As a consequence, the positively charged, basic amino group localizes closely to the membrane surface where it can repel protons, leading to a local pH increase. ascribes the pathogenesis of the disease to a cascade of events, starting with the generation and accumulation of the amyloid peptide (A), a proteolytic fragment of the amyloid precursor protein (APP) (Hardy and Selkoe, 2002). Formation of A requires APP to be proteolytically processed by the two proteases, – and -secretase. A third protease, -secretase, cleaves APP within the A domain and precludes A generation. -Secretase is the membrane-bound aspartyl protease BACE1 and cleaves APP at the N terminus of the A domain (Rossner et al., 2006; Cole and Vassar, 2008). This cleavage generates the soluble APPs and a C-terminal fragment (C99), which undergoes a second cleavage at the C terminus of the A domain by -secretase. -Secretase belongs to the GxGD-family of aspartyl proteases and cleaves APP within its hydrophobic transmembrane domain. -Secretase is a heterotetrameric protein complex consisting of presenilin, nicastrin, PEN-2, and APH-1 (Steiner et al., 2008). -Secretase mainly cleaves after amino acid 40 of the A sequence, resulting in the generation of the A40 peptide. To a lower extent, -secretase also generates A38 and A42. Although a minor product, A42 is considered the key player in AD pathogenesis and is the main constituent of the amyloid plaques found in AD brains (Hardy and Selkoe, 2002). -Secretase is an obvious drug target for AD, but most -secretase inhibitors do not reach sufficiently high concentrations in the brain (Ghosh et al., 2008; Meredith et al., 2008). It is therefore essential to identify alternative strategies for reducing -secretase cleavage. One possibility is the addition of a membrane anchor to a -secretase inhibitor, which has been shown to increase its local membrane concentration, resulting in a more efficient -secretase inhibition than the free inhibitor (Rajendran et al., 2008). Here we tested the possibility of targeting the cellular environment of -secretase cleavage instead of the -secretase enzyme itself. -Secretase has an acidic pH optimum and mostly cleaves APP in the mildly acidic endosomes (Rossner et al., 2006), where it can be blocked by the weak base ammonium chloride (Haass et al., 1993; Schrader-Fischer and Paganetti, 1996). Interestingly, several drugs in clinical use contain weakly basic amino groups and thus have the potential to raise the endosomal pH. We screened such compounds for an inhibition of -secretase cleavage and identified the drugs bepridil and amiodarone as novel inhibitors of -secretase cleavage. Both compounds are calcium channel blockers and calmodulin antagonists and are used for the treatment of heart disease. Here we found them to inhibit -secretase cleavage and A generation independently of their calcium channel- and calmodulin-blocking activities. The inhibition of -secretase cleavage occurred at therapeutically achievable and nontoxic concentrations by raising the endosomal, membrane-proximal pH. Surprisingly, both compounds additionally modulated the cleavage specificity of -secretase in an inverse manner. Thus, bepridil and amiodarone define a new class of drugsthe dual modulatorswhich may be used for drug development targeting – and -secretase, either individually or simultaneously. Materials and Methods Antibodies and reagents. The following antibodies were used: 192wt and 192swe (APPs wild-type and Swedish mutations, respectively, Elan Pharmaceuticals), W02 (APPs) and 22C11 [full-length APP (fl.APP), provided by K. Beyreuther, University of Heidelberg, Heidelberg, Germany], 6687 (fl.APP, Eurogentec), 6E10 (A1-16, Santa Cruz Biotechnology), 3552 (A, Eurogentec), 2D8 (A1-16, provided by E. Kremmer, Helmholtz Institute, Munich, Germany), -A38 MSD-Tag (Meso Scale Discovery), -A40 BAP24-TAG and -A42 BAP24-TAG (provided by M. Brockhaus, Roche, Basel, Switzerland), -calmodulin (Millipore), and –actin (Sigma). The reagents used were as follows: bepridil hydrochloride, amiodarone hydrochloride, chloroquine, nimodipine, and tamoxifen (Sigma); AMI-AcOH (provided by H.