Novel 5-aryloxypyrimidine SEN1576 as a candidate for the treatment of Alzheimer's disease.

Prefibrillar assembly of amyloid-ß (Aß) is a major event underlying the development of neuropathology and dementia in Alzheimer's disease (AD). This study determined the neuroprotective properties of an orally bioavailable Aß synaptotoxicity inhibitor, SEN1576. Binding of SEN1576 to monomeric Aß 1-42 was measured using surface plasmon resonance. Thioflavin-T and MTT assays determined the ability of SEN1576 to block Aß 1-42-induced aggregation and reduction in cell viability, respectively. In vivo long-term potentiation (LTP) determined effects on synaptic toxicity induced by intracerebroventricular (i.c.v.) injection of cell-derived Aß oligomers. An operant behavioural schedule measured effects of oral administration following i.c.v. injection of Aß oligomers in normal rats. SEN1576 bound to monomeric Aß 1-42, protected neuronal cells exposed to Aß 1-42, reduced deficits in in vivo LTP and behaviour. SEN1576 exhibits the necessary features of a drug candidate for further development as a disease modifying treatment for the early stages of AD-like dementia.

Dose-dependent improvements in learning and memory deficits in APPPS1-21 transgenic mice treated with the orally active Aß toxicity inhibitor SEN1500.

In the Alzheimer's disease (AD) brain, accumulation of Aß1-42 peptides is suggested to initiate a cascade of pathological events. To date, no treatments are available that can reverse or delay AD-related symptoms in patients. In the current study, we introduce a new Aß toxicity inhibitor, SEN1500, which in addition to its block effect on Aß1-42 toxicity in synaptophysin assays, can be administered orally and cross the blood-brain barrier without adverse effects in mice. In a different set of animals, APPPS1-21 mice were fed with three different doses of SEN1500 (1 mg/kg, 5 mg/kg and 20 mg/kg) for a period of 5 months. Cognition was assessed in a variety of behavioral tests (Morris water maze, social recognition, conditioned taste aversion and passive avoidance). Results suggest a positive effect on cognition with 20 mg/kg SEN1500 compared to control APPPS1-21 mice. However, no changes in soluble or insoluble Aß1-40 and Aß1-42 were detected in the brains of SEN1500-fed mice. SEN1500 also attenuated the effect of Aß1-42 on synaptophysin levels in mouse cortical neurons, which indicated that the compound blocked the synaptic toxicity of Aß1-42. In vitro and in vivo effects presented here suggest that SEN1500 could be an interesting AD therapeutic.

Orally bioavailable small molecule drug protects memory in Alzheimer's disease models.

Oligomers of beta-amyloid (Aß) are implicated in the early memory impairment seen in Alzheimer's disease before to the onset of discernable neurodegeneration. Here, the capacity of a novel orally bioavailable, central nervous system-penetrating small molecule 5-aryloxypyrimidine, SEN1500, to prevent cell-derived (7PA2 [conditioned medium] CM) Aß-induced deficits in synaptic plasticity and learned behavior was assessed. Biochemically, SEN1500 bound to Aß monomer and oligomers, produced a reduction in thioflavin-T fluorescence, and protected a neuronal cell line and primary cortical neurons exposed to synthetic soluble oligomeric Aß(1-42). Electrophysiologically, SEN1500 alleviated the in vitro depression of long-term potentiation induced by both synthetic Aß(1-42) and 7PA2 CM, and alleviated the in vivo depression of long-term potentiation induced by 7PA2 CM, after systemic administration. Behaviorally, oral administration of SEN1500 significantly reduced memory-related deficits in operant responding induced after intracerebroventricular injection of 7PA2 CM. SEN1500 reduced cytotoxicity, acute synaptotoxicity, and behavioral deterioration after in vitro and in vivo exposure to synthetic Aß and 7PA2 CM, and shows promise for development as a clinically viable disease-modifying Alzheimer's disease treatment.

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a tetrameric intracellular Ca(2+) channel, which mediates the release of Ca(2+) from the endoplasmic reticulum in response to many different extracellular stimuli. We present a 3D structure of the type 1 IP(3)R obtained by electron microscopy and single-particle analysis that reveals its domain organization. The IP(3)R has a flower-like appearance with fourfold symmetry and is made up of three distinct domains connected by slender links. By relating the organization of the structural domains to secondary-structure predictions and biochemical data we develop a model in which structural domains are mapped onto the amino acid sequence to deduce the location of the channel region and the cytoplasmic inositol 1,4,5-trisphosphate-binding and modulatory subdomains. The structure of the IP(3)R is compared with that of other tetrameric cation channels. The channel domain is similar in size and shape to its counterparts in the ryanodine receptor and the Shaker voltage-gated K(+) channel.

