. hC10.2 represents an excellent candidate for a hyperphosphorylation-selective therapeutic tau antibody for the treatment of AD and primary tauopathies.

Debenzylative Cycloetherification as a Synthetic Tool in the Diastereoselective Synthesis of 3,6-Disubstituted Hexahydro-2H-furo[3,2-b]pyrroles, PDE1 Enzyme Inhibitors with an Antiproliferative Effect on Melanoma Cells.

Two series of novel chiral hexahydro-2H-furo[3,2-b]pyrroles, 4-(7,8-dimethoxyquinazolin-4-yl) series A and 4-(6,7- dimethoxyquinazolin-4-yl) series B, were synthesized in enantiomerically pure form and evaluated for their inhibitory effects on phosphodiesterase 1 (PDE1) and phosphodiesterase 4 (PDE4) as well as for their inhibitory activity on cell proliferation in A375 melanoma and 3T3 fibroblast cells in vitro. Key steps of synthesis were (i) diastereoselective nucleophilic addition of vinylmagnesium bromide to N-allylimine derived from conveniently protected d-glyceraldehyde, (ii) ring-closing metathesis, (iii) debenzylative cycloetherification, and (iv) aromatic nucleophilic substitution. Some of the obtained compounds were proven to be active as inhibitors of PDE1 isoforms, with IC50 values in the high nanomolar/low micromolar concentration range, and showed antiproliferative activity on A375 melanoma cells.

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum.

The calcium-regulated phosphodiesterase 1 (PDE1) family is highly expressed in the brain, but its functional role in neurones is poorly understood. Using the selective PDE1 inhibitor Lu AF64196 and biosensors for cyclic nucleotides including a novel biosensor for cGMP, we analyzed the effect of PDE1 on cAMP and cGMP in individual neurones in brain slices from male newborn mice. Release of caged NMDA triggered a transient increase of intracellular calcium, which was associated with a decrease in cAMP and cGMP in medium spiny neurones in the striatum. Lu AF64196 alone did not increase neuronal cyclic nucleotide levels, but blocked the NMDA-induced reduction in cyclic nucleotides indicating that this was mediated by calcium-activated PDE1. Similar effects were observed in the prefrontal cortex and the hippocampus. Upon corelease of dopamine and NMDA, PDE1 was shown to down-regulate the D1-receptor mediated increase in cAMP. PDE1 inhibition increased long-term potentiation in rat ventral striatum, showing that PDE1 is implicated in the regulation of synaptic plasticity. Overall, our results show that PDE1 reduces cyclic nucleotide signaling in the context of glutamate and dopamine coincidence. This effect could have a therapeutic value for treating brain disorders related to dysfunctions in dopamine neuromodulation.

Elucidation of fluorine's impact on pKa and in vitro Pgp-mediated efflux for a series of PDE9 inhibitors.

P-Glycoprotein (Pgp)-mediated cellular efflux is recognized as a common challenge in CNS drug discovery. In this study, the influence of replacing a hydrogen atom with fluorine on the pKa and Pgp-mediated efflux is elucidated for a series of PDE9 inhibitors. The PDE9 inhibitors with and without fluorine were synthesized using a novel condensation-oxidation approach, providing access to several analogues, all from the same stereoenriched aldehyde building block. The incorporation of fluorine was found to influence two acid-base functionalities concomitantly, both of which were involved in Pgp-recognition. By methylating the acidic functionality, it was possible to isolate the effect responsible for lowering the Pgp-mediated efflux.

A mouse model of the schizophrenia-associated 1q21.1 microdeletion syndrome exhibits altered mesolimbic dopamine transmission.

1q21.1 hemizygous microdeletion is a copy number variant leading to eightfold increased risk of schizophrenia. In order to investigate biological alterations induced by this microdeletion, we generated a novel mouse model (Df(h1q21)/+) and characterized it in a broad test battery focusing on schizophrenia-related assays. Df(h1q21)/+ mice displayed increased hyperactivity in response to amphetamine challenge and increased sensitivity to the disruptive effects of amphetamine and phencyclidine hydrochloride (PCP) on prepulse inhibition. Probing of the direct dopamine (DA) pathway using the DA D1 receptor agonist SKF-81297 revealed no differences in induced locomotor activity compared to wild-type mice, but Df(h1q21)/+ mice showed increased sensitivity to the DA D2 receptor agonist quinpirole and the D1/D2 agonist apomorphine. Electrophysiological characterization of DA neuron firing in the ventral tegmental area revealed more spontaneously active DA neurons and increased firing variability in Df(h1q21)/+ mice, and decreased feedback reduction of DA neuron firing in response to amphetamine. In a range of other assays, Df(h1q21)/+ mice showed no difference from wild-type mice: gross brain morphology and basic functions such as reflexes, ASR, thermal pain sensitivity, and motor performance were unaltered. Similarly, anxiety related measures, baseline prepulse inhibition, and seizure threshold were unaltered. In addition to the central nervous system-related phenotypes, Df(h1q21)/+ mice exhibited reduced head-to tail length, which is reminiscent of the short stature reported in humans with 1q21.1 deletion. With aspects of both construct and face validity, the Df(h1q21)/+ model may be used to gain insight into schizophrenia-relevant alterations in dopaminergic transmission.

