Aspartyl Protease Inhibitor Research Articles

Harnessing Nature’s Insight: Design of Aspartyl Protease Inhibitors from Treatment of Drug-Resistant HIV to Alzheimer’s Disease

Historically, nature has provided an incredible variety of structurally complex and biologically important molecules. Indeed, a great many of today’s clinical medicines are obtained directly from natural products or from their derivatives.1 Natural products continue to be a very important source for modern drug discovery and development.2 The seemingly limitless structural features coupled with their novel biological properties have provided enormous inspiration for the development of new reactions and methodologies en route to natural product synthesis and structural variations for drug-discovery.3 Beginning in the fall of 1994, we initiated a research program aimed at synthesizing bioactive natural products with interesting structural features. This endeavor resulted in the total synthesis of numerous targets, covering over two dozen or so different structural families. Some notable examples of our accomplished bioactive targets include novel and exceedingly potent microtubule stabilizing agents, laulimalide (1)4 and peloruside A (2)5 a potent microtubule destabilizing agent cryptophycin 52 (3)6; anticancer agents amphidinolide T (4)7, amphidinolide W (5)8, and lasonolide A (6)9; antibiotic agent, madumycin II (7)10; pancreatic lipase inhibitor, tetrahydrolipstatin (8)11; novel actin inhibitory agents doliculide (9)12 and jasplakinolide (10)13; novel antibacterial agent platensimycin (11)14; and the histone deacytelase inhibitor, largazole (12)15 (see Figure 1). The unique structural features of these natural products required the development of new synthetic tools and methodologies for their synthesis. In the context of our synthesis of various bioactive targets, we have developed a variety of new and practical asymmetric reactions based upon intermolecular and intramolecular metal chelation.16 Notable carbon-carbon bond forming synthetic methodologies include highly diastereoselective syn-and anti-aldol reactions17; asymmetric inter and intramolecular Diels-Alder and hetero Diels-Alder reactions18, and asymmetric multicomponent reactions.19 The scope and utility of these methodologies have been demonstrated through the synthesis of a variety of bioactive molecules. Open in a separate window Figure 1 Structures of Recent Bioactive Targets

Construction of a Complementary DNA Library of Parelaphostrongylus tenuis and Identification of a Potentially Sero-Diagnostic Recombinant Antigen

Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

Cathepsin E regulates the presentation of tetanus toxin C-fragment in PMA activated primary human B cells

Processing of antigens by proteases in the endocytic compartments of antigen presenting cells (APC) is essential to make them suitable for presentation as antigenic peptides to T lymphocytes. Several proteases of the cysteine, aspartyl and serine classes are involved in this process. It has been speculated, that the aspartyl protease cathepsin E (CatE) is involved in antigen processing in B cell line, monocyte-derived dendritic cells (DC) and murine DC. Here we show the expression of CatE in primary human B cells and DC, which was only elevated in B cells after induction with phorbol 12-myristate 13-acetate (PMA), resulted in enhanced presentation of tetanus toxin C-fragment (TTC) to the respective T cells. Inhibition of aspartyl proteases using pepstatin-A-penetratin (PepA-P), a highly efficient, cell-permeable aspartyl protease inhibitor, reduced significantly T cell activation in PMA activated B cells but not in PMA activated myeloid DC (mDC). Thus we suggest that CatE is important in the processing of TTC in primary human B cells.

Discovery and X-ray Crystallographic Analysis of a Spiropiperidine Iminohydantoin Inhibitor of β-Secretase

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Autophagy Induction and Autophagosome Clearance in Neurons: Relationship to Autophagic Pathology in Alzheimer's Disease

Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimer's disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, because newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR (mammalian target of rapamycin) kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. In contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autolysosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double-membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway.

Synthesis and Antimalarial Activity of Novel Hydroxyethylamines,Potential Aspartyl Protease Inhibitors

Synthesis and Antimalarial Activity of Novel Hydroxyethylamines,Potential Aspartyl Protease Inhibitors

Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir

BackgroundAspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, although specific mechanisms are poorly understood. Recently, PIs, particularly Nelfinavir, were reported to disrupt Notch signaling in the HIV-related endothelial cell neoplasm, Kaposi's sarcoma. Given the importance of maintaining proper cerebral endothelial cell signaling at the blood brain barrier during HIV infection, we considered potential signaling pathways such as Notch, that may be vulnerable to dysregulation during exposure to PI-based anti-retroviral regimens. Notch processing by γ-secretase results in cleavage of the notch intracellular domain that travels to the nucleus to regulate expression of genes such as vascular endothelial cell growth factor and NFκB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on γ-secretase substrate pathways in cerebral endothelial cell signaling have not been addressed, we sought to determine the effects of HIV PIs on Notch and amyloid precursor protein.ResultsExposure to reported physiological levels of Saquinavir, Indinavir, Nelfinavir and Ritonavir, significantly increased reactive oxygen species in cerebral endothelial cells, but had no effect on cell survival. Likewise, PIs decreased Notch 4-protein expression, but had no effect on Notch 1 or amyloid precursor protein expression. On the other hand, only Nelfinavir increased significantly Notch 4 processing, Notch4 intracellular domain nuclear localization and the expression of notch intracellular domain targets NFκB and matrix metalloproteinase 2. Pre-treatment with the antioxidant Vitamin E prevented PI-induced reactive oxygen species generation and partially prevented Nelfinavir-induced changes in both Notch 4 processing, and cellular localization patterns. Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity.ConclusionNelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFκB and matrix metalloproteinase 2 in cerebral endothelial cells.

Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH(4)Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH(4)Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% +/- 110%, 1,212% +/- 34%, and 1,100% +/- 179%, respectively, in porcine kidney (PK-15) cells; by 128% +/- 7%, 158% +/- 3%, and 142% +/- 11% in swine kidney cells; by 160% +/- 28%, 446% +/- 50%, and 162% +/- 56% in swine testicle (ST) cells; and by 313% +/- 25%, 611% +/- 86%, and 352% +/- 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification.

Cover Picture: Design and Synthesis of 2,3,5‐Substituted Imidazolidin‐4‐one Inhibitors of BACE‐1 (ChemMedChem 7/2007)

The cover picture shows a close-up view of a modified hydroxyethylamine aspartyl protease inhibitor bound in the BACE-1 active site. The imidazolidinone ring serves as a rigid scaffold for orienting substituents into the S1' and S2' sites of the enzyme while making hydrogen bond contacts to the catalytic aspartate groups (highlighted in red) and the flap (colored in blue). Based on this X-ray crystal structure, additional analogues were designed to exploit the imidazolidinone heterocycle as a template for potent BACE-1 inhibitors. For details, see the Communication by J. Barrow et al. on p. 995 ff.

Design and Synthesis of 2,3,5‐Substituted Imidazolidin‐4‐one Inhibitors of BACE‐1

Structure-based drug design was used to incorporate the traditional hydroxyethyl amine aspartyl protease inhibitor motif into 2,3,5-substituted imidazolidin-4-one structures with good BACE-1 enzyme inhibitory potency. These compounds represent a promising drug target for Alzheimer′s disease-modifying therapy and are therefore of interest to the medicinal chemistry community.

Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry

Gastrointestinal nematode infections of livestock animals are prevalent and costly problems worldwide. Currently, infections are controlled by anthelmintic chemicals but increasing drug resistance has prompted research interest to shift towards alternative methods of control such as vaccine development and selection of worm-resistant animals. The present study analyses proteins from Trichostrongylus colubriformis infective L3s that are recognised by IgG of immune sheep. Following protein separation via two-dimensional electrophoresis and Western blot probing with plasma from sheep resistant to T. colubriformis, mass spectrometry-based proteomic analyses were used to identify immuno-reactive protein spots. We were able to identify 28 immune targets, including aspartyl protease inhibitor, enolase, chaperone proteins, galectin, glycolytic enzymes, kinase, phosphatase and structural muscle proteins such as myosin, paramyosin, calponin and DIM-1. The data suggest that immune responses to T. colubriformis are dispersed over a relatively large number of parasite antigens, including several cytoplasmically expressed proteins. The results have new implications for understanding the molecular mechanisms that underpin host–parasite interaction during gastrointestinal nematode infections.

Calpain Inhibition Is Sufficient to Suppress Aggregation of Polyglutamine-expanded Ataxin-3

The formation of intraneuronal inclusions is a common feature of neurodegenerative polyglutamine disorders, including Spinocerebellar ataxia type 3. The mechanism that triggers inclusion formation in these typically late onset diseases has remained elusive. However, there is increasing evidence that proteolytic fragments containing the expanded polyglutamine segment are critically required to initiate the aggregation process. We analyzed ataxin-3 proteolysis in neuroblastoma cells and in vitro and show that calcium-dependent calpain proteases generate aggregation-competent ataxin-3 fragments. Co-expression of the highly specific cellular calpain inhibitor calpastatin abrogated fragmentation and the formation of inclusions in cells expressing pathological ataxin-3. These findings suggest a critical role of calpains in the pathogenesis of Spinocerebellar ataxia type 3.