Interactions of inositol 1,4,5-trisphosphate (IP(3)) receptors with synthetic poly(ethylene glycol)-linked dimers of IP(3) suggest close spacing of the IP(3)-binding sites.

The distances between the inositol 1,4,5-trisphosphate (IP(3))-binding sites of tetrameric IP(3) receptors were probed using dimers of IP(3) linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated (45)Ca(2+) release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP(3) receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3-4-fold more potent than IP(3). In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP(3). The affinity of monomeric IP(3) with only the PEG attached was 2-fold weaker than IP(3), confirming that the increased affinity of the dimers requires the presence of both IP(3) motifs. The increased affinity of the long dimer probably results from the linked IP(3) molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP(3) to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP(3) to purified receptors. IP(3) and the IP(3) dimers, irrespective of their length, bound with similar affinity to a monomeric IP(3)-binding domain of the type 1 IP(3) receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP(3)-binding sites of an IP(3) receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 x 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP(3)-binding sites of the IP(3) receptor.

Determinants of adenophostin A binding to inositol trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP(3)) receptors from cerebellum and recombinant type 1 IP(3) receptors expressed in Sf9 cells had indistinguishable affinities for IP(3) ( K (d)=6.40+/-0.48 nM) and adenophostin A ( K (d)=0.89+/-0.05 nM). In cytosol-like medium, each of the three mammalian IP(3) receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP(3). It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [(3)H]IP(3) from type 1 IP(3) receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [(3)H]IP(3) binding to the 1-604 protein, but abolished binding to the 224-604 protein. The 1-604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP(3), suggesting that C-terminal residues selectively inhibit IP(3) binding. The 224-604S1(+) fragment bound IP(3) and adenophostin A with increased affinity, but as with the 1-604 fragment it bound adenophostin A with only 2-fold greater affinity than IP(3). High-affinity binding of adenophostin A may be partially determined by its 2'-phosphate interacting more effectively than the 1-phosphate of IP(3) with residues within the IP(3)-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP(3) for the minimal IP(3)-binding domain. In addition we suggest that C-terminal residues, which impede access of IP(3), may selectively interact with adenophostin A to allow it unhindered access to the IP(3)-binding domain.

A novel Ca2+-induced Ca2+ release mechanism mediated by neither inositol trisphosphate nor ryanodine receptors.

Members of both major families of intracellular Ca(2+) channels, ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, are stimulated by substantial increases in cytosolic free Ca(2+) concentration ([Ca(2+)]c). They thereby mediate Ca(2+)-induced Ca(2+) release (CICR), which allows amplification and regenerative propagation of intracellular Ca(2+) signals. In permeabilized hepatocytes, increasing [Ca(2+)]c to 10 microM stimulated release of 30+/-1% of the intracellular stores within 60 s; the EC(50) occurred with a free [Ca(2+)] of 170+/-29 nM. This CICR was abolished at 2 degrees C. The same fraction of the stores was released by CICR before and after depletion of the IP3-sensitive stores, and CICR was not blocked by antagonists of IP3 receptors. Ryanodine, Ruthenium Red and tetracaine affected neither the Ca(2+) content of the stores nor the CICR response. Sr(2+) and Ba(2+) (EC(50)=166 nM and 28 microM respectively) mimicked the effects of increased [Ca(2+)] on the intracellular stores, but Ni(2+) blocked the passive leak of Ca(2+) without blocking CICR. In rapid superfusion experiments, maximal concentrations of IP3 or Ca(2+) stimulated Ca(2+) release within 80 ms. The response to IP3 was complete within 2 s, but CICR continued for tens of seconds despite a slow [half-time (t(1/2))=3.54+/-0.07 s] partial inactivation. CICR reversed rapidly (t(1/2)=529+/-17 ms) and completely when the [Ca(2+)] was reduced. We conclude that hepatocytes express a novel temperature-sensitive, ATP-independent CICR mechanism that is reversibly activated by modest increases in [Ca(2+)], and does not require IP3 or ryanodine receptors or reversal of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase. This mechanism may both regulate the Ca(2+) content of the intracellular stores of unstimulated cells and allow even small intracellular Ca(2+) signals to be amplified by CICR.