-R. Ha, University Hospital, Zurich, Switzerland); GL-189 (provided by K. Maskos and W. Bode, Max Plank Institute for Biochemistry, Martinsried, Germany); egg phosphatidylcholine (PC) (Avanti Polar Lipids); fluorescein phosphatidylethanolamine (FPE) (Invitrogen); and siRNA pools targeting Calmodulin genes and and nontargeting control pools (Dharmacon). Cell culture, compound treatment, and immunoblots. Human embryonic kidney 293EBNA (HEK293) cells were cultured as described previously (Kuhn et.This suggests that a mild rise in the endosomal pH may be a clinically acceptable strategy. an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit -secretase. Surprisingly, bepridil and amiodarone also modulated -secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target – and -secretase through distinct molecular mechanisms. In addition to Alzheimer’s disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins. Introduction Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. The amyloid hypothesis ascribes the pathogenesis of the disease to a cascade of events, starting with the generation and accumulation of the amyloid peptide (A), a proteolytic fragment of the amyloid precursor protein (APP) (Hardy and Selkoe, 2002). Formation of A requires APP to be proteolytically processed by the two proteases, – and -secretase. A third protease, -secretase, cleaves APP within the A domain and precludes A generation. -Secretase is the membrane-bound aspartyl protease BACE1 and cleaves APP at the N terminus of the A domain (Rossner et al., 2006; Cole and Vassar, 2008). This cleavage generates the soluble APPs and a C-terminal fragment (C99), which undergoes a second cleavage at the C terminus of the A domain by -secretase. -Secretase belongs to the GxGD-family of aspartyl proteases and cleaves APP within its hydrophobic transmembrane domain. -Secretase is a heterotetrameric protein complex consisting of presenilin, nicastrin, PEN-2, and APH-1 (Steiner et al., 2008). -Secretase mainly cleaves after amino acid 40 of the A sequence, resulting in the generation of the A40 peptide. To a lower extent, -secretase also generates A38 and A42. Although a minor product, A42 is considered the key player in AD pathogenesis and is the main constituent of the amyloid plaques found in AD brains (Hardy and Selkoe, 2002). -Secretase is an obvious drug target for AD, but most -secretase inhibitors do not reach sufficiently high concentrations in the brain (Ghosh et al., 2008; Meredith et al., 2008). It is therefore essential to identify alternative strategies for reducing -secretase cleavage. One possibility is the addition of a membrane anchor to a -secretase inhibitor, which has been shown to increase its local Goat polyclonal to IgG (H+L)(HRPO) membrane concentration, resulting in a more efficient -secretase inhibition than the free inhibitor (Rajendran et al., 2008). Here we tested the possibility of targeting the cellular environment of -secretase cleavage instead of the -secretase enzyme itself. -Secretase has an acidic pH optimum and mostly cleaves APP in the mildly acidic endosomes (Rossner et al., 2006), where it can be blocked by the weak base ammonium chloride (Haass et al., 1993; Schrader-Fischer and Paganetti, 1996). Interestingly, several drugs in clinical use contain weakly basic amino groups and thus have the potential to raise the endosomal pH. We screened such compounds for an inhibition of -secretase cleavage and identified the drugs bepridil and amiodarone as novel inhibitors of -secretase cleavage. Both compounds are calcium channel blockers and calmodulin antagonists and are used for the treatment of heart disease. Here we found them to inhibit -secretase cleavage and A generation independently of their calcium channel- and calmodulin-blocking activities. The inhibition of -secretase cleavage occurred at therapeutically achievable and nontoxic concentrations