Janet Greeson

Identification of naturally occurring spirostenols preventing β-amyloid-induced neurotoxicity

22R-Hydroxycholesterol is an intermediate in the steroid biosynthesis pathway shown to exhibit a neuroprotective property against beta-amyloid (1-42) (Abeta) toxicity in rat PCl2 and human NT2N neuronal cells by binding and inactivating Abeta. In search of potent 22R-hydroxycholesterol derivatives, we assessed the ability of a series of naturally occurring entities containing the 22R-hydroxycholesterol structure to protect PC12 cells against Abeta-induced neurotoxicity, determined by measuring changes in membrane potential, mitochondrial diaphorase activity, ATP levels and trypan blue uptake. 22R-Hydroxycholesterol derivatives sharing a common spirost-5-en-3-ol or a furost-5-en-3-ol structure were tested. Although some of these compounds were neuroprotective against 0.1 microM Abeta, only three protected against the 1-10 microM Abeta-induced toxicity and, in contrast to 22R-hydroxycholesterol, all were devoid of steroidogenic activity. These entities shared a common structural feature, a long chain ester in position 3 and common stereochemistry. The neuroprotective property of these compounds was coupled to their ability to displace radiolabeled 22R-hydroxycholesterol from Abeta, suggesting that the Abeta-22R-hydroxycholesterol physicochemical interaction contributes to their beneficial effect. In addition, a 22R-hydroxycholesterol derivative inhibited the formation of neurotoxic amyloid-derived diffusible ligands. Computational docking simulations of 22R-hydroxycholesterol and its derivatives on Abeta identified two binding sites. Chemical entities, as 22R-hydroxycholesterol, seem to bind preferentially only to one site. In contrast, the presence of the ester chain seems to confer the ability to bind to both sites on Abeta, leading to neuroprotection against high concentrations of Abeta. In conclusion, these results suggest that spirost-5-en-3-ol naturally occurring derivatives of 22R-hydroxycholesterol might offer a new approach for Alzheimers disease therapy.

22R-Hydroxycholesterol protects neuronal cells from β-amyloid-induced cytotoxicity by binding to β-amyloid peptide

22R‐hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimers disease (AD) hippocampus and frontal cortex tissue specimens compared to age‐matched controls. β‐Amyloid (Aβ) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R‐hydroxycholesterol was found to protect, in a dose‐dependent manner, against Aβ‐induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R‐hydroxycholesterol was found to be stereospecific because its enantiomer 22S‐hydroxycholesterol failed to protect the neurons from Aβ‐induced cell death. Moreover, the effect of 22R‐hydroxycholesterol was specific for Aβ‐induced cell death because it did not protect against glutamate‐induced neurotoxicity. The neuroprotective effect of 22R‐hydroxycholesterol was seen when using Aβ1−42 but not the Aβ25−35 peptide. To investigate the mechanism of action of 22R‐hydroxycholesterol we examined the direct binding of this steroid to Aβ using a novel cholesterol‐protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R‐hydroxycholesterol to Aβ1−42 and Aβ17−40, but not Aβ25−35, was observed. These data suggest that 22R‐hydroxycholesterol binds to Aβ and the formed 22R‐hydroxycholesterol/Aβ complex is not toxic to rodent and human neurons. We propose that 22R‐hydroxycholesterol offers a new means of neuroprotection against Aβ toxicity by inactivating the peptide.

Beta-amyloid and oxidative stress jointly induce neuronal death, amyloid deposits, gliosis, and memory impairment in the rat brain.

Infusion of Fe2+, Aβ42, and buthionine-sulfoximine (FAB), but not Aβ42 alone or in combination with Fe2+, into the left cerebral ventricle of Long-Evans rats for 4 weeks induced memory impairment that was accompanied by increased hyperphosphorylated Tau protein levels in the CSF. FAB-infused animals displayed thioflavin-S-positive amyloid deposits, hyperphosphorylated Tau protein, neuronal loss, and gliosis. Animals treated with Aβ42, Fe2+, or buthionine-sulfoximine alone or in combination failed to show the histological modifications seen with FAB. This data suggests that Aβ42 is not sufficient to induce an Alzheimer’s disease-like symptomatology, and it supports a model whereby a decrease in the brain’s antioxidant defense system leads to the Aβ42-independent oxidative stress necessary for the peptide to induce histopathological changes and memory loss.

The spirostenol (22R, 25R)-20α-spirost-5-en-3β-yl hexanoate blocks mitochondrial uptake of Aβ in neuronal cells and prevents Aβ-induced impairment of mitochondrial function

Abeta(1-42) has been shown to uncouple the mitochondrial respiratory chain and promote the opening of the membrane permeability transition (MPT) pore, leading to cell death. We have previously reported that the spirostenol derivative (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate (SP-233) protects neuronal cells against Abeta(1-42) toxicity by binding to and inactivating the peptide. Picomolar concentrations of Abeta(1-42) decreased the mitochondrial respiratory coefficient in mitochondria isolated from the rat forebrain, and this decrease was partially reversed by SP-233. SP-233 abolished the uncoupling of oxidative phosphorylation induced by carbonyl cyanide 3-chlorophenylhydrazone on isolated mitochondria. These results are consistent with a direct effect of SP-233 on the MPT. Moreover, SP-233 displayed a neuroprotective effect on SK-N-AS human neuroblastoma cells treated with the MPT promoter, phenylarsine oxide. Treatment of SK-N-AS cells with Abeta(1-42) resulted in an accumulation of the peptide in the mitochondrial matrix; SP-233 completely scavenged Abeta(1-42) from the matrix. In addition, SP-233 protected the cells against mitochondrial toxins targeting complexes IV and V of the respiratory chain. These results indicate that Abeta(1-42) and SP-233 exert direct effects on mitochondrial function and SP-233 protects neuronal cells against Abeta-induced toxicity by targeting Abeta directly.

Caprospinol: moving from a neuroactive steroid to a neurotropic drug

In search of new drugs for Alzheimers disease, we departed from the classic concepts and investigated the ability of normal and Alzheimers disease brain to convert cholesterol to steroids, otherwise known as neurosteroids. We identified 22R-hydroxycholesterol to be present in much lower levels in the hippocampus and frontal cortex of Alzheimers disease than in tissue from age-matched controls. 22R-hydroxycholesterol was shown to protect against β-amyloid (Aβ42)-induced neurotoxicity and block the formation of Aβ oligomers. In search of a 22R-hydroxycholesterol stable analog, we identified the naturally occurring heterospirostenol, (22R,25R)-20α-spirost-5-en-3β-yl hexanoate (caprospinol). The mechanism of action underlying the neuroprotective properties of caprospinol involves, first, the ability of the compound to bind Aβ42 and, second, its interaction with components of the mitochondria respiratory chain. Samaritan Pharmaceuticals is developing caprospinol as a disease-modifying drug for the treatment of Alzheimers disease. Samaritan Pharmaceuticals filed for an Investigational New Drug application with the FDA in 2006. The pharmacokinetic and pharmacodynamic parts of the application were found satisfactory, and the FDA has requested that additional information is submitted in support of caprospinols safety prior to initiating the Phase I clinical study.

Dimethyl-Carbamic Acid 2,3-Bis-Dimethylcarbamoyloxy-6-(4-Ethyl-Piperazine- 1-Carbonyl)-Phenyl Ester: A Novel Multi-Target Therapeutic Approach to Neuroprotection

We report herein the synthesis and biological evaluation of dimethyl-carbamic acid 2,3-bis-dimethylcarbamoyloxy-6-(4-ethyl-piperazine-1-carbonyl)-phenyl ester (SP-04), a new drug candidate that is designed to offer a multi-target therapeutic neuroprotective approach as a treatment for Alzheimers disease (AD). SP-04 inhibits acetylcholinesterase (AchE) activity both in vitro and in vivo, and induces a dose-dependent increase in Ach levels. SP-04 releases the metabolite 4-(4-ethyl-piperazin-1-yl)-1-(2,3,4-trihydroxy-phenyl)-butan-1-one (SP-04m). Both SP-04 and SP-04m are s1-receptor antagonists supporting their interest in relieving symptoms related to psychosis, a non-cognitive condition often associated with AD. SP-04m displays important antioxidant properties and both SP-04 and SP-04m offers neuroprotection against Ab42 toxicity in various neuronal cell lines. In addition, both SP-04 and SP-04m protect neuronal cells and rat brain mitochondria exposed to various mitochondrial respiratory chain complex toxins. Taken together these data suggest that the SP-04 multi-targeting approach might offer a novel therapeutic strategy for the treatment of AD.

Local Anesthetic Procaine Protects Rat Pheochromocytoma PC12 Cells against β-Amyloid-Induced Neurotoxicity

Alzheimer’s disease (AD) is the most common dementia occurring in elderly. We report herein the neuroprotective properties of procaine and other anesthetic agents against β-amyloid-induced neurotoxicity. Procaine displayed strong neuroprotective properties against the amyloid peptide Aβ1–42 and preserved Aβ1–42-induced ATP depletion on rat pheochromocytoma PC12 cells. Procaine also inhibited the neurotoxic effect that glutamate displayed on PC12 cells, suggesting that the reduction of glutamate-induced neurotoxicity may be the mechanism by which these compounds exert their ‘antiamyloid’ effects. In search of a mechanism of action we observed that procaine is a ligand for the σ1 receptor, a protein which ligands have been shown to protect mitochondrial function and to exert antidepressant properties. Procaine binds also to muscarinic receptors but the true meaning of this feature needs to be clarified. In conclusion, these data suggest that procaine exerts neuroprotective properties and may serve either as a treatment for AD or as a starting point for the development of novel therapies for AD.

