Transition State Analogue Inhibitors Research Articles

Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (K(d) = 56 pM) is driven by large negative enthalpy values (DeltaH = -21.2 kcal/mol) with a substantial entropic (-TDeltaS) penalty. The tightest-binding inhibitors (K(d) = 5-9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP.inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the 17 Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. Via introduction of flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.

Intramembrane-cleaving Proteases

In the past decade or so, membrane-embedded proteases that carry out hydrolysis on the transmembrane region of their substrates have been discovered. These I-CLiPs2 (1) somehow create an environment for water and the hydrophilic residues needed for catalysis and bend or unwind their helical substrates to make the amide bonds susceptible to hydrolysis. Despite the distinction of being membrane-embedded and cleaving TMDs, the residues essential for catalysis by these I-CLiPs are virtually the same as those found in aqueous proteases, clear examples of convergent evolution toward a common mechanism. Described herein are the different types of I-CLiPs and an update on their structural and mechanistic features and biological roles.

Biochemical And Structural Characterization Of Intramembrane Proteases

In regulated intramembrane proteolysis membrane proteins are cleaved within their transmembrane region, resulting in soluble fragments that regulate cell physiology. The intramembrane proteases responsible for cleavage are widespread with important roles in human biology and disease. Rhomboids catalyze the activation of epidermic growth factor receptor ligands in D. melanogaster. How rhomboids recognize their substrates and select which peptide bond to cleave is not understood. We have studied the substrate specificity and peptide bond selectivity of purified rhomboids from several organisms using chimeric transmembrane substrates. Analysis of the proteolytic products by mass spectrometry reveals that cleavage occurs in the membrane/water interface at sites shared by both eukaryotic and prokaryotic rhomboids. Mutagenesis of the substrates reveals a helical amino acid motif that is crucial for substrate recognition and peptide bond selection. With insight from computational data a model for the substrate-enzyme complex will be presented.Gamma-Secretase catalyzes the production of amyloid beta-peptides involved in Alzheimer's disease. Using negative-stain single-particle electron microscopy we have determined the structure of a native-like 500kDa gamma-secretase complex comprising presenilin, nicastrin, APH-1, and PEN-2 that is fully catalytically active. Antibody labeling of the extracellular domain of nicastrin was employed to ascertain the topology of the reconstruction. Active site labeling with a gold-coupled transition state analog inhibitor demonstrates that gamma-secretase contains a single active site facing a large conical internal cavity. This cavity, surrounded by a ~35A thick transmembrane protein wall, extends from the extracellular side of the membrane to past the membrane centre, where it narrows to finally close at the cytoplasmic side. Based on our structure we suggest a model for gamma-secretase function, in which a hydrophobic transmembrane helix substrate is hydrolyzed by catalytic aspartyl moieties at the interface of a water-accessible internal cavity away from the surrounding lipid environment.

Structure Of Gamma-secretase Reveals An Active Site Facing An Internal Membrane Cavity

The integral membrane protein complex gamma-secretase catalyzes the final step in the production of amyloid beta-peptides involved in the pathogenesis of Alzheimer's disease. Using negative-stain single-particle electron microscopy we have determined the structure of a native-like 500kDa gamma-secretase complex comprising presenilin, nicastrin, APH-1, and PEN-2 that is fully catalytically active. Antibody labeling of the extracellular domain of nicastrin was employed to ascertain the topology of the reconstruction. Active site labeling with a gold-coupled transition state analog inhibitor demonstrates that gamma-secretase contains a single active site facing a large conical internal cavity. This cavity, surrounded by a ∼35A thick transmembrane protein wall, extends from the extracellular side of the membrane to past the membrane centre, where it narrows to finally close at the cytoplasmic side. Based on our structure we suggest a model for gamma-secretase function, in which a hydrophobic transmembrane helix substrate is hydrolyzed by catalytic aspartyl moieties at the interface of a water-accessible internal cavity away from the surrounding lipid environment.

Purification, Pharmacological Modulation, and Biochemical Characterization of Interactors of Endogenous Human γ-Secretase

Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.

Thiasugars: Potential Glycosidase Inhibitors

The inhibitory activities against glycosidases of synthetic thiasugars, the ring sulfur analogs of carbohydrate, were surveyed with a special emphasis on our own studies. 5-Thio-L-fucose, the ring sulfur analog of L-fucose, was the first thiopyran that shows a K(i) value in the micromolar range against a glycosidase. The structure-activity relationship studies for the fucosidase inhibition by 5-thio-L-fucose disclosed that a hydrophobic interaction between the ring sulfur atom and the enzyme is responsible for the strong binding. The syntheses and activities of di- and trisaccharide analogs incorporating thiasugars are also outlined. These oligosaccharide analogs are glycosidase-resistant and some of them show strong binding to antibodies or lectins. We created a sulfylimine compound having a thiafuran structure as a transition-state analog inhibitor of glucosidases and found that it has a weak inhibition against a glucosidase. The same thiafuran structure could be found in a natural product, salacinol, which was isolated and elucidated to be a strong glucosidase inhibitor by another group. A study of the structure-activity relationship using the synthetic analogs of salacinol indicated the relevance of both the sulfonium ion in the thiafuran ring and its intramolecular counter anion, sulfate, to the inhibition activity.

Plasmodium falciparum Purine Nucleoside Phosphorylase Is Critical for Viability of Malaria Parasites

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

BCX-4208 (RO5092888), a Purine Nucleoside Phosphorylase (PNP) Inhibitor, Is a Novel, Potent Orally Active Anti-T-Cell and B-Cell Agent.

The profound suppression of T-cell immunity seen in patients with an inherited PNP deficiency supports the potential application of inhibitors of this purine salvage enzyme in the therapy of T-cell malignancies and T-cell mediated autoimmune diseases. About thirty percent of PNP deficient patients also show evidence of B-cell dysfunction. BCX-4208 is a novel potent transition state analog inhibitor of PNP (IC50 ~ 0.0005 μM) and in the presence of 10 μM deoxyguanosine (dGuo), inhibits human lymphocyte proliferation induced by MLR, IL-2 or Con-A with IC50s of 0.159, 0.26 and 0.73 μM, respectively. The IC50 for dGuo in the same assays in the presence of 1 μM BCX-4208 ranges from 1–3 μM. Neither BCX-4208 alone nor dGuo alone inhibits proliferation of lymphocytes. In the presence of PNP inhibitor, dGuo is converted to dGMP and then to dGTP. Accumulation of dGTP results in the alteration of deoxynucleotide (dNTP) pools, causing death of cells via a mechanism characteristic of apoptosis. In vitro data demonstrates that following exposure to BCX-4208 and dGuo, dGTP in human lymphocytes is elevated and a 5–8 fold increase in dGTP results in 50% inhibition of lymphocyte proliferation. Flow cytometric analyses of human lymphocytes using annexin-V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis not only in T cells (CD3+), but also in B cells (CD20+; CD19+) (Table 1). BCX-4208 is orally bioavailable in mice, can achieve maximal inhibition of PNP, and elevates plasma dGuo levels to 3–5 μM (predose levels < 0.004 μM), which is similar to levels seen in PNP deficient patients and to levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of not only T-cell mediated diseases but also B-cell mediated diseases. BCX- 4208 is currently undergoing early clinical investigation in patients with psoriasis.Table 1.Cell subsets% Apoptotic cells mean ± SEM (n = 4–7)VehicleTreatment groupCD3+7.2 ± 0.918.3 ± 3.7*CD20+24.0 ± 3.641.8 ± 6.2*CD19+25.0 ± 3.151.6 ± 7.4**p ≤ 0.01 compared to vehicle

Distinct Pharmacological Effects of Inhibitors of Signal Peptide Peptidase and γ-Secretase

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Transition state analogues in quorum sensing and SAM recycling

Transition state structures can be derived from kinetic isotope effects and computational chemistry. Molecular electrostatic potential maps of transition states serve as blueprints to guide synthesis of transition state analogue inhibitors of target enzymes. 5'- Methylthioadenosine phosphorylase (MTAP) functions in the polyamine pathway by recycling methylthioadenosine (MTA) and maintaining cellular S-adenosylmethionine (SAM). Its transition state structure was used to guide synthesis of MT-DADMe-ImmA, a picomolar inhibitor that shows anticancer effects against solid tumors. Biochemical and genomic analysis suggests that MTAP inhibition acts by altered DNA methylation and gene expression patterns. A related bacterial enzyme, 5'-methylthioadenosine nucleosidase (MTAN), functions in pathways of quorum sensing involving AI-1 and AI-2 molecules. Transition states have been solved for several bacterial MTANs and used to guide synthesis of powerful inhibitors with dissociation constants in the femtomolar to picomolar range. BuT-DADMe-ImmA blocks quorum sensing in Vibrio cholerae without changing bacterial growth rates. Transition state analogue inhibitors show promise as anticancer and antibacterial agents.

Immucillins in custom catalytic-site cavities

Neighboring-group participation in the reaction catalyzed by purine nucleoside phosphorylase involves a compression mode between the 5′- and 4′-ribosyl oxygens, facilitated by His257. The His257Gly mutant opens a space in the catalytic site. Hydrophobic 5′-substituted Immucillins are transition-state analogue inhibitors of this mutant enzyme. Dissociation constants as low as 2 pM are achieved, with K m/ K d as high as 400,000,000.

Selective Modulation of Matrix Metalloproteinase 9 (MMP-9) Functions via Exosite Inhibition

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.

Antihypertensive and Renoprotective Mechanisms of Renin Inhibition in Diabetic Rats

Diabetic nephropathy, the most common cause of end-stage renal disease (ESRD) in the Western world, is characterized by suppression of the systemic but activation of the intrarenal tissue renin-angiotensin system (RAS).1 Blockade of the RAS with angiotensin converting enzyme (ACE) inhibitors or angiotensin type-1 receptor blockers (ARBs) retards but does not eliminate the progression of diabetic nephropathy to ESRD in prospective, randomized, controlled clinical trials.2–4 Studies have demonstrated that a major component of tissue protection from RAS blockers derives from reduction in blood pressure.5 However, a possibility for failure to prevent completely the decline in renal function attributable to tissue damage is that the activity of the intrarenal RAS may be inadequately suppressed by these drugs. Renin, an aspartyl protease cleaving angiotensinogen (Agt) at the origin of the angiotensin peptide cascade, has been a logical target for RAS inhibition. Since 1982, when the first peptide analogues of Agt were designed to block its catalytic cleavage by renin,6 multiple attempts have been made to inhibit renin, first by dipeptide transition-state analogue inhibitors of the renin active site and most recently by nonpeptide compounds, of which aliskiren is the most successful example.7 Aliskiren, approved for the treatment …

The Catalytic Mechanism of an Aspartic Proteinase Explored with Neutron and X-ray Diffraction

Hydrogen atoms play key roles in enzyme mechanism, but as this study shows, even high-quality X-ray data to a resolution of 1 A cannot directly visualize them. Neutron diffraction, however, can locate deuterium atoms even at resolutions around 2 A. Both neutron and X-ray diffraction data have been used to investigate the transition state of the aspartic proteinase endothiapepsin. The different techniques reveal a different part of the story, revealing the clearest picture yet of the catalytic mechanism by which the enzyme operates. Room temperature neutron and X-ray diffraction data were used in a newly developed joint refinement software package to visualize deuterium atoms within the active site of the enzyme when a gem-diol transition state analogue inhibitor is bound at the active site. These data were also used to estimate their individual occupancy, while analysis of the differences between the bond lengths of the catalytic aspartates was performed using atomic resolution X-ray data. The two methods are in agreement on the protonation state of the active site with a transition state analogue inhibitor bound confirming the catalytic mechanism at which the enzyme operates.

&amp;#947;-Secretase Inhibition and Modulation for Alzheimers Disease

Gamma-secretase is a multi-protein complex that proteolyzes the transmembrane region of the amyloid beta-peptide (Abeta) precursor (APP), producing the Abeta peptide implicated in the pathogenesis of Alzheimer's disease (AD). This protease has been a top target for AD, and various inhibitors have been identified, including transition-state analogue inhibitors that interact with the active site, helical peptides that interact with the initial substrate docking site, and other less peptide-like, more drug-like compounds. Although one gamma-secretase inhibitor has advanced into late-phase clinical trials, concerns about inhibiting this protease remain. The protease complex cleaves a number of other substrates, and in vivo toxicities observed with gamma-secretase inhibitors are apparently due to blocking one particularly important substrate, the Notch receptor. Thus, the potential of gamma-secretase as therapeutic target likely depends on the ability to selectively inhibit Abeta production without hindering Notch proteolysis (i.e., modulation rather than inhibition). The discovery of gamma-secretase modulators has revived gamma-secretase as an attractive target and has so far resulted in one compound in late-phase clinical trials. The identification of other modulators in a variety of structural classes raise the hope that more promising agents will soon be in the pipeline.

Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. 3H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas 3H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine–guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.

Azetidine Based Transition State Analogue Inhibitors of N-Ribosyl Hydrolases and Phosphorylases

N-ribosyl phosphorylases and hydrolases catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon from the fixed purine to phosphate and water nucleophiles, respectively. As the lysis reaction progresses along the reaction coordinate, the distance between the purine and carbocation increases and the distance between carbocation and nucleophile decreases. Immucillin-H and DADMe-immucillin-H have been shown previously to be potent inhibitors of purine nucleoside phosphorylases and lie more toward the reactant and products side of this reaction coordinate, respectively. Both these enzyme inhibitors, which are currently in human clinical trials for different indications, are chiral and expensive to manufacture. We now report the synthesis of azetidine analogues of the DADMe-immucillins, which, despite their lack of stereochemical complexity, remain potent inhibitors (equilibrium dissociation constants as low as 229 pM) of purine nucleoside phosphorylase (PNP), methylthioadenosine phosphorylase (MTAP), and methylthioadenosine nucleosidase (MTAN), with potential utility as drug candidates.

DFT and Docking Study of Potential Transition State Analogue Inhibitors of Glycosyltransferases

Conformational behavior of the [(2S,3R,4R,5S)-3,4,5-trihydroxy-2-(phenylsulfanyl)tetrahydrofuran-2-yl]methyl sulfate anion (2), which is the potential transition state (TS) analogue of the inverting glycosyltransferases, was studied by means of two-dimensional potential-energy maps, using a density functional theory method at the B3LYP/6-31+G* level. The maps revealed the presence of eight low-energy domains which were refined at the B3LYP/6-311++G** level and led to six conformers in vacuum. In aqueous solution, two conformers dominate at equilibrium. The preferred conformers superimpose well with the transition state structure, as determined previously for glycosyltransferase GnT-I. The conformations of 2 in the active site of glycosyltransferase GnT-I were obtained by docking methods. It was found that one of the two best docking poses mimics the binding mode of TS. These results suggest that the proposed TS mimics 2 have the potential to be used as a scaffold for the design of TS analogue inhibitors.

Aβ46 Is Processed to Aβ40 and Aβ43, but Not to Aβ42, in the Low Density Membrane Domains

Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites referred to as gamma-, epsilon-, and zeta-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide gamma-secretase inhibitor, causes differential accumulation of longer amyloid beta-proteins (Abetas) within Chinese hamster ovary cells co-expressing beta C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Abeta46 localized exclusively to low density membrane (LDM) domains. Incubating the Abeta46-accumulating LDM domains at 37 degrees C produced Abeta40, Abeta42, Abeta43, and beta-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented beta-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Abeta46 and the concomitant appearance of Abeta40 and Abeta43 but not Abeta42. Further addition of DAPT suppressed the production of Abeta40/43 and abolished the decrease in the amount of Abeta46. These data indicate that preaccumulated Abeta46 is processed by gamma-secretase to Abeta40/43 but not to Abeta42 in the LDM domains. The amount of newly produced Abeta40 and Abeta43 was roughly equivalent to the decrease in the amount of Abeta46. Temporal profiles did not show a maximal concentration for Abeta43, suggesting that Abeta46 is processed to Abeta40 and Abeta43 through a nonsuccessive process.