Lisa M. Kopcho

P-Glycoprotein Efflux and Other Factors Limit Brain Amyloid β Reduction by β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitors in Mice

Alzheimers disease (AD) is a progressive neurodegenerative disease. Amyloid β (Aβ) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Aβ. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Aβ formation in cultured cells with IC50 values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Aβ peptides by mass spectrometry showed that these inhibitors decreased Aβ by inhibiting BACE1. An assay for Aβ1–40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Aβ1–40, but not brain Aβ1–40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Aβ1–40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Aβ1–40 and brain Aβ1–40 dose responses for these three compounds revealed differences in relative ED50 values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Potency, selectivity and prolonged binding of saxagliptin to DPP4: maintenance of DPP4 inhibition by saxagliptin in vitro and ex vivo when compared to a rapidly-dissociating DPP4 inhibitor.

BackgroundDipeptidylpeptidase 4 (DPP4) inhibitors have clinical benefit in patients with type 2 diabetes mellitus by increasing levels of glucose-lowering incretin hormones, such as glucagon-like peptide -1 (GLP-1), a peptide with a short half life that is secreted for approximately 1 hour following a meal. Since drugs with prolonged binding to their target have been shown to maximize pharmacodynamic effects while minimizing drug levels, we developed a time-dependent inhibitor that has a half-life for dissociation from DPP4 close to the duration of the first phase of GLP-1 release.ResultsSaxagliptin and its active metabolite (5-hydroxysaxagliptin) are potent inhibitors of human DPP4 with prolonged dissociation from its active site (Ki = 1.3 nM and 2.6 nM, t1/2 = 50 and 23 minutes respectively at 37°C). In comparison, both vildagliptin (3.5 minutes) and sitagliptin ( < 2 minutes) rapidly dissociated from DPP4 at 37°C. Saxagliptin and 5-hydroxysaxagliptin are selective for inhibition of DPP4 versus other DPP family members and a large panel of other proteases, and have similar potency and efficacy across multiple species.Inhibition of plasma DPP activity is used as a biomarker in animal models and clinical trials. However, most DPP4 inhibitors are competitive with substrate and rapidly dissociate from DPP4; therefore, the type of substrate, volume of addition and final concentration of substrate in these assays can change measured inhibition. We show that unlike a rapidly dissociating DPP4 inhibitor, inhibition of plasma DPP activity by saxagliptin and 5-hydroxysaxagliptin in an ex vivo assay was not dependent on substrate concentration when substrate was added rapidly because saxagliptin and 5-hydroxysaxagliptin dissociate slowly from DPP4, once bound. We also show that substrate concentration was important for rapidly dissociating DPP4 inhibitors.ConclusionsSaxagliptin and its active metabolite are potent, selective inhibitors of DPP4, with prolonged dissociation from its active site. They also demonstrate prolonged inhibition of plasma DPP4 ex vivo in animal models, which implies that saxagliptin and 5-hydroxysaxagliptin would continue to inhibit DPP4 during rapid increases in substrates in vivo.

Synthesis and SAR of indole-and 7-azaindole-1,3-dicarboxamide hydroxyethylamine inhibitors of BACE-1.

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aβ levels, but did not lower rat brain Aβ due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.

Comparative studies of active site–ligand interactions among various recombinant constructs of human β-amyloid precursor protein cleaving enzyme

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimers disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.

Novel Inhibition of Porcine Pepsin by a Substituted Piperidine PREFERENCE FOR ONE OF THE ENZYME CONFORMERS

Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site. Simulation of the stopped-flow fluorescence transients provided estimates of the K d values of 1.4 ± 0.2 μm and 39 ± 2 nm for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H. (1964) Arch. Biochem. Biophys. 106, 234–251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data set to the appropriate equation yielded an α factor of 8 ± 1. The magnitude of this factor (∞ > α > 1) can be explained by a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values. Inhibition by the piperidine was maximal when a group with pK 4.8 ± 0.2 was deprotonated and another group with pK 5.9 ± 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the substituted piperidine.

[3H]BMS-599240--a novel tritiated ligand for the characterization of BACE1 inhibitors.