Identification, design, synthesis, and pharmacological activity of (4-ethyl-piperazin-1-yl)-phenylmethanone derivatives with neuroprotective properties against β-amyloid-induced toxicity

In search of novel therapeutic approaches for Alzheimers disease (AD), we report herein the identification, design, synthesis, and pharmacological activity of (4-ethyl-piperaz-1-yl)-phenylmethanone derivatives with neuroprotective properties against beta-amyloid-induced toxicity. (4-ethyl-piperaz-1-yl)-phenylmethanone is a common substructure shared by molecules isolated from plants of the Asteraceae genus, traditionally used as restorative of lost or declining mental functions. (4-Ethyl-piperaz-1-yl)-phenylmethanone displayed strong neuroprotective properties against Abeta1-42 and reversed Abeta1-42-induced ATP depletion on neuronal cells, suggesting a mitochondrial site of action. Abeta1-42 has been described to induce a hyperactivity of the glutamate network in neuronal cells. (4-Ethyl-piperaz-1-yl)-phenylmethanone also inhibited the neurotoxic effect that glutamate displayed on PC12 cells, suggesting that the reduction of glutamate-induced neurotoxicity may be one of the mechanisms by which this compound exerts its neuroprotective properties against the deleterious effects of the Abeta1-42. These data suggest that the identified (4-ethyl-piperaz-1-yl)-phenylmethanone chemical entity exerts neuroprotective properties and may serve as a lead compound for the development of novel therapies for AD.

The benzamide derivative N-(1-(7-tert-Butyl-1H-indol-3- ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin- 1-yl)-2-oxo-ethyl)-4-nitro-benzamide (SP-10) reduces HIV-1 infectivity in vitro by modifying actin dynamics

Current treatments for patients infected with HIV are suboptimal. There is a need for new HIV therapies that act through different mechanisms than current treatments. We investigated the in vitro efficacy, safety and mechanism of action of the benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10), a potential new HIV treatment. When HIV-1-responsive engineered HeLa cells were pre-incubated for 48 h with either SP-10 or zidovudine (AZT), SP-10 was able to inhibit viral replication at much lower concentrations (IC50=0.036 nM) than AZT (IC50=27.4 nM). In contrast to AZT, SP-10 also inhibited replication of the multidrug-resistant HIV-1 strain MDR-769 in the HeLa cell model. In co-incubation experiments, SP-10 also inhibited the CCR5-sensitive HIV-1 BaL virus replication in human peripheral blood mononuclear cells. SP-10 displayed very low toxicity compared with current antiviral treatments. Confocal laser scanning microscopy and immunoprecipitation studies showed that SP-10 reduced the expression of CD4 and CCR5 on the surface of the host cell. SP-10 also reduced the level of gp120 binding to the cell surface. Confocal laser scanning microscopy studies showed that SP-10 blocked the formation of actin filaments (F-actin) and altered actin accumulation near the cell surface. These promising results suggest that SP-10 has a novel mechanism of action that enables effective inhibition of HIV-1 binding and cell entry. Further development of SP-10 as a new HIV treatment appears warranted.

Identification of a benzamide derivative that inhibits stress-induced adrenal corticosteroid synthesis.

Elevated serum glucocorticoid levels contribute to the progression of many diseases, including depression, Alzheimer’s disease, hypertension, and acquired immunodeficiency syndrome. Here we show that the benzamide derivative N-[2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-1-(tert-butyl-1H-indol-3-yl-methyl)-2-oxo-ethyl]-4-nitrobenzamide (SP-10) inhibits dibutyryl cyclic AMP (dbcAMP)-induced corticosteroid synthesis in a dose-dependent manner in Y-1 adrenal cortical mouse tumor cells, without affecting basal steroid synthesis and reduced stress-induced corticosterone increases in rats without affecting the physiological levels of the steroid in blood. SP-10 did not affect cholesterol transport and metabolism by the mitochondria but was unexpectedly found to increase 3-hydroxy-3-methylglutaryl-coenzyme A, low density lipoprotein receptor, and scavenger receptor class B type I (SR-BI) expression. However, it also markedly reduced dbcAMP-induced NBD-cholesterol uptake, suggesting that this is a compensatory mechanism aimed at maintaining cholesterol levels. SP-10 also induced a redistribution of filamentous (F-) and monomeric (G-) actin, leading to decreased actin levels in the submembrane cytoskeleton suggesting that SP-10-induced changes in actin distribution might prevent the formation of microvilli– cellular structures required for SR-BI-mediated cholesterol uptake in adrenal cells.