Characterisation of Lu AF33241: A novel, brain-penetrant, dual inhibitor of phosphodiesterase (PDE) 2A and PDE10A.

Here, we present a preliminary pharmacological characterisation of Lu AF33241, a novel, brain penetrant phosphodiesterase inhibitor of (PDE) 2A and 10A tool compound, in in vitro/in vivo assays indicative of PDE2A and/or PDE10A inhibition, and in vivo models/assays relevant to cognitive processing and antipsychotic-like activity. An assay was also included to investigate potential effects on motor activity. The in vitro selectivity of Lu AF33241 was determined against a panel of PDE enzymes. Lu AF33241 potently inhibited both full-length recombinant hPDE2A (Ki=4.2nM) and hPDE10A (Ki=42nM). The compound moderately inhibited both hPDE1C (Ki=1200nM), hPDE7B (Ki=890nM), and hPDE11A (Ki=1800nM). Lu AF33241 displayed a Ki above 5000nM against all other tested members of the PDE family. Albeit within a narrow dose range, Lu AF33241 attenuated sub-chronic phencyclidine-induced deficits in novel object recognition (3 and 10mg/kg), displayed antipsychotic-like activity in the conditioned avoidance response paradigm (10mg/kg), and did not induce catalepsy within a dose-range of 2-6mg/kg. Further catalepsy studies are needed to investigate a predictive safety window. Lu AF33241 represents a novel PDE2A/PDE10A inhibitor tool compound that may serve to further the understanding of the roles played by these enzymes in various CNS disorders.

Novel aza-analogous ergoline derived scaffolds as potent serotonin 5-HT6 and dopamine D2 receptor ligands.

By introducing distal substituents on a tetracyclic scaffold resembling the ergoline structure, two series of analogues were achieved exhibiting subnanomolar receptor binding affinities for the dopamine D2 and serotonin 5-HT6 receptor subtype, respectively. While the 5-HT6 ligands were antagonists, the D2 ligands displayed intrinsic activities ranging from full agonism to partial agonism with low intrinsic activity. These structures could potentially be interesting for treatment of neurological diseases such as schizophrenia, Parkinson's disease, and cognitive deficits.

In vitro and in vivo characterisation of Lu AF64280, a novel, brain penetrant phosphodiesterase (PDE) 2A inhibitor: potential relevance to cognitive deficits in schizophrenia.

Here, we present the pharmacological characterisation of Lu AF64280, a novel, selective, brain penetrant phosphodiesterase (PDE) 2A inhibitor, in in vitro/in vivo assays indicative of PDE2A inhibition, and in vivo models/assays relevant to cognitive processing or antipsychotic-like activity. The in vitro selectivity of Lu AF64280 was determined against a panel of PDE enzymes and 3',5'-cyclic guanosine monophosphate (cGMP) levels in the hippocampus were determined using in vivo microdialysis. Lu AF64280 potently inhibited hPDE2A (Ki = 20 nM), 50-fold above moderate inhibition of both hPDE9A (Ki = 1,000 nM) and hPDE10A (Ki = 1,800 nM), and displayed a >250-fold selectivity over all other full-length human recombinant PDE family members (Ki above 5,000 nM). Lu AF64280 (20 mg/kg) significantly increased cGMP levels in the hippocampus (p < 0.01 versus vehicle-treated mice), attenuated sub-chronic phencyclidine-induced deficits in novel object exploration in rats (10 mg/kg, p < 0.001 versus vehicle-treated), blocked early postnatal phencyclidine-induced deficits in the intradimensional/extradimensional shift task in rats (1 and 10 mg/kg, p < 0.001 versus vehicle-treated) and attenuated spontaneous P20-N40 auditory gating deficits in DBA/2 mice (20 mg/kg, p < 0.05 versus vehicle-treated). In contrast, Lu AF64280 failed to attenuate phencyclidine-induced hyperactivity in mice, and was devoid of antipsychotic-like activity in the conditioned avoidance response paradigm in rats, at any dose tested. Lu AF64280 represents a novel tool compound for selective PDE2A inhibition that substantiates a critical role of this enzyme in cognitive processes under normal and pathological conditions.

Synthesis and SAR study of a novel series of dopamine receptor agonists.