by raising the endosomal, membrane-proximal pH. Surprisingly, both compounds additionally modulated the cleavage specificity of -secretase in an inverse manner. Thus, bepridil and amiodarone define a new class of drugsthe dual modulatorswhich may be used for drug development targeting – and -secretase, either individually or simultaneously. Materials and Methods Antibodies and reagents. The following antibodies were used: 192wt and 192swe (APPs wild-type and Swedish mutations, respectively, Elan Pharmaceuticals), W02 (APPs) and 22C11 [full-length APP (fl.APP), provided by K. Beyreuther, University of Heidelberg, Heidelberg, Germany], 6687 (fl.APP, Eurogentec), 6E10 (A1-16, Santa Cruz Biotechnology), 3552 (A, Eurogentec), 2D8 (A1-16, provided by E. Kremmer, Helmholtz Institute, Munich, Germany), -A38 MSD-Tag (Meso Scale Discovery), -A40 BAP24-TAG and -A42 BAP24-TAG (provided by M. Brockhaus, Roche, Basel, Switzerland), -calmodulin (Millipore), and –actin (Sigma). The reagents used were as follows: bepridil hydrochloride, amiodarone hydrochloride, chloroquine, nimodipine, and tamoxifen (Sigma); AMI-AcOH (provided by H.-R. Ha, University Hospital, Zurich, Switzerland); GL-189 (provided by K. Maskos and W. Bode, Max Plank Institute for Biochemistry, Martinsried, Germany); egg phosphatidylcholine (PC) (Avanti Polar Lipids); fluorescein phosphatidylethanolamine (FPE) (Invitrogen); and siRNA pools targeting Calmodulin genes and and nontargeting control pools (Dharmacon). Cell culture, compound treatment, and immunoblots. Human embryonic kidney 293EBNA (HEK293) cells were cultured.This system has been successfully used in the past for the measurement of changes in surface potential and pH in the membrane-proximal zone (Teissie et al., 1985; Simard et al., 2008). disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins. Introduction Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. The amyloid hypothesis ascribes the pathogenesis of the disease to a cascade of events, starting with the generation and accumulation of the amyloid peptide (A), a proteolytic fragment of the amyloid precursor protein (APP) (Hardy and Selkoe, 2002). Formation of A requires APP to be proteolytically processed by the two proteases, – and -secretase. A third protease, -secretase, cleaves APP within the A domain and precludes A generation. -Secretase is the membrane-bound aspartyl protease BACE1 and cleaves APP at the N terminus of the A domain (Rossner et al., 2006; Cole and Vassar, 2008). This cleavage generates the soluble APPs and a C-terminal fragment (C99), which undergoes a second cleavage in the C terminus of the A domain by -secretase. -Secretase belongs to the GxGD-family of aspartyl proteases and cleaves APP within its hydrophobic transmembrane domain. -Secretase is a heterotetrameric protein complex consisting of presenilin, nicastrin, PEN-2, and APH-1 (Steiner et al., 2008). -Secretase mainly cleaves after amino acid 40 of the A sequence, resulting in the generation of the A40 peptide. To a lower extent, -secretase also generates A38 and A42. Although a minor product, A42 is considered the key player in AD pathogenesis and is the main constituent of the amyloid plaques found in AD brains (Hardy and Selkoe, 2002). -Secretase is an obvious drug target for AD, but most -secretase inhibitors do not reach sufficiently high concentrations in the brain (Ghosh et al., 2008; Meredith et al., 2008). It is therefore essential to identify alternative strategies for reducing -secretase cleavage. One possibility is the addition of a membrane anchor to a -secretase inhibitor, which has been shown to increase its local membrane concentration, resulting in a more efficient -secretase inhibition than the free inhibitor (Rajendran et al., 2008). Here we tested Echinacoside the possibility of targeting the cellular environment of -secretase cleavage instead of the -secretase enzyme itself. -Secretase has an acidic pH optimum and mostly cleaves APP in the mildly acidic endosomes (Rossner et al., 2006), where it can be blocked from the weak base ammonium chloride (Haass et al., 1993; Schrader-Fischer and Paganetti, 1996). Interestingly, several drugs in clinical use contain weakly basic amino groups and thus have the potential to raise the endosomal pH. We screened such compounds for an inhibition of -secretase cleavage and identified the drugs bepridil and amiodarone as novel inhibitors of -secretase cleavage. Both compounds are calcium channel blockers and calmodulin antagonists and are used for the treatment of heart disease. Here we found them to inhibit -secretase cleavage and A generation independently of their calcium channel- and calmodulin-blocking activities. The inhibition of -secretase cleavage occurred at therapeutically achievable and nontoxic concentrations by raising the endosomal, membrane-proximal pH. Surprisingly, both compounds additionally modulated the cleavage specificity of -secretase in an inverse manner. Thus, bepridil and amiodarone define a new class of drugsthe dual modulatorswhich may be used for drug development targeting – and -secretase, either individually or simultaneously. Materials and Methods Antibodies and reagents. The following antibodies were used: 192wt and 192swe (APPs wild-type and Swedish mutations, respectively, Elan Pharmaceuticals), W02 (APPs) and 22C11 [full-length APP (fl.APP), provided by K. Beyreuther, University of Heidelberg, Heidelberg, Germany], 6687 (fl.APP, Eurogentec), 6E10 (A1-16, Santa Cruz Biotechnology), 3552 (A, Eurogentec), 2D8 (A1-16, provided by E. Kremmer, Helmholtz Institute, Munich, Germany), -A38 MSD-Tag (Meso Scale Discovery), -A40 BAP24-TAG and -A42 BAP24-TAG (provided by M. Brockhaus, Roche, Basel, Switzerland), -calmodulin (Millipore), and –actin (Sigma). The reagents used were as follows: bepridil hydrochloride, amiodarone hydrochloride, chloroquine, nimodipine, and tamoxifen (Sigma);.A third protease, -secretase, cleaves APP within the A domain and precludes A generation. inhibit -secretase. Surprisingly, bepridil and amiodarone also modulated -secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target – and -secretase through distinct molecular mechanisms. In addition to Alzheimer’s disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins. Introduction Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. The amyloid hypothesis ascribes the pathogenesis of the disease to a cascade of events, starting with the generation and accumulation of the amyloid peptide (A), a proteolytic fragment of the amyloid precursor protein (APP) (Hardy and Selkoe, 2002). Formation of A requires APP to be proteolytically processed by the two proteases, – and -secretase. A third protease, -secretase, cleaves APP within the A domain and precludes A generation. -Secretase is the membrane-bound aspartyl protease BACE1 and cleaves APP in the N terminus of the A domain (Rossner et al., 2006; Cole and Vassar, 2008). This cleavage generates the soluble APPs and a C-terminal fragment (C99), which undergoes a second cleavage in the C terminus of the A domain by -secretase. -Secretase belongs to the GxGD-family of aspartyl proteases and cleaves APP within its hydrophobic transmembrane domain. -Secretase is a heterotetrameric protein complex consisting of presenilin, nicastrin, PEN-2, and APH-1 (Steiner et al., 2008). -Secretase mainly cleaves after amino acid 40 of the A sequence, resulting in the generation of the A40 peptide. To a lower extent, -secretase also generates A38 and A42. Although a minor product, A42 is considered the key player in AD pathogenesis and is the main constituent of the amyloid plaques found in AD brains (Hardy and Selkoe, 2002). -Secretase is an obvious drug target for AD, but most -secretase inhibitors do not reach sufficiently high concentrations in the brain (Ghosh et al., 2008; Meredith et al., 2008). It is therefore essential to identify alternative strategies for reducing -secretase cleavage. One possibility is the addition of a membrane anchor to a -secretase inhibitor, which has been shown to increase its local membrane concentration, resulting in a more efficient -secretase inhibition than the free inhibitor (Rajendran et al., 2008). Here we tested the possibility of targeting the cellular environment of -secretase Echinacoside cleavage instead of the -secretase enzyme itself. -Secretase has an acidic pH optimum and mostly cleaves APP in the mildly acidic Echinacoside endosomes (Rossner et al., 2006), where it can be blocked from the weak base ammonium chloride (Haass et al., 1993; Schrader-Fischer and Paganetti, 1996). Interestingly, several drugs in clinical use contain weakly basic amino groups and thus have the potential to raise the endosomal pH. We screened such compounds for an inhibition of -secretase cleavage and identified the drugs bepridil and amiodarone as novel inhibitors of -secretase cleavage. Both compounds are calcium channel blockers and calmodulin antagonists and are used for the treatment of heart disease. Here we found them to inhibit -secretase cleavage and A generation independently of their calcium channel- and calmodulin-blocking activities. The inhibition of -secretase cleavage occurred at therapeutically achievable and nontoxic concentrations by raising the endosomal, membrane-proximal pH. Surprisingly, both compounds additionally modulated the cleavage specificity of -secretase in an inverse manner. Thus, bepridil and amiodarone define a new class of drugsthe dual modulatorswhich may be used for drug development targeting – and -secretase, either individually or simultaneously. Materials and Methods Antibodies and reagents. The following antibodies were used: 192wt and 192swe (APPs wild-type and Swedish mutations, respectively, Elan Pharmaceuticals), W02 (APPs) and 22C11 [full-length APP (fl.APP), provided by K. Beyreuther, University of Heidelberg, Heidelberg, Germany], 6687 (fl.APP, Eurogentec), 6E10 (A1-16, Santa Cruz Biotechnology), 3552 (A, Eurogentec), 2D8 (A1-16, provided by E. Kremmer, Helmholtz Institute, Munich, Germany), -A38 MSD-Tag (Meso Scale Discovery), -A40 BAP24-TAG and -A42 BAP24-TAG (provided by M. Brockhaus, Roche, Basel, Switzerland), -calmodulin (Millipore), and –actin (Sigma). The reagents used were as follows: bepridil hydrochloride, amiodarone hydrochloride, chloroquine, nimodipine, and tamoxifen (Sigma); AMI-AcOH (provided by H.-R. Ha, University Hospital, Zurich, Switzerland); GL-189 (provided by K. Maskos and W. Bode, Max Plank Institute for Biochemistry, Martinsried, Germany); egg phosphatidylcholine (PC) (Avanti Polar Lipids); fluorescein phosphatidylethanolamine (FPE) (Invitrogen); and siRNA pools targeting Calmodulin genes and and nontargeting control pools (Dharmacon). Cell culture, compound treatment, and immunoblots. Human embryonic kidney 293EBNA (HEK293) cells were cultured as described previously (Kuhn et al., 2007). Clonal H4 and U373 cells stably expressing APP751 from your vector pRC/CMV hAPPwt were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 200 g/ml G418 (G418-medium) or supplemented with 15% FCS, respectively. Clonal HEK293 cells stably expressing APP695 from your vector pCEP4-APP695 (HEK293-APP) and polyclonal.Brain sections were collected and maintained in culture plate inserts (Millicell CM, 30 mm diameter; Millipore) in DMEM/Ham’s F12 medium containing 25% horse serum, 0.2% d-glucose, 2 mm l-glutamine, and 0.3 mm HEPES. most common neurodegenerative disorder. The amyloid hypothesis ascribes the pathogenesis of the disease to a cascade of events, starting with the generation and accumulation of the amyloid peptide (A), a proteolytic fragment of the amyloid precursor protein (APP) (Hardy and Selkoe, 2002). Formation of A requires APP to be proteolytically processed by the two proteases, – and -secretase. A third protease, -secretase, cleaves APP within the A website and precludes A generation. -Secretase is the membrane-bound aspartyl protease BACE1 and cleaves APP in the N terminus of the A website (Rossner et al., 2006; Cole and Vassar, 2008). This cleavage