Distinguishing Specific and Nonspecific Complexes of Alkyladenine DNA Glycosylase.

Human alkyladenine DNA glycosylase (AAG) recognizes many alkylated and deaminated purine lesions and excises them to initiate the base excision DNA repair pathway. AAG employs facilitated diffusion to rapidly scan nonspecific sites and locate rare sites of damage. Nonspecific DNA binding interactions are critical to the efficiency of this search for damage, but little is known about the binding footprint or the affinity of AAG for nonspecific sites. We used biochemical and biophysical approaches to characterize the binding of AAG to both undamaged and damaged DNA. Although fluorescence anisotropy is routinely used to study DNA binding, we found unexpected complexities in the data for binding of AAG to DNA. Systematic comparison of different fluorescent labels and different lengths of DNA allowed binding models to be distinguished and demonstrated that AAG can bind with high affinity and high density to nonspecific DNA. Fluorescein-labeled DNA gave the most complex behavior but also showed the greatest potential to distinguish specific and nonspecific binding modes. We suggest a unified model that is expected to apply to many DNA binding proteins that exhibit affinity for nonspecific DNA. Although AAG strongly prefers to excise lesions from duplex DNA, nonspecific binding is comparable for single- and double-stranded nonspecific sites. The electrostatically driven binding of AAG to small DNA sites (∼5 nucleotides of single-stranded and ∼6 base pairs of duplex) facilitates the search for DNA damage in chromosomal DNA, which is bound by nucleosomes and other proteins.

A sensitive aß oligomer assay discriminates Alzheimer's and aged control cerebrospinal fluid.

A hallmark of Alzheimer's disease (AD) brain is the amyloid ß (Aß) plaque, which is comprised of Aß peptides. Multiple lines of evidence suggest that Aß oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aß oligomers over Aß monomers with a limit of detection ∼ 0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aß multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aß oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aß oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aß oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

Discovery of pyrrolidine-based ß-secretase inhibitors: lead advancement through conformational design for maintenance of ligand binding efficiency.

We have developed a novel series of pyrrolidine derived BACE-1 inhibitors. The potency of the weak initial lead structure was enhanced using library-based SAR methods. The series was then further advanced by rational design while maintaining a minimal ligand binding efficiency threshold. Ultimately, the co-crystal structure was obtained revealing that these inhibitors interacted with the enzyme in a unique fashion. In all, the potency of the series was enhanced by 4 orders of magnitude from the HTS lead with concomitant increases in physical properties needed for series advancement. The progression of these developments in a systematic fashion is described.

High throughput monitoring of amyloid-ß(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-ß (Aß)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aß(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aß(42). Modeling suggests that oligomerized Aß(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aß(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aß(42) oligomerization. Using ELISA and AlphaLISA, we found that Aß(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aß(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aß(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aß(42) oligomerization.

Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.

Inhibition of calcineurin-mediated endocytosis and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors prevents amyloid beta oligomer-induced synaptic disruption.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (Abeta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Abeta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.

Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase initiates the repair of a wide variety of alkylated and deaminated purine lesions in DNA. In this study, we take advantage of the natural fluorescence of the 1,N(6)-ethenoadenosine (epsilonA) lesion and report a kinetic analysis of binding, nucleotide flipping, base excision, and product release. The transient changes in the fluorescence of epsilonA revealed the existence of two distinct complexes that are formed prior to the hydrolysis step. An initial recognition complex forms rapidly and is characterized by partial disruption of the stacking interactions of the lesioned base. Subsequently, a very stable extrahelical complex is formed in which the epsilonA lesion is strongly quenched by interactions in the AAG active site pocket. Our results indicate that DNA binding and base flipping take place on the millisecond to second time scale. N-Glycosidic bond cleavage is much slower, taking place on the minute time scale. A pulse-chase experiment was used to demonstrate that even for the tightly bound epsilonA substrate, the extrahelical complex is not fully committed to excision. Nevertheless, flipping of epsilonA is highly favorable, and we calculate that the equilibrium constant for flipping is approximately 1300. This kinetic mechanism has important biological implications. First, two-step binding provides multiple opportunities to discriminate between damaged and undamaged nucleotides. Second, a rapid equilibrium flipping mechanism maximizes specificity for damaged versus undamaged bases, since undamaged bases generally form stronger base pairs than damaged bases. Finally, the highly favorable equilibrium for flipping of epsilonA ensures that epsilonA removal is independent of sequence context and highly efficient despite the relatively slow rate of N-glycosidic bond hydrolysis.