The synthesis of a novel series of dopamine receptor agonists are described as well as their in vitro potency and efficacy on dopamine D1 and D2 receptors. This series was designed from pergolide and (4aR,10aR)-1-propyl-1,2,3,4,4a,5,10,10a-octahydro-benzo[g]quinolin-6-ol (PHBQ) and resulted in the synthesis of (2R,4aR,10aR)-2-methylsulfanylmethyl-4-propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol (compound 27), which has a D1 and D2 receptor profile similar to that of the most recently approved drug for Parkinson's disease, rotigotine.

Discovery of novel α1-adrenoceptor ligands based on the antipsychotic sertindole suitable for labeling as PET ligands.

The synthesis and in vitro preclinical profile of a series of 5-heteroaryl substituted analogs of the antipsychotic drug sertindole are presented. Compounds 1-(4-fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-(pyrimidin-5-yl)-1H-indole (Lu AA27122, 3i) and 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(1-methylpiperidin-4-yl)-1H-indole (3l) were identified as high affinity α(1A)-adrenoceptor ligands with K(i) values of 0.52 and 0.16 nM, respectively, and with a >100-fold selectivity versus dopamine D(2) receptors. Compound 3i showed almost equal affinity for α(1B)- (K(i)=1.9 nM) and α(1D)-adrenoceptors (K(i)=2.5 nM) as for α(1A), as well as moderate affinity for 5-HT(1B) (K(i)=13 nM) and 5-HT(6) (K(i)=16 nM) receptors, whereas 3l showed >40-fold selectivity toward all other targets tested. Based on in vitro assays for assessment of permeability rates and extent, it is predicted that both compounds enter the brain of rats, non-human primates, as well as humans, and as such are good candidates to be carried forward for further evaluation as positron emission tomography (PET) ligands.

Exploring the neuroleptic substituent in octoclothepin: potential ligands for positron emission tomography with subnanomolar affinity for α(1)-adrenoceptors.

A series of 1-(10,11-dihydrodibenzo[b,f]thiepin-10-yl)-4-methylpiperazine analogues substituted in the 8-position of the 10,11-dihydrodibenzo[b,f]thiepine scaffold with aryl, heteroaryl, amine, and amide substituents are described. The compounds were designed using the previously reported Liljefors-Bøgesø pharmacophore model for dopamine D(2) and α(1)-adrenoceptor antagonists, with the aim of obtaining selective α(1)-adrenoceptor antagonists suitable for development as radioligands for imaging of central α(1)-adrenoceptors by positron emission tomography. Sixteen aryl and heteroaryl substituted octoclothepin analogues were prepared by a convergent synthesis via coupling of 1-methyl-4-(8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazine with aryl and heteroaryl halides under palladium catalysis. The most selective compound obtained, (S)-N-((11-(4-methylpiperazin-1-yl)-10,11-dihydrodibenzo[b,f]thiepin-2-yl)methyl)isobutyramide (S)-35, showed a similar subnanomolar affinity compared to α(1a), α(1b), and α(1d)-adrenoceptors and a selectivity ratio of 20, 440, and 20 with respect to D(2), 5-HT(2C), and H(1) receptors, respectively.

Discovery of N-{1-[3-(3-oxo-2,3-dihydrobenzo[1,4]oxazin-4-yl)propyl]piperidin-4-yl}-2-phenylacetamide (Lu AE51090): an allosteric muscarinic M1 receptor agonist with unprecedented selectivity and procognitive potential.

The discovery and structure-activity relationship (SAR) of a series of allosteric muscarinic M(1) receptor agonists are described. Compound 17 (Lu AE51090) was identified as a representative compound from the series, based on its high selectivity as an agonist at the muscarinic M(1) receptor across a panel of muscarinic receptor subtypes. Furthermore, 17 displayed a high degree of selectivity when tested in a broad panel of G-protein-coupled receptors, ion channels, transporters, and enzymes, and 17 showed an acceptable pharmacokinetic profile and sufficient brain exposure in rodents in order to characterize the compound in vivo. Hence, in a rodent model of learning and memory, 17 reversed delay-induced natural forgetting, suggesting a procognitive potential of 17.

Design, synthesis and biological evaluation of novel bicyclo[1.1.1]pentane-based omega-acidic amino acids as glutamate receptors ligands.

A novel series of bicyclo[1.1.1]pentane-based omega-acidic amino acids, including (2S)- and (2R)-3-(3'-carboxybicyclo[1.1.1]pentyl)alanines (8 and 9), (2S)- and (2R)-2-(3'-carboxymethylbicyclo[1.1.1]pentyl)glycines (10 and 11), and (2S)- and (2R)-3-(3'-phosphonomethylbicyclo[1.1.1]pentyl)glycines (12 and 13), were synthesized and evaluated as glutamate receptor ligands. Among them, (2R)-3-(3'-phosphonomethylbicyclo[1.1.1]pentyl)glycine (13) showed relatively high affinity and selectivity at the NMDA receptor. The results are also discussed in light of pharmacophoric modelling studies of NMDA agonists and antagonists.