produces the soluble APPs and a C-terminal fragment (C99), which undergoes a second cleavage in the C terminus of the A website by -secretase. -Secretase belongs to the GxGD-family of aspartyl proteases and cleaves APP within its hydrophobic transmembrane website. -Secretase is definitely a heterotetrameric protein complex consisting of presenilin, nicastrin, PEN-2, and APH-1 (Steiner et al., 2008). -Secretase primarily cleaves after amino acid 40 of the A sequence, resulting in the generation of the A40 peptide. To a lower degree, -secretase also produces A38 and A42. Although a minor product, A42 is considered the key player in AD pathogenesis and is the main constituent of the amyloid plaques found in AD brains (Hardy and Selkoe, 2002). -Secretase is an obvious drug target for AD, but most -secretase inhibitors do not reach sufficiently high concentrations in the brain (Ghosh et al., 2008; Meredith et al., 2008). It is therefore essential to identify alternative strategies for reducing -secretase cleavage. One possibility is the addition of a membrane anchor to a -secretase inhibitor, which has been shown to increase its local membrane concentration, resulting in a more efficient -secretase inhibition than the free inhibitor (Rajendran et al., 2008). Here we tested the possibility of targeting the cellular environment of -secretase cleavage instead of the -secretase enzyme itself. -Secretase has an acidic pH optimum and mostly cleaves APP in the mildly acidic endosomes (Rossner et al., 2006), where it can be blocked by the poor base ammonium chloride (Haass et al., 1993; Schrader-Fischer and Paganetti, 1996). Interestingly, several drugs in clinical use contain weakly basic amino groups and thus have the potential to raise the endosomal pH. We screened such compounds for an inhibition of -secretase cleavage and recognized the drugs bepridil and amiodarone as novel inhibitors of -secretase cleavage. Both compounds are calcium channel blockers and calmodulin antagonists and are used for the treatment of heart disease. Here we found them to inhibit -secretase cleavage and A generation independently of their calcium channel- and calmodulin-blocking activities. The inhibition of -secretase cleavage occurred at therapeutically achievable and nontoxic concentrations by raising the endosomal, membrane-proximal pH. Surprisingly, both compounds additionally modulated the cleavage specificity of -secretase in an inverse manner. Thus, bepridil and amiodarone define a new class of drugsthe dual modulatorswhich may be used for drug development targeting – and -secretase, either individually or simultaneously. Materials and Methods Antibodies and reagents. The following antibodies were used: 192wt and 192swe (APPs wild-type and Swedish mutations, respectively, Elan Pharmaceuticals), W02 (APPs) and 22C11 [full-length APP (fl.APP), provided by K. Beyreuther, University of Heidelberg, Heidelberg, Germany], 6687 (fl.APP, Eurogentec), 6E10 (A1-16, Santa Cruz Biotechnology), 3552 (A, Eurogentec), 2D8 (A1-16, provided by E. Kremmer, Helmholtz Institute, Munich, Germany), -A38 MSD-Tag (Meso Scale Discovery), -A40 BAP24-TAG and -A42 BAP24-TAG (provided by M. Brockhaus, Roche, Basel, Switzerland), -calmodulin (Millipore), and –actin (Sigma). The reagents used were as follows: bepridil hydrochloride, amiodarone hydrochloride, chloroquine, nimodipine, and tamoxifen (Sigma); AMI-AcOH (provided by H.-R. Ha, University Hospital, Zurich, Switzerland); GL-189 (provided by K. Maskos and W. Bode, Max Plank Institute for Biochemistry, Martinsried, Germany); egg phosphatidylcholine (PC) (Avanti Polar Lipids); fluorescein phosphatidylethanolamine (FPE) (Invitrogen); and siRNA pools targeting Calmodulin genes and and nontargeting control pools (Dharmacon). Cell culture, compound treatment, and immunoblots. Human embryonic kidney 293EBNA (HEK293) cells were cultured as described previously (Kuhn et al., 2007). Clonal H4 and U373 cells stably expressing APP751 from the vector pRC/CMV hAPPwt were cultured.