Parallel synthesis of arrays of 1,4,5-trisubstituted 1-(4-piperidyl)-imidazoles by IMCR: A novel class of aspartyl protease inhibitors

The synthesis of a novel class of potential aspartyl-protease inhibitors is described. Arrays of 1,4,5-trisubstituted 1-(4-piperidyl)-imidazoles have been assembled by parallel synthesis using an isocyanide-based multi component reaction (vL-3CR).

Metalloprotease inhibitors block release of soluble CD27 and enhance the immune stimulatory activity of chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) B cells from most patients express both membrane-bound CD27 (mCD27) and soluble CD27 (sCD27). Expression of sCD27 inhibits CD27-dependent T-cell or CLL-cell activation mediated by its ligand, CD70. In this study, we evaluated whether protease inhibitors could inhibit the release of sCD27 from CLL cells and enhance T-cell activation mediated by CD27-CD70 interaction. CLL cells exposed to hydroxamic acid-based matrix metalloprotease (MMP) inhibitors were evaluated for the release of sCD27 by sandwich enzyme-linked immunosorbent assay and immunoprecipitation. We examined for phenotypic changes in CLL cells treated with MMP inhibitors by flow cytometry and T-cell activation by CLL cells was assessed by [(3)H] thymidine incorporation assay and the production of interferon-gamma. Treatment of CLL cells with MMP inhibitors blocked the release of sCD27 to the culture supernatant. In contrast, a non-hydroxamic acid control compound or inhibitors of other proteases, including serine, cysteine, and aspartyl proteases, were ineffective. Furthermore, CLL cells treated with MMP inhibitors expressed significantly higher levels of accessory molecules, such as CD54, CD80, and CD95. Consistent with such changes, we found that CLL cells treated with MMP inhibitors, but not control treated cells, could stimulate allogeneic and autologous T cells in mixed lymphocyte reactions. These data reveal that metalloprotease inhibitors can block production of sCD27, which can interfere with mCD27-CD70 interactions that induce expression of immune costimulatory molecules on CLL B cells. Conceivably, treatment of CLL cells with metalloprotease inhibitors may enhance their potential for stimulating cellular immune recognition of leukemia-associated antigens.

The Proteolytic System of Candida dubliniensis

Proteases of Candida dubliniensis have been scarcely studied, these enzymes may play an important role in nitrogen metabolism, post-translational processing, nutritional stress, dimorphism, virulence, etc. In this work, we report the presence of five different intracellular proteases and one extracellular proteolytic activity. The intracellular proteases are: aminopeptidase ycdAPE, carboxypeptidase ycdCP, dipeptidyl aminopeptidase ycdDAP, proteinases ycdPrA and ycdPrB, and extracellular protease Sap activity, measured under several nutritional conditions. C. dubliniensis produced the highest level of intracellular proteolytic enzymes, i.e., ycdAPE, ycdCP, ycdDAP, ycdPrA and ycdPrB in media with peptone during stationary growth phase. Chelating agents affected mainly APE activity; whereas ycdCp, ycdDAP, and ycdPrB were affected by serine protease inhibitors; ycdPrA was affected by pepstatin, an aspartyl protease inhibitor. We found Sap activity in C. dubliniensis in YCB-SBA medium, this activity was inhibited by pepstatin inhibitor. Southern analysis revealed the presence of at least four genes encoding Sap in the C. dublinienisis genome (using as probes SAP1, SAP2, SAP3, and SAP4-6 genes from C. albicans).

The Low-Barrier Double-Well Potential of the O(δ)(1)-H-O(δ)(1) Hydrogen Bond in Unbound HIV Protease: A QM/MM Characterization.

The presence of a low-barrier hydrogen bond (LBHB) in aspartyl proteases and its implications in drug design have been the subject of intense study. Here, we present a combined quantum mechanical/molecular mechanical (QM/MM)-Numerov procedure and use it to characterize the O(δ)(1)-H-O(δ)(1) hydrogen bond (HB) in unbound HIV protease. The QM/MM scheme fully traces the shape of the HB's potential energy curve. The potential is used to obtain numerical solutions to the wave functions and vibrational energies of hydrogen, deuterium, and tritium. The vibrational eigenfunctions are used to compute expectation values for interatomic distances and vibrationally and thermally averaged spectroscopic properties of the O(δ)(1)-H-O(δ)(1) HB. Our work corroborates previous results by Piana and Carloni who found a LBHB via an ab initio molecular dynamics simulation (Piana, S.; Carloni, P. Proteins 2000, 39, 26-36). Our predictions of isotope effects on the chemical shift of unbound HIV protease are consistent with experimental measurements in similar HBs. These results support the predictive power of this method and its potential use in screening inhibitors of aspartyl proteases.

