Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells.

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.

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, synthesis, X-ray studies, and biological evaluation of novel BACE1 inhibitors with bicyclic isoxazoline carboxamides as the P3 ligand.

We describe the design, synthesis, X-ray studies, and biological evaluation of novel BACE1 inhibitors containing bicyclic isoxazoline carboxamides as the P3 ligand in combination with methyl cysteine, methylsulfonylalanine and Boc-amino alanine as P2 ligands. Inhibitor 3a displayed a BACE1 Ki value of 10.9 nM and EC50 of 343 nM. The X-ray structure of 3a bound to the active site of BACE1 was determined at 2.85 Šresolution. The structure revealed that the major molecular interactions between BACE1 and the bicyclic tetrahydrofuranyl isoxazoline heterocycle are van der Waals in nature.

Design, synthesis, and X-ray structural studies of BACE-1 inhibitors containing substituted 2-oxopiperazines as P1'-P2' ligands.

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1' and P2' ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (Ki=2nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23nM and cellular EC50 of 80nM.

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.

Prospects of ß-Secretase Inhibitors for the Treatment of Alzheimer's Disease.

ß-Secretase continues to be an attractive drug discovery target for the therapeutic intervention of Alzheimer's disease (AD). This enzyme plays a critical role in the production of neurotoxic ß-amyloid (Aß) peptides in the brain. Over the years, extensive research efforts have led to the development of many promising classes of inhibitors against this protease. Many small-molecule, peptidomimetic, and nonpeptide ß-secretase inhibitors have now overcome the key challenging development hurdles such as selectivity and brain penetration. A number of inhibitors have also shown further promise in reducing brain Aß and rescuing cognitive decline in animal models. Recently, several ß-secretase inhibitors have entered into preclinical and phase I studies, and at least one of these inhibitors has advanced to phase II/III human trials. The outlook on ß-secretase inhibitor drugs for the treatment of AD patients is discussed herein.

Structure-based design, synthesis and biological evaluation of novel ß-secretase inhibitors containing a pyrazole or thiazole moiety as the P3 ligand.

We describe structure-based design, synthesis, and biological evaluation of a series of novel inhibitors bearing a pyrazole (compounds 3a-h) or a thiazole moiety (compounds 4a-e) as the P3 ligand. We have also explored Boc-ß-amino-l-alanine as a novel P2 ligand. A number of inhibitors have displayed ß-secretase inhibitory potency. Inhibitor 4c has shown potent BACE1 inhibitory activity, Ki=0.25nM, cellular EC50 of 194nM, and displayed good selectivity over BACE2. A model of 4c was created based upon the X-ray structure of 2-bound ß-secretase which revealed critical interactions in the active site.

Beneficial effects of the ß-secretase inhibitor GRL-8234 in 5XFAD Alzheimer's transgenic mice lessen during disease progression.

The ß-secretase enzyme BACE1, which initiates the cleavage of amyloid precursor protein (APP) into the amyloid-ß (Aß) peptide, is a prime therapeutic target for Alzheimer's disease (AD). However, recent investigations using genetic animal models raise concern that therapeutic BACE1 inhibition may encounter the dramatic reduction of efficacy in ameliorating AD-like pathology and memory deficits during disease progression. Here, we compared the effects of the potent and selective small-molecule BACE1 inhibitor GRL-8234 in different pathological stages of AD mouse model. Specifically, we administered GRL-8234 (33.4 mg/kg, i.p.) once daily for 2 months to 5XFAD transgenic mice, which showed modest (4 months) and massive (10 months of age) Aß plaque deposition at starting points. Chronic treatments with GRL-8234 reversed memory impairments, as tested by the spontaneous alternation Y-maze task, in the younger 5XFAD group concomitant with significant reductions in cerebral Aß42 levels. In contrast, only marginal reductions of Aß42 were observed in 12-month-old 5XFAD mice treated with GRL-8234 and their memory function remained impaired. We found that not only BACE1 but also full-length APP expression was significantly elevated with progressive Aß accumulation in 5XFAD mice, while GRL-8234 failed to affect these detrimental mechanisms that further accelerate plaque growth in brains of older 5XFAD mice. Therefore, our results provide important insights into the mechanisms by which Aß accumulation and related memory impairments become less responsive to rescue by BACE1 inhibition during the course of AD development.

