Gerald Koelsch

Proteolytic activation of recombinant pro-memapsin 2 (Pro-beta-secretase) studied with new fluorogenic substrates

Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 2 suggests that it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease [Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein, and trypsin increased the activity of pro-memapsin 2. Clostripain activation was accompanied by the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2. Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4-(4-dimethylaminophe nyl azo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of pro-memapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu(33p)-memapsin 2 observed in the cells expressing this enzyme [Vassar et al. (1999) Science 286, 735-741; Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptide Glu(33p)-Arg(44p) may serve a regulatory role.

Multiple functions of pro-parts of aspartic proteinase zymogens.

The importance of aspartic proteinases in human pathophysiology continues to initiate extensive research. With burgeoning information on their biological functions and structures, the traditional view of the role of activation peptides of aspartic proteinases solely as inhibitors of the active site is changing. These peptide segments, or pro‐parts, are deemed important for correct folding, targeting, and control of the activation of aspartic proteinase zymogens. Consequently, the primary structures of pro‐parts reflect these functions. We discuss guidelines for formation of hypotheses derived from comparing the physiological function of aspartic proteinases and sequences of their pro‐parts.

β-Secretase inhibitor GRL-8234 rescues age-related cognitive decline in APP transgenic mice

Alzheimer disease is intimately linked to an excess amount of amyloid‐β (Aβ) in the brain. Thus, therapeutic inhibition of Aβ production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of β‐secretase, GRL‐8234, rescues the age‐related cognitive decline. We demonstrated that the injected GRL‐8234 effectively enters the brain and rapidly decreases soluble Aβ in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long‐term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid‐β plaque load. We also found no accumulation of amyloid‐β precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Aβ accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Aβ reduction therapy as a treatment of AD.—Chang, W.‐P., Huang, X., Downs, D., Cirrito, J. R., Koelsch, G., Holtzman, D. M. Ghosh, A. K., Tang, J. β‐Secretase inhibitor GRL‐8234 rescues age‐related cognitive decline in APP transgenic mice. FASEB J. 25, 775–784 (2011). www.fasebj.org

Potent Memapsin 2 (β-Secretase) Inhibitors: Design, Synthesis, Protein-Ligand X-ray Structure and in vivo Evaluation

Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic beta-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K(i) 1.8 nM) and cellular (IC(50)=1 nM in Chinese hamster ovary cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma A beta) in transgenic mice. The X-ray structure of protein-ligand complex of memapsin 2 revealed critical interactions in the memapsin 2 active site.

Localization of β-Secretase Memapsin 2 in the Brain of Alzheimer's Patients and Normal Aged Controls

Chronic accumulation of beta-amyloid in the brain has been shown to result in complex molecular and cellular changes that accompany neurodegeneration in Alzheimers disease (AD). In this study, we examined the expression of a newly identified beta-secretase, memapsin 2 (M2) or beta-site APP cleaving enzyme in deparaffinized sections from 10 AD patients and 10 aged matched controls and in frozen samples of parietal cortex from 11 AD and 8 controls. M2 is mainly expressed in neurons, with high levels in CA4 to CA2 regions and transentorhinal cortex and low or intermediate levels in CA1, subiculum, and granule cells of the dentate gyrus. The majority of AD brains showed an increase of M2 expression in the CA1, but a decrease in the transentorhinal cortex. A subset of controls and AD patients had high M2 expression in parietal neocortex. Double-staining revealed that senile plaques are not directly associated with the soma of M2-expressing neurons. Neurofibrillary tangles were associated with lower M2 expression in AD. These data indicate that beta-secretase M2 may not be straightforwardly involved in amyloid plaque formation in AD brain.

Memapsin 2 (Beta-Secretase) Inhibitor Drug, between Fantasy and Reality

A major strategy for the development of a disease-modifying therapy against Alzheimers disease is pharmacological intervention designed to reduce levels of beta-amyloid in the brain. Among various ways of reducing beta-amyloid production, the inhibition of beta-secretase (memapsin 2, BACE) is particularly attractive. Not only does beta-secretase initiates the amyloid cascade, it also is an aspartic protease, a class of proteases for which successful inhibitor drugs have been developed to treat AIDS patients. Extensive efforts in research and development of a beta-secretase inhibitor drug have taken place in many laboratories during the past few years. However, no drug candidate is currently in clinical trials. In spite of the lack of obvious success, much progress has been made to incorporate the drug-like properties in the evolution of better inhibitors. The inhibitors from more recent generations are indeed similar in characteristics to other protease inhibitor drugs. This progress permits optimism that development of clinical candidates of beta-secretase inhibitor drugs is a realistic goal.

Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

Memapsin 2 (β-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-β (Aβ) peptide and the onset of Alzheimers disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2ED) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2ED into endosomes with increased Aβ production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2ED. The internalization of M2ED was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2ED internalization. These results suggest that exogenously added M2ED interacts with the ectodomain of APP on the cell surface leading to the internalization of M2ED, supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the β-secretase site of APPsw had an effect on the uptake of M2ED. In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.

Study of memapsin 2 (β-secretase) and strategy of inhibitor design

The discovery that β-secretase is a membrane-anchored aspartic protease memapsin 2 has stimulated much interest in the design and testing of its inhibitors for the treatment of Alzheimer’s disease. This article discusses the strategy for the development of such inhibitor drugs. Enzymology and structural determination tools have permitted the design of memapsin 2 inhibitors with high potency and in a size range possible for penetration of the blood-brain barrier. Transgenic Alzheimer’s mice have been used to show that when memapsin 2 inhibitors are transported to the brain, they effectively reduce the production of amyloid β. Although development of a clinical candidate of memapsin 2 inhibitor drug remains a very challenging undertaking, the progress so far lends some optimism for future prospects.

In vivo inhibition of Aβ production by memapsin 2 (β-secretase) inhibitors: Inhibition of Aβ production in vivo

We have previously reported structure‐based design of memapsin 2 (β‐secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a ‘carrier peptide’ penetrated the plasma membrane in cultured cells and inhibited the production of β‐amyloid (Aβ). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimers mice (Tg2576) resulted in a significant decrease of Aβ level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Aβ reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.

Studies on Pepsin Mutagenesis and Recombinant Rhizopuspepsinogen

During the past fifteen years, many chemical and three-dimensional structures of aspartic proteases have been determined. It is now clear that, in spite of some diverse substrate specificities, these enzymes are alike in many ways, including their folding patterns, active center structures, and mechanisms of zymogen activation. Because the catalytic apparatuses of these enzymes are nearly identical, it is obvious that this group of enzymes shares a common catalytic mechanism. Although a number of catalytic mechanisms have been proposed for the aspartic proteases after consideration of kinetic and structural results (Fruton, 1976; James et al, 1977; Davies, 1990), a consensus on the mechanism has not emerged. One of the reasons for the uncertainty is that static structural information suggests, but does not provide, direct evidence for a mechanism of catalysis. On the other hand, kinetic experiments suggest reaction schemes but do not pinpoint structural components which are operational. It seems clear that additional experimental information is necessary to link structural information to reaction schemes.