Highly Selective and Potent Human ß-Secretase†2 (BACE2) Inhibitors against Type†2 Diabetes: Design, Synthesis, X-ray Structure and Structure-Activity Relationship Studies.

Herein we present the design, synthesis, and biological evaluation of potent and highly selective ß-secretase 2 (memapsin 1, beta-site amyloid precursor protein cleaving enzyme 2, or BACE 2) inhibitors. BACE2 has been recognized as an exciting new target for type 2 diabetes. The X-ray structure of BACE1 bound to inhibitor 2 a {N3 -[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)propyl]amino]propyl]-5-[methyl(methylsulfonyl)amino]-N1 -[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide} containing a hydroxyethylamine isostere was determined. Based on this structure, a computational docking study was performed which led to inhibitor 2 a-bound BACE2 models. These were used to optimize the potency and selectivity of inhibitors. A systematic structure-activity relationship study led to the identification of determinants of the inhibitors' potency and selectivity toward the BACE2 enzyme. Inhibitors 2 d [N3 -[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)pentyl]amino]propyl]-N1 -methyl-N1 -[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide; Ki =0.031 nm, selectivity over BACE1: ≈174 000-fold] and 3 l [N1 -((2S,3R)-3-hydroxy-1-phenyl-4-((3-(trifluoromethyl)benzyl)amino)butan-2-yl)-N3 ,5-dimethyl-N3 -((R)-1-phenylethyl)isophthalamide; Ki =1.6 nm, selectivity over BACE1: >500-fold] displayed outstanding potency and selectivity. Inhibitor 3 l is nonpeptide in nature and may pave the way to the development of a new class of potent and selective BACE2 inhibitors with clinical potential.

Design of Potent and Highly Selective Inhibitors for Human ß-Secretase 2 (Memapsin 1), a Target for Type 2 Diabetes.

Design, synthesis and evaluation of very potent and selective ß-Secretase 2 (memapsin 1, BACE 2) inhibitors are described. The inhibitors were designed specifically to interact with the S2'-site of ß-secretase 2 to provide >170,000-fold selectivity over ß-secretase (BACE 1) and >15,000-fold selectivity over cathepsin D. BACE 2 is implicated in Type 2 diabetes. The studies serve as an important guide to selective BACE 2 inhibitors.

Candida albicans secreted aspartic proteases 4-6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism.

Systemic infection by the pathogenic yeast Candida albicans produces high mortality in immune-compromised people. Such infection starts with the penetration of the organism at the mucosal surfaces, facilitated by the secreted aspartic proteases (Saps) 4, 5, and 6. The functional mechanism of these virulence factors is unclear. We discovered that Saps 4-6 each contains amino acid motifs RGD/KGD to bind integrins on epithelial cell A549 and are internalized to endosomes and lysosomes. These processes are inhibited by RGD-containing peptides or by substituting RGD motifs of these Saps. The internalization of Saps 4-6 results in partial permeabilization of lysosomal membranes, measured by the redistribution of the lysosomal tropic dye acridine orange to the cytosol, and the triggering of apoptosis via caspase activation. Sap 2 and mutated Saps 4-6 contain no RGD motif, are ineffective in these processes, and a proteolytic inhibitor abolished Sap 4 activity in lysosome permeabilization. Same results were also seen for human tongue keratinocyte SCC-15 cells. Mucosal lesions from this fundamental new mechanism may permit C. albicans to enter the body and may be used to attack cells in immune defense during systemic infections. RGD-motif may also be incorporated in Sap inhibitors for Candidiasis drugs targeting to lysosomes.

Beta-secretase inhibitor GRL-8234 rescues age-related cognitive decline in APP transgenic mice.

Alzheimer disease is intimately linked to an excess amount of amyloid-ß (Aß) in the brain. Thus, therapeutic inhibition of Aß 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 ß-secretase, GRL-8234, rescues the age-related cognitive decline. We demonstrated that the injected GRL-8234 effectively enters the brain and rapidly decreases soluble Aß 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-ß plaque load. We also found no accumulation of amyloid-ß precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Aß accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Aß reduction therapy as a treatment of AD.

Amyloid-beta reduction by memapsin 2 (beta-secretase) immunization.

Memapsin 2 (beta-secretase, BACE1) is the protease that initiates cleavage of beta-amyloid precursor protein leading to the production of amyloid-beta (Abeta) and the onset of Alzheimer's disease (AD). Reducing Abeta by targeting memapsin 2 is a major strategy in developing new AD therapy. Here, in a proof-of-concept study, we show that immunization of transgenic AD mice (Tg2576) with memapsin 2 resulted in Abeta reduction and cognitive improvement. To study the basis of this therapy, we demonstrated that anti-memapsin 2 (anti-M2) antibodies were rapidly internalized and reduced Abeta production in cultured cells. These antibodies also effectively crossed the blood-brain barrier to reach the brain. Two- and 10-month Tg2576 mice were immunized and monitored over 10 and 6 months, respectively. We observed a significant decrease of plasma and brain Abeta40 and Abeta42 (approximately 35%) in the immunized mice as compared to controls. Immunized mice also showed better cognitive performance than controls in both cohorts. Brain histological analyses found no evidence of T cell/microglia/astrocyte activation in the immunized mice, suggesting the absence of inflammatory responses. These results suggest that memapsin 2 immunization in Tg2576 was effective in reducing Abeta production and improving cognitive function and that the current approach warrants further investigation as a therapy for AD.

In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors.

We have previously reported structure-based design of memapsin 2 (beta-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 beta-amyloid (Abeta). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimer's mice (Tg2576) resulted in a significant decrease of Abeta level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Abeta reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.

Enzymic properties of recombinant BACE2.

BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.