In vivo inhibition of Aβ production by memapsin 2 (β‐secretase) inhibitors

Abstract

We have previously reported structure-based design of memapsin 2 (b-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 b-amyloid (Ab). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimer's mice (Tg2576) resulted in a significant decrease of Ab level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Ab reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition. The pathogenesis of Alzheimer's disease (AD) is intimately related to the presence of the neurotoxic amyloid-b peptide (Ab) in the brain (Selkoe 1999;Walsh et al. 2002). Reduction of brain Ab, which improved cognitive functions in transgenic AD mice (Morgan et al. 2000;Dodart et al. 2002), is therefore an important strategy for AD therapy (Dominguez and De Strooper 2002). Ab, a peptide of 40/42 amino acid residues, is generated by the proteolysis of b-amyloid precursor protein (APP), first by the membraneassociated aspartic protease memapsin 2 (b-secretase or BACE; Lin et al. 2000;Sinha et al. 1999;Vassar et al. 1999;Yan et al. 1999), then by the intramembrane proteolysis of c-secretase (Selkoe 1999). Both proteases are therefore therapeutic targets for the treatment of AD. The development of inhibitor drugs for memapsin 2 is encouraged by the lack of a deleterious response after gene deletion in mice (Cai et al. 2001;Luo et al. 2001;Roberds et al. 2001). However, to attain clinically useful memapsin 2 inhibitors is generally thought to be very challenging because they must be small enough (generally < 500 Da) to penetrate the blood-brain barrier (BBB) yet manifest high potency and other desirable pharmaceutical properties. Toward the development of such inhibitor drugs, it is important to demonstrate in cellular and animal models that memapsin 2 inhibitors can in fact achieve Ab reduction. Not only would such results validate memapsin 2 as a therapeutic target, the cellular and animal models may also serve as future tools for drug development. Although several potent memapsin 2 inhibitors with K i values in the nanomolar range have been reported (Ghosh et al. 2000;Ghosh et al. 2001;Turner et al. 2001; Ghosh Received October 31, 2003; revised

In Vivo β-Secretase 1 Inhibition Leads to Brain Aβ Lowering and Increased α-Secretase Processing of Amyloid Precursor Protein without Effect on Neuregulin-1

“…Our results are consistent with previous studies in which intraperitoneal administration of a hydroxyethylene isostere inhibitor conjugated to a peptide carrier (Chang et al, 2004) led to a dose-and time-dependent reduction of plasma and brain A␤40. Intrahippocampal injection of a different BACE1 inhibitor (cell IC 50 ϳ 10 M) showed in vivo lowering of brain A␤40, A␤42, and C99, but no change in C83 was observed in mice (Asai et al, 2006).…”

“…Thus, a mechanism-based effect on peripheral or central myelination could potentially affect BACE1 inhibitor development as an AD therapeutic. Although BACE1 inhibitors have shown acute in vivo efficacy to lower brain A␤ levels in murine models when administered peripherally (Chang et al, 2004;Stachel et al, 2006;Hussain et al, 2007) or via direct intracranial injection (Asai et al, 2006;Nishitomi et al, 2006), their poor pharmacokinetic properties have prevented effective evaluation of subchronic to chronic BACE1 inhibition on brain APP processing and NRG-1 processing in adult mice.…”

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

“…Although these inhibitors are very active on the enzyme, their in vivo utility has been limited by poor membrane permeability and susceptibility to P-gp transport and thus poor brain penetration. Brain A␤ lowering has been demonstrated previously in mice models with transition state isosteres via direct intracranial administration (Asai et al, 2006;Nishitomi et al, 2006;Sankaranarayanan et al, 2008), parenteral administration (Chang et al, 2004;Stachel et al, 2006), or via oral codosing with P-gp inhibitors (Hussain et al, 2007), but none of these compounds were optimal in pharmacokinetic or brain exposure properties for testing in higher animals. TC-1 is a novel BACE1 inhibitor in which the previously reported isonicotinamide Stauffer et al, 2007) and isophthalate oxadiazole (Rajapakse et al, 2006) BACE1 inhibitors were merged to create a new structural class of inhibitors (Lindsley et al, 2007;Moore et al, 2007).…”

“…The structural requirements for binding also impart high susceptibility to P-glycoprotein (P-gp) transport, thus reducing brain concentrations of BACE-1 inhibitors (Mahar Doan et al, 2002). A few BACE1 inhibitors have been shown to lower brain A␤ levels after direct intracranial administration (Asai et al, 2006;Nishitomi et al, 2006;Sankaranarayanan et al, 2008) or via peripheral administration at relatively high doses in murine models (Chang et al, 2004;Stachel et al, 2006;Hussain et al, 2007;Stanton et al, 2007). However, the lack of potent inhibitors that also possess good pharmacokinetic and brain penetration properties have prevented evaluation of BACE1 inhibitors in larger animals, such as nonhuman primates.…”

Design, Synthesis and X-ray Structure of Protein−Ligand Complexes: Important Insight into Selectivity of Memapsin 2 (β-Secretase) Inhibitors

“…They have shown an average cellular IC 50 value of 1.4 μM and 1.7 μM respectively compared to an IC 50 value of 45 μM for 1. 13 In conclusion, our structure-based design led to the development of very potent and highly selective memapsin 2 inhibitors. Furthermore, our X-ray structural analysis of protein-inhibitor complexes has uncovered potentially important molecular interactions useful in the design of selectivity against other aspartyl proteases.…”