Structure-activity relationship of memapsin 2: implications on physiological functions and Alzheimer's disease.

Memapsin 2 (BACE1, ß-secretase), a membrane aspartic protease, functions in the cleavage of the type I transmembrane protein, ß-amyloid precursor protein (APP), leading to the production of amyloid ß (Aß) in the brain. Since Aß is closely associated with the pathogenesis of Alzheimer's disease, understanding the biological function, particularly the catalytic activities of memapsin 2, would assist in a better understanding of the disease and the development of its inhibitors. The transmembrane and cytosolic domains of memapsin 2 function in cellular transport and localization, which are important regulatory mechanisms for its activity. The catalytic ectodomain contains a long substrate cleft that is responsible for substrate recognition, specificity, and peptide bond hydrolysis. The substrate cleft accommodates 11 residues of the substrate in separate binding subsites. Besides APP, a number of membrane proteins have been reported to be substrates of memapsin 2. The elucidation for the specificity of these subsites and the amino acid sequences surrounding the memapsin 2 cleavage site in these proteins has led to the establishment of a predictive model that can quantitatively estimate the efficiency of cleavage for any potential substrates. Such tools may be employed for future studies of memapsin 2 about its biological function. Herein, we review the current knowledge on the structure-function relationship of memapsin 2 and its relationship in the biological function.

Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP).

Processing of ß-amyloid precursor protein (APP) by ß- and γ-secretases in neurons produces amyloid-ß (Aß), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aß, whose level was reduced by inhibitors of ß- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by ß-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.

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.

Structure-based design of highly selective ß-secretase inhibitors: synthesis, biological evaluation, and protein-ligand X-ray crystal structure.

The structure-based design, synthesis, and X-ray structure of protein-ligand complexes of exceptionally potent and selective ß-secretase inhibitors are described. The inhibitors are designed specifically to interact with S(1)' active site residues to provide selectivity over memapsin 1 and cathepsin D. Inhibitor 5 has exhibited exceedingly potent inhibitory activity (K(i) = 17 pM) and high selectivity over BACE 2 (>7000-fold) and cathepsin D (>250000-fold). A protein-ligand crystal structure revealed important molecular insight into these selectivities. These interactions may serve as an important guide to design selectivity over the physiologically important aspartic acid proteases.

Structure-based design, synthesis, and biological evaluation of dihydroquinazoline-derived potent ß-secretase inhibitors.

Structure-based design, synthesis, and biological evaluation of a series of dihydroquinazoline-derived ß-secretase inhibitors incorporating thiazole and pyrazole-derived P2-ligands are described. We have identified inhibitor 4f which has shown potent enzyme inhibitory (K(i)=13 nM) and cellular (IC(50)=21 nM in neuroblastoma cells) assays. A model of 4f was created based upon the X-ray structure of 3a-bound ß-secretase. The model suggested possible interactions in the active site.

Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group.

The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH(2)-(D: -Arg)(9)-Val-Leu-Arg-CO-NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin's lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), -N(CH(3))-O-CH(3). R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.

Developing ß-secretase inhibitors for treatment of Alzheimer's disease.

ß-Secretase (memapsin 2; BACE-1) is the first protease in the processing of amyloid precursor protein leading to the production of amyloid-ß (Aß) in the brain. It is believed that high levels of brain Aß are responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, ß-secretase is a major therapeutic target for the development of inhibitor drugs. During the past decade, steady progress has been made in the evolution of ß-secretase inhibitors toward better drug properties. Recent inhibitors are potent, selective and have been shown to penetrate the blood-brain barrier to inhibit Aß levels in the brains of experimental animals. Moreover, continuous administration of a ß-secretase inhibitor was shown to rescue age-related cognitive decline in transgenic AD mice. A small number of ß-secretase inhibitors have also entered early phase clinical trials. These developments offer some optimism for the clinical development of a disease-modifying drug for AD.