Development of an aequorin luminescence calcium assay for high-throughput screening using a plate reader, the LumiLux.

A luminescence assay using a new plate reader, the LumiLux (PerkinElmer, Waltham, MA), has been validated for high-throughput screening (HTS). In this study, we compared the aequorin luminescence-based calcium mobilization assay to the fluorescence-based calcium assay. A cell line stably co-expressing apo-aequorin, a chimeric G-protein, and a G-protein-coupled dopamine receptor was used to screen a collection of 8,106 compounds using the Hamamatsu Photonics (Bridgewater, NJ) FDSS6000 and LumiLux as the plate readers. The assay parameters evaluated included hit rate correlation, signal-to-noise ratio, and overall assay performance calculated by Z and standard deviation. The average Z values and hit rates were comparable between assay platforms;however, the standard deviation for the agonist aequorin assay was significantly smaller. There was also a significant decrease in the number of false-positives with the aequorin assay. These results suggest that the aequorin assay in combination with the new plate reader, LumiLux, provides a simple, cost-effective, robust, and sensitive assay for HTS

Lu 35-138 ((+)-(S)-3-{1-[2-(1-acetyl-2,3-dihydro-1H-indol-3-yl)ethyl]-3,6-dihydro-2H-pyridin-4-yl}-6-chloro-1H-indole), a dopamine D4 receptor antagonist and serotonin reuptake inhibitor: characterisation of its in vitro profile and pre-clinical antipsychotic potential.

The present study describes the pharmacological profile of the putative antipsychotic drug Lu 35-138 ((+)-(S)-3-{1-[2-(1-acetyl-2,3-dihydro-1H-indol-3-yl)ethyl]-3,6-dihydro-2H-pyridin-4-yl}-6-chloro-1H-indole). The in vitro receptor profile of Lu 35-138 revealed high affinity (K(i)=5 nM) and competitive antagonism (K(b)=8 nM) at dopamine D(4) receptors combined with potent 5-HT uptake inhibition (IC(50)=3.2 nM) and moderate alpha(1)-adrenoceptor affinity (K(i)=45 nM). In vivo, Lu 35-138 selectively counteracted hyperlocomotion induced by d-amphetamine (0.5 mg/kg; ED(50)=4.0 mg/kg, s.c.) in rats and phencyclidine (PCP; 2.5 mg/kg; ED(50)=13 mg/kg, s.c.) in mice. Lu 35-138 was unable to affect hyperlocomotion induced by a high dose of d-amphetamine (2.0 mg/kg), which indicates a preferential action on limbic versus striatal structures. A similar limbic selectivity of Lu 35-138 was indicated in voltammetric measure of dopamine output in the core and shell subdivisions of the nucleus accumbens in rats. Furthermore, a relatively large dose of Lu 35-138 (18 mg/kg, s.c.) counteracted d-amphetamine-induced disruption of pre-pulse inhibition in rats and repeated administration of Lu 35-138 (0.31 or 1.25 mg/kg, p.o. once daily for 3 weeks) reduced the number of spontaneously active dopamine neurones in the ventral tegmental area, underlining its antipsychotic-like profile. Lu 35-138 failed to induce catalepsy in rats or dystonia in Cebus apella monkeys and did not deteriorate spatial memory in rats as assessed by water maze performance. Collectively, these results suggest that Lu 35-138 possesses antipsychotic activity combined with a low extrapyramidal and cognitive side effect liability.

IRS-4 mediated mitogenic signalling by insulin and growth hormone in LB cells, a murine T-cell lymphoma devoid of IGF-I receptors.

Insulin and growth hormone (GH) induce mitogenic and metabolic signals in cells, GH either directly or indirectly via IGF-I production. We have studied a spontaneous murine T-cell lymphoma (LB cells) devoid of IGF-1 receptors in which proliferation is maintained by insulin [Int. J. Cancer 50 (1992) 80], and show that GH is more potent than insulin, with both GH and insulin dose-response curves for thymidine incorporation being bell-shaped. Binding showed somatogenic rather than lactogenic GH receptors. Insulin stimulated phosphorylation of the insulin receptor and of a 160-kDa protein, identified as the IRS-4 protein. This phosphorylated IRS-4 associated with PI3-kinase, which was activated along with the downstream p70(S6) kinase, whereas the Ras-MAPK pathway was not. Using selective inhibitors, the PI3-kinase, but not p70(S6) kinase or MEK, was found to be involved in insulin-stimulated DNA synthesis. GH induced tyrosine phosphorylation of IRS-4 and nuclear translocation of STAT5. The LB cells constitute a new model for studying GH and insulin signalling without interference of IGF-1 receptors.