In this report we describe a novel radioligand, [(3)H](S)-2-((S)-3-Acetylamino-3-sec-butyl-2-oxo-pyrrolidin-1-yl)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-methoxy-benzylamino)-propyl]-4-phenyl-butyramide ([(3)H]BMS-599240), that exhibits robust specific binding in homogenates from cell cultures overexpressing beta-site amyloid precursor protein cleaving enzyme-1 (BACE1). Radioligand binding exhibited high affinity, K(d)=2 nM, commensurate with its inhibitory potency against BACE1. Inhibition of radioligand binding in the presence of a range of different BACE1 inhibitors exhibited the same rank order of potency as for inhibition of BACE1 enzymatic activity. BACE1-dependent binding of the radioligand was also demonstrated in mouse brain homogenates, where genetic ablation of BACE1 eliminated high affinity binding. Thus, the radioligand [(3)H]BMS-599240 is a novel tool potentially useful for evaluation of BACE1 enzyme in biological samples, and for evaluation of inhibitor binding to BACE1.

Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.

To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes.

Crystal structures of apo and inhibitor-bound TGFβR2 kinase domain: insights into TGFβR isoform selectivity

The cytokine TGF-β modulates a number of cellular activities and plays a critical role in development, hemostasis and physiology, as well as in diseases including cancer and fibrosis. TGF-β signals through two transmembrane serine/threonine kinase receptors: TGFβR1 and TGFβR2. Multiple structures of the TGFβR1 kinase domain are known, but the structure of TGFβR2 remains unreported. Wild-type TGFβR2 kinase domain was refractory to crystallization, leading to the design of two mutated constructs: firstly, a TGFβR1 chimeric protein with seven ATP-site residues mutated to their counterparts in TGFβR2, and secondly, a reduction of surface entropy through mutation of six charged residues on the surface of the TGFβR2 kinase domain to alanines. These yielded apo and inhibitor-bound crystals that diffracted to high resolution (<2 Å). Comparison of these structures with those of TGFβR1 reveal shared ligand contacts as well as differences in the ATP-binding sites, suggesting strategies for the design of pan and selective TGFβR inhibitors.

Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders.

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.

P2-346: PGP efflux and other factors limit brain Aβ reduction by BACE1 inhibitors in mice

one protein located on the endoplasmic reticulum (ER) membrane, precisely at focal contacts between the ER and mitochondria (Hayashi & Su, Cell 131:596, 2007). The protein regulates the activity of different ER proteins, like IP3 receptors or ER stress sensors (GRP78/BiP, PERK, ATF-6). The 1 chaperone has the unique particularity to be sensitive to synthetic ligands, which therefore allow a very focal regulation of intracellular calcium homeostasis at ER and/or mitochondria contacts. As a consequence, 1 activators/agonists have been shown to induce acute modulation of transduction pathways, effective at the behavioral level. 1 Activators are indeed anti-amnesic, antidepressant and neuroprotective compounds. We validate new 1 activator compounds as neuroprotective agents against amyloid toxicity and analyze their mechanism of action. Methods: PRE-084 is a morpholine piperidine derivative acting as a high affinity and selective 1 activator. ANAVEX1-41 is a tetrahydro-furanmethanamine that shows high affinity for M1, M2, M4 muscarinic acetylcholine receptors and 1 protein. Both compounds are potent anti-amnesic drugs alleviating learning impairments observed in mice after the central (i.c.v.) injection of amyloid 25-35 peptide (A 25-35). Results: Central administration of A 25-35 induces within one week histological and biochemical changes, memory deficits, oxidative stress and ER stress in sensitive brain structures (hippocampus, cortex), highly reminiscent of the amyloid toxicity observed in Alzheimer’s disease. A 25-35 also provokes the induction of intracellular pro-apoptotic caspases and Bax-related proteins, markers of the induction of apoptosis. At the morphological level, A 25-35 induces a marked glial (astroglia, microglia) reaction and cell loss quantifiable in pyramidal layers of the hippocampus. Pre-administration of PRE-084 or ANAVEX1-41 prevents significantly all these pathological changes, showing that 1 activators are effective neuroprotectants. Part of the mechanism involves regulation of the expression of activity of IP3 receptors or ER stress sensors, in relation with the massive calcium overload induced by A 25-35. Conclusions: 1 Activators/agonists show potent neuroprotective and putatively disease-modifying activity against amyloid toxicity. Moreover, ANAVEX1-41 is active at 30-100 g/kg i.p., suggesting a cooperative action between muscarinic and 1 targets.