RESUMEN
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 Alzheimer's 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 Abï±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.
Asunto(s)
Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/farmacología , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Receptores sigma/antagonistas & inhibidores , Acetilcolinesterasa , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Antioxidantes/síntesis química , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Carbamatos/síntesis química , Proteínas Ligadas a GPI/antagonistas & inhibidores , Ácido Gálico/análogos & derivados , Ácido Gálico/síntesis química , Células HEK293 , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Células PC12 , Fragmentos de Péptidos/metabolismo , Piperazinas/síntesis química , Profármacos/síntesis química , Ratas , Ratas Long-Evans , Receptor Sigma-1RESUMEN
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 SRBI-mediated cholesterol uptake in adrenal cells.
Asunto(s)
Corticoesteroides/biosíntesis , Glándulas Suprarrenales/metabolismo , Benzamidas/farmacología , Estrés Fisiológico/efectos de los fármacos , Actinas/metabolismo , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/enzimología , Animales , Benzamidas/química , Bucladesina/farmacología , Antígenos CD36/genética , Antígenos CD36/metabolismo , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Corticosterona/sangre , Sistema Enzimático del Citocromo P-450 , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
In search of new drugs for Alzheimer's disease, we departed from the classic concepts and investigated the ability of normal and Alzheimer's 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 Alzheimer's disease than in tissue from age-matched controls. 22R-hydroxycholesterol was shown to protect against beta-amyloid (A beta(42))-induced neurotoxicity and block the formation of A beta oligomers. In search of a 22R-hydroxycholesterol stable analog, we identified the naturally occurring heterospirostenol, (22R,25R)-20 alpha-spirost-5-en-3beta-yl hexanoate (caprospinol). The mechanism of action underlying the neuroprotective properties of caprospinol involves, first, the ability of the compound to bind A beta(42) 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 Alzheimer's 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 caprospinol's safety prior to initiating the Phase I clinical study.
Asunto(s)
Diosgenina/análogos & derivados , Neuronas/efectos de los fármacos , Esteroides/farmacología , Amiloide/metabolismo , Animales , Sitios de Unión , Caproatos , Diosgenina/química , Diosgenina/farmacología , Descubrimiento de Drogas , Humanos , Mitocondrias/metabolismo , Neuronas/metabolismo , Espirostanos , Esteroides/químicaRESUMEN
Three-dimensional quantitative structure-activity relationship (3D QSAR) studies were performed on acetylcholinesterase (AChE) inhibitors, based on molecular docking scores obtained by using FlexX and FlexiDock and comparative molecular field analysis (CoMFA). The docking scores were used as molecular descriptors along with the steric and electrostatic field values of CoMFA, for partial least square (PLS) analysis. The high leave one out (LOO) cross-validated correlation coefficient (q(2)=0.714) reveals that the model is a useful tool for the prediction of test set as well as newly designed structures against AChE activity. The superimposed CoMFA models on the receptor site of AChE are guiding the design of potential inhibitory structures directed against AChE activity.
Asunto(s)
Acetilcolinesterasa/química , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/farmacología , Relación Estructura-Actividad Cuantitativa , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/química , Modelos Moleculares , Conformación Proteica , Electricidad Estática , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Compuestos de Espiro/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Caproatos/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Proteínas Portadoras/metabolismo , Ciclosporina/farmacología , Humanos , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Mitocondriales , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Neuroblastoma/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Ratas , Respiración/efectos de los fármacos , Espirostanos/farmacología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Infusion of Fe2+, Abeta42, and buthionine-sulfoximine (FAB), but not Abeta42 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 Abeta42, Fe2+, or buthionine-sulfoximine alone or in combination failed to show the histological modifications seen with FAB. This data suggests that Abeta42 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 Abeta42-independent oxidative stress necessary for the peptide to induce histopathological changes and memory loss.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Encéfalo/efectos de los fármacos , Butionina Sulfoximina/farmacología , Compuestos Ferrosos/farmacología , Trastornos de la Memoria/inducido químicamente , Fragmentos de Péptidos/farmacología , Amiloide/metabolismo , Péptidos beta-Amiloides/administración & dosificación , Animales , Encéfalo/patología , Butionina Sulfoximina/administración & dosificación , Muerte Celular , Ventrículos Cerebrales , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Compuestos Ferrosos/administración & dosificación , Gliosis/inducido químicamente , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo , Fragmentos de Péptidos/administración & dosificación , Perfusión , Ratas , Ratas Long-Evans , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
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.
Asunto(s)
Actinas/fisiología , Fármacos Anti-VIH/farmacología , Benzamidas/farmacología , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/toxicidad , Benzamidas/síntesis química , Benzamidas/uso terapéutico , Benzamidas/toxicidad , Células Cultivadas , Farmacorresistencia Viral Múltiple , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Unión Proteica , Receptores CCR5/metabolismo , Replicación Viral/efectos de los fármacos , Zidovudina/farmacologíaRESUMEN
In search of novel therapeutic approaches for Alzheimer's 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.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Fitoestrógenos/síntesis química , Fitoestrógenos/farmacología , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Animales , Supervivencia Celular/efectos de los fármacos , Cromatografía en Capa Delgada/métodos , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Radicales Libres/metabolismo , Células PC12 , Fragmentos de Péptidos/toxicidad , Piperazinas/síntesis química , Piperazinas/farmacología , Preparaciones de Plantas , Ratas , Sales de Tetrazolio , TiazolesRESUMEN
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 beta-amyloid-induced neurotoxicity. Procaine displayed strong neuroprotective properties against the amyloid peptide Abeta(1-42) and preserved Abeta(1-42)-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 sigma1 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.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Anestésicos Locales/farmacología , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Procaína/farmacología , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Radicales Libres/metabolismo , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Células PC12 , Feocromocitoma/patología , Ensayo de Unión Radioligante , Ratas , Receptores de Droga/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Neuropéptido/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Sodio/metabolismoRESUMEN
Spirostenol steroids have been found to inhibit beta-amyloid-induced neurotoxicity. We have evaluated in parallel experimental and molecular-modeling studies the relative effectiveness of 17 (22R)-hydroxycholesterol derivatives in binding to the target peptide. Our results support the previous evidence that beta-amyloid offers multiple docking sites for these steroids. Molecular modeling allowed for the correlation of spirostenol candidate structural differences with a choice of proposed active sites. A multi-site identification technique based on a Site-Identifier Matrix (SIM) was developed that clearly showed the uniqueness of our lead (maximum neurotoxicity inhibition) candidate SP233, with a nearly equal docking affinity for two sites.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Modelos Moleculares , Proteínas/química , Espirostanos/química , Péptidos beta-Amiloides/toxicidad , Ligandos , Inhibición Neural/fisiología , Unión Proteica/fisiología , Proteínas/metabolismoRESUMEN
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 Alzheimer's disease therapy.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Hidroxicolesteroles , Hidroxicolesteroles/farmacología , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/tratamiento farmacológico , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/toxicidad , Péptidos beta-Amiloides/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Simulación por Computador , Humanos , Hidroxicolesteroles/química , Ratones , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fármacos Neuroprotectores/química , Síndromes de Neurotoxicidad/etiología , Fragmentos de Péptidos/metabolismo , Estructuras de las Plantas/química , Plantas/química , Ratas , Células Tumorales CultivadasRESUMEN
Elevated glucocorticoid levels are associated with many diseases, including age-related depression, hypertension, Alzheimer's disease, and acquired immunodeficiency syndrome. Cortisol-lowering agents could provide useful complementary therapy for these disorders. We examined the effect of procaine and procaine in a pharmaceutical formulation on adrenal cortical steroid formation. Procaine inhibited dibutyryl cyclic AMP (dbcAMP)-induced corticosteroid synthesis by murine Y1 and human H295R adrenal cells in a dose-dependent manner without affecting basal steroid formation. Treatment of rats with the procaine-based formulation reduced circulating corticosterone levels. This steroidogenesis-inhibiting activity of procaine was not observed in Leydig cells, suggesting that the effect was specific to adrenocortical cells. In search of the mechanism underlying this inhibitory effect on cAMP-induced corticosteroidogenesis, procaine was found to affect neither the cAMP-dependent protein kinase activity nor key proteins involved in cholesterol transport into mitochondria, cytochrome P450 side chain cleavage enzyme expression, and enzymatic activities associated with cholesterol metabolism to final steroid products. However, procaine reduced in a dose-dependent manner the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) activity and the dbcAMP-induced HMG-CoA reductase mRNA levels by affecting mRNA stability. These data suggest that the inhibitory effect of procaine on cAMP-induced corticosteroid formation is due to the reduced synthesis of cholesterol. This modulatory effect of procaine on HMG-CoA reductase mRNA expression was also seen in dbcAMP-stimulated Hepa1-6 mouse liver hepatoma cells. Taken together, these results suggest that procaine may provide a pharmacological means for the control of hormone-induced HMG-CoA reductase mRNA expression and hypercortisolemia.
Asunto(s)
Corteza Suprarrenal/metabolismo , Anestésicos Locales/farmacología , AMP Cíclico/fisiología , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Procaína/farmacología , ARN Mensajero/biosíntesis , Esteroides/metabolismo , Corteza Suprarrenal/efectos de los fármacos , Neoplasias de las Glándulas Suprarrenales/metabolismo , Animales , Bucladesina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Immunoblotting , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Luciferasas/genética , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
22R-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age-matched controls. beta-Amyloid (Abeta) 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 Abeta-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 Abeta-induced cell death. Moreover, the effect of 22R-hydroxycholesterol was specific for Abeta-induced cell death because it did not protect against glutamate-induced neurotoxicity. The neuroprotective effect of 22R-hydroxycholesterol was seen when using Abeta1-42 but not the Abeta25-35 peptide. To investigate the mechanism of action of 22R-hydroxycholesterol we examined the direct binding of this steroid to Abeta using a novel cholesterol-protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R-hydroxycholesterol to Abeta1-42 and Abeta17-40, but not Abeta25-35, was observed. These data suggest that 22R-hydroxycholesterol binds to Abeta and the formed 22R-hydroxycholesterol/Abeta complex is not toxic to rodent and human neurons. We propose that 22R-hydroxycholesterol offers a new means of neuroprotection against Abeta toxicity by inactivating the peptide.