Discovery of aminoheterocycles as a novel beta-secretase inhibitor class: pH dependence on binding activity part 1.

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK(a) of these inhibitors and the pH of the BACE-1 enzyme binding assay.

Discovery and X-ray crystallographic analysis of a spiropiperidine iminohydantoin inhibitor of beta-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.

Interaction of benzoate pyrimidine analogues with class 1A dihydroorotate dehydrogenase from Lactococcus lactis.

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate to orotate in the only redox reaction in pyrimidine biosynthesis. The pyrimidine binding sites are very similar in all structurally characterized DHODs, suggesting that the prospects for identifying a class-specific inhibitor directed against this site are poor. Nonetheless, two compounds that bind specifically to the Class 1A DHOD from Lactococcus lactis, 3,4-dihydroxybenzoate (3,4-diOHB) and 3,5-dihydroxybenzoate (3,5-diOHB), have been identified [Palfey et al. (2001) J. Med. Chem. 44, 2861-2864]. The mechanism of inhibitor binding to the Class 1A DHOD from L. lactis has now been studied in detail and is reported here. Titrations showed that 3,4-diOHB binds more tightly at higher pH, whereas the opposite is true for 3,5-diOHB. Isothermal titration calorimetry and absorbance spectroscopy showed that 3,4-diOHB ionizes to the phenolate upon binding to the enzyme, but 3,5-diOHB does not. The charge-transfer band that forms in the 3,4-diOHB complex allowed the kinetics of binding to be observed in stopped-flow experiments. Binding was slow enough to observe from pH 6 to pH 8 and was (minimally) a two-step process consisting of the rapid formation of a complex that isomerized to the final charge-transfer complex. Orotate and 3,5-diOHB bind too quickly to follow directly, but their dissociation kinetics were studied by competition and described adequately with a single step. Crystal structures of both inhibitor complexes were determined, showing that 3,5-diOHB binds in the same orientation as orotate. In contrast, 3,4-diOHB binds in a twisted orientation, enabling one of its phenolic oxygens to form a very strong hydrogen bond to an asparagine, thus stabilizing the phenolate and causing charge-transfer interactions with the pi-system of the flavin, resulting in a green color.

Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells.

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.

Compounds that bind APP and inhibit Abeta processing in vitro suggest a novel approach to Alzheimer disease therapeutics.

Extracellular deposits of aggregated amyloid-beta (Abeta) peptides are a hallmark of Alzheimer disease; thus, inhibition of Abeta production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Abeta requires processing of the amyloid precursor protein (APP) by both beta- and gamma-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for beta-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Abeta by BACE2 and selectively inhibit the production of Abeta(42) species by gamma-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Abeta domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Abeta production.

Design and synthesis of 8-hydroxy-[1,6]naphthyridines as novel inhibitors of HIV-1 integrase in vitro and in infected cells.

Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 microM. It does not exhibit cytotoxicity in cell culture at < or =12.5 microM and shows a good pharmacokinetic profile when dosed orally to rats. The antiviral activity of 7 and its effect on integration were confirmed using viruses with specific integrase mutations.

Inhibition of HIV-1 ribonuclease H by a novel diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid.

Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC(50) = 3.2 microm) but has no effect on DNA polymerization (IC(50) > 50 microm). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the alpha-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn(2+) implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H.

Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes.

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.