Symbiosis: Chemical Biology at Wisconsin

C hemical biology has been percolating at the University of Wisconsin (UW)–Madison for most of the university’s history (www.chemicalbiology. wisc.edu; 1–4). UW–Madison played a predominant role in early research on vitamins—those small molecules that are essential in trace amounts for human life. There, Harry Steenbock developed the irradiation process for the production of vitamin D and thereby eliminated the scourge of rickets (5, 6). Vitamin A was discovered at Wisconsin, as were the vitamin B complex and the hormonal form of vitamin D. Karl Paul Link isolated the potent anticoagulant dicoumarol there and then synthesized warfarin, an analogue that is still a common chemotherapeutic agent. Microbial fermentation methods developed at UW–Madison enabled the large-scale biosynthesis of penicillin and other antibiotics. H. Gobind Khorana carried out the first chemical synthesis of a gene there (7), andW. S. Johnson and Eugene van Tamelen devised synthetic routes to steroids inspired by the biosynthesis of this critical class of natural products (8). The use of natural protease inhibitors to devise highly effective inhibitors of aspartyl proteases was an insight that arose atWisconsin andwas used to design potent HIV protease inhibitors (9). These and other triumphs demonstrate an early symbiosis of chemistry and biology, which fostered the training of eminent interdisciplinary scientists such as Carl Djerassi and Ralph Hirschmann. Seeking to coalesce the well-established success in research at the interface between chemistry and biology, Dan Rich sought one of the initial Chemistry–Biology Interface (CBI) Training Grants from the National Institutes of Health (NIH) in 1993. His vision was to provide graduate students with a multitude of opportunities for interdisciplinary training in chemistry and biology. The process of preparing the grant application launched the UW–Madison program in chemical biology (www.chemical biology.wisc.edu). The now long-standing NIH training grant remains a core component of graduate training in chemical biology at the university. Since the inception of the training program, UW–Madison’s commitment to graduate-student training in chemical biology has increased. For example, the university has made the recruitment of faculty members with research interests in chemical biology a priority. As a result, graduate students have a wide variety of research options from which to choose. In addition, a powerful infrastructure for conducting chemical biology research has been built. Third, courses in chemical biology have been developed that employ innovative teaching methods. These courses are designed not only to introduce students to concepts in chemical biology but also to build their skills in critical thinking, creative problem selection, and communication. Lastly, students affiliated with *Corresponding author, kiessling@chem.wisc.edu.

Unexpected Novel Binding Mode of Pyrrolidine‐Based Aspartyl Protease Inhibitors: Design, Synthesis and Crystal Structure in Complex with HIV Protease

At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25'. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.

Synthesis of New Substituted 4,5-Dihydro-3H-spiro[1,5]-benzoxazepine-2,4′-piperidine and Biological Properties

The reduction of substituted spiro-piperidinyl chromanone oximes with DIBAH reagents has been known to afford the corresponding substituted 4,5-dihydro-3H-spiro[1,5]-benzoxazepine-2,4′-piperidine. The position and electronic effects of the substituents on the aryl moiety control the observed rearrangement. Spiro-benzoxazepine analogue 5j represents a key intermediate for the creation of a library of diverse potential bioactive drugs. With three functional groups that could be selectively and orthogonally protected, many different substituents can be introduced. The obtained analogues were assayed as the possible aspartyl protease inhibitors HIV protease (HIV-1), and β-secretase (BACE-1).

Recent Developments of Structure Based β-Secretase Inhibitors for Alzheimers Disease

The amyloid-beta (Abeta) peptide is the principal components of the senile plaques found in the brains of patients with Alzheimer's disease (AD). The poorly soluble 40-42 amino acid peptide, formed from the cleavage of the Abeta precursor protein (APP) by two proteases, is believed to play a central role in the pathogenesis of AD. Beta-secretase (memapsin 2, BACE1), a membrane-anchored aspartic protease, is responsible for the initial step of APP cleavage leading to the generation of Abeta. Identification and structural determination of beta-secretase have established it to be a primary drug target for AD therapy and stimulated active studies on the inhibitors of this protease. Here we review more recent developments in the design and testing of structure-based beta-secretase inhibitors.