Subsite specificity of anthrax lethal factor and its implications for inhibitor development.

The lethal factor of Bacillus anthracis is a major factor for lethality of anthrax infection by this bacterium. With the aid of the protective antigen, lethal factor gains excess to the cell cytosol where it manifests toxicity as a metalloprotease. For better understanding of its specificity, we have determined its residue preferences of 19 amino acids in six subsites (from P3 to P3') as relative k(cat)/K(m) values (specificity constants). These results showed that lethal factor has a broad specificity with preference toward hydrophobic residues, but not charged or branched residues. The most preferred residues in these six subsites are, from P1 to P3', Trp, Leu, Met, Tyr, Pro, and Leu. The result of residue preference was used to design new substrates with superior hydrolytic characteristics and inhibitors with high potency. For better use of the new findings for inhibitor design, we have modeled the most preferred residues in the active site of lethal factor. The observed interactions provide new insights to future inhibitor designs.

Treating transgenic Alzheimer mice with a ß-secretase inhibitor, what have we learned?

ß-Secretase is an attractive target of amyloid-reduction therapy for Alzheimer's disease. Currently, no efficacy data is available from clinical trials of ß-secretase inhibitors. Treating young transgenic Tg2576 mice with a brain-penetrating ß-secretase inhibitor reduced brain amyloid-ß by about 50% and rescued the age-related cognitive decline. Implications from these model studies on the design of clinical trials are discussed herein.

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.

Predicting memapsin 2 (ß-secretase) hydrolytic activity.

Memapsin 2 (BACE1, ß-secretase), a membrane aspartic protease, functions in the cleavage of brain ß-amyloid precursor protein (APP) leading to the production of ß-amyloid. Because the excess level of ß-amyloid in the brain is a leading factor in Alzheimer's disease (AD), memapsin 2 is a major therapeutic target for inhibitor drugs. The substrate-binding cleft of memapsin 2 accommodates 12 subsite residues, from P(8) to P(4)'. We have determined the hydrolytic preference as relative k(cat)/K(M) (preference constant) in all 12 subsites and used these data to establish a predictive algorithm for substrate hydrolytic efficiency. Using the sequences from 12 reported memapsin 2 protein substrates, the predicted and experimentally determined preference constants have an excellent correlation coefficient of 0.97. The predictive model indicates that the hydrolytic preference of memapsin 2 is determined mainly by the interaction with six subsites (from P(4) to P(2)'), a conclusion supported by the crystal structure B-factors calculated for the various residues of transition-state analogs bound to different memapsin 2 subsites. The algorithm also predicted that the replacement of the P(3), P(2), and P(1) subsites of APP from Val, Lys, and Met, respectively, to Ile, Asp, and Phe, respectively, (APP(IDF)) would result in a highest hydrolytic rate for ß-amyloid-generating APP variants. Because more ß-amyloid was produced from cells expressing APP(IDF) than those expressing APP with Swedish mutations, this designed APP variant may be useful in new memapsin 2 substrates or transgenic mice for AD studies.

Design, synthesis, protein-ligand X-ray structure, and biological evaluation of a series of novel macrocyclic human immunodeficiency virus-1 protease inhibitors to combat drug resistance.

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic macrocyclic HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic pocket in the S1'-S2' subsites and retain all major hydrogen bonding interactions with the protein backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution, and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in a reduction of potency. Both inhibitors 14b and 14c exhibited marked enzyme inhibitory and antiviral activity, and they exerted potent activity against multidrug-resistant HIV-1 variants. Protein-ligand X-ray structures of inhibitors 2 and 14c provided critical molecular insights into the ligand-binding site interactions.