Limits...
Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus

Specific inhibitory effect of aptamer A1 on BACE1 activity in vitro.(A-B) Effects of different concentrations of a standard inhibitor (A), A1 (straight line in B), Gp30 (dotted line in B), and U31 (dashed line in B) on fluorescence intensity determined by FRET assay are shown. (C-D) Inhibition profile of BACE1 activity by a standard inhibitor (C) and aptamer A1 (D) is shown. The data are represented as the mean±SD of three independent experiments. *P<0.05 and **P<0.01 compared with the positive control group.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608735&req=5

pone.0140733.g003: Specific inhibitory effect of aptamer A1 on BACE1 activity in vitro.(A-B) Effects of different concentrations of a standard inhibitor (A), A1 (straight line in B), Gp30 (dotted line in B), and U31 (dashed line in B) on fluorescence intensity determined by FRET assay are shown. (C-D) Inhibition profile of BACE1 activity by a standard inhibitor (C) and aptamer A1 (D) is shown. The data are represented as the mean±SD of three independent experiments. *P<0.05 and **P<0.01 compared with the positive control group.

Mentions: Next, a fluorescence resonance energy transfer assay was conducted to confirm the inhibitory effect of A1 on BACE1 activity in vitro. The BACE1 substrate is linked to a fluorescent donor on one end and a quenching acceptor group on the other end. As a result of intramolecular energy transfer to the quenching acceptor, fluorescence from the donor can be quenched by the acceptor. Following substrate cleavage via BACE1 enzymes, the energy transfer is disturbed, which results in fluorescent signal enhancement. The fluorescence of the substrate is significantly reduced when the substrate cleavage is blocked by an inhibitor. Based on this principle, various concentrations of A1 were added into a reaction system that contained BACE1 and its fluorescent substrate; the changes in the fluorescence intensity were subsequently detected. A BACE1 specific inhibitor ([Asn670, Sta671, Val672]-Amyloid β/A4 Precursor Protein 770 Fragment 662–675), Gp30, and U31 served as controls. Following increases in the concentration, the fluorescence intensity in the standard inhibitor group gradually decreased, with a significant decrease at a concentration of 500 nM compared with the positive control (P<0.01). The fluorescence intensity in the A1 group significantly decreased at a concentration of 250 nM (P<0.01). No significant decrease in the fluorescence intensities occurred in the Gp30 or U31 groups (Fig 3A and 3B). The data of BACE1 activity were converted from percent inhibition to probit values, which were drawed versus the log of the BACE1 specific inhibitor or aptamer A1 concentration. A1 inhibited BACE1 activity with an IC50 value of 139.81 nM. Under the same experimental conditions, standard inhibitor inhibited the activity of BACE1 in a concentration-dependent manner (IC50 = 242.50 nM; Fig 3C and 3D). These findings demonstrate that A1 can act as a potent inhibitor of BACE1 activity in vitro.


Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Specific inhibitory effect of aptamer A1 on BACE1 activity in vitro.(A-B) Effects of different concentrations of a standard inhibitor (A), A1 (straight line in B), Gp30 (dotted line in B), and U31 (dashed line in B) on fluorescence intensity determined by FRET assay are shown. (C-D) Inhibition profile of BACE1 activity by a standard inhibitor (C) and aptamer A1 (D) is shown. The data are represented as the mean±SD of three independent experiments. *P<0.05 and **P<0.01 compared with the positive control group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608735&req=5

pone.0140733.g003: Specific inhibitory effect of aptamer A1 on BACE1 activity in vitro.(A-B) Effects of different concentrations of a standard inhibitor (A), A1 (straight line in B), Gp30 (dotted line in B), and U31 (dashed line in B) on fluorescence intensity determined by FRET assay are shown. (C-D) Inhibition profile of BACE1 activity by a standard inhibitor (C) and aptamer A1 (D) is shown. The data are represented as the mean±SD of three independent experiments. *P<0.05 and **P<0.01 compared with the positive control group.
Mentions: Next, a fluorescence resonance energy transfer assay was conducted to confirm the inhibitory effect of A1 on BACE1 activity in vitro. The BACE1 substrate is linked to a fluorescent donor on one end and a quenching acceptor group on the other end. As a result of intramolecular energy transfer to the quenching acceptor, fluorescence from the donor can be quenched by the acceptor. Following substrate cleavage via BACE1 enzymes, the energy transfer is disturbed, which results in fluorescent signal enhancement. The fluorescence of the substrate is significantly reduced when the substrate cleavage is blocked by an inhibitor. Based on this principle, various concentrations of A1 were added into a reaction system that contained BACE1 and its fluorescent substrate; the changes in the fluorescence intensity were subsequently detected. A BACE1 specific inhibitor ([Asn670, Sta671, Val672]-Amyloid β/A4 Precursor Protein 770 Fragment 662–675), Gp30, and U31 served as controls. Following increases in the concentration, the fluorescence intensity in the standard inhibitor group gradually decreased, with a significant decrease at a concentration of 500 nM compared with the positive control (P<0.01). The fluorescence intensity in the A1 group significantly decreased at a concentration of 250 nM (P<0.01). No significant decrease in the fluorescence intensities occurred in the Gp30 or U31 groups (Fig 3A and 3B). The data of BACE1 activity were converted from percent inhibition to probit values, which were drawed versus the log of the BACE1 specific inhibitor or aptamer A1 concentration. A1 inhibited BACE1 activity with an IC50 value of 139.81 nM. Under the same experimental conditions, standard inhibitor inhibited the activity of BACE1 in a concentration-dependent manner (IC50 = 242.50 nM; Fig 3C and 3D). These findings demonstrate that A1 can act as a potent inhibitor of BACE1 activity in vitro.

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus