H
O
-oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalytic cysteine residues (C
(SH) undergo rapid S-glutathionylation. Restoration of the enzyme activity is accomplished by ...thiol/disulfide S
2 displacement (directly or enzymatically) forming glutathione disulfide (G(SS)G) and active enzyme, a process that should be facile as C
(SH) reside on the subunit surface. As S-glutathionylated GAPDH accumulates following ischemic and/or oxidative stress, in vitro/silico approaches have been employed to address this paradox. C
(SH) residues were selectively oxidized and S-glutathionylated. Kinetics of GAPDH dehydrogenase recovery demonstrated that glutathione is an ineffective reactivator of S-glutathionylated GAPDH compared to dithiothreitol. Molecular dynamic simulations (MDS) demonstrated strong binding interactions between local residues and S-glutathione. A second glutathione was accommodated for thiol/disulfide exchange forming a tightly bound glutathione disulfide G(SS)G. The proximal sulfur centers of G(SS)G and C
(SH) remained within covalent bonding distance for thiol/disulfide exchange resonance. Both these factors predict inhibition of dissociation of G(SS)G, which was verified by biochemical analysis. MDS also revealed that both S-glutathionylation and bound G(SS)G significantly perturbed subunit secondary structure particularly within the S-loop, region which interacts with other cellular proteins and mediates NAD(P)
binding specificity. Our data provides a molecular rationale for how oxidative stress elevates S-glutathionylated GAPDH in neurodegenerative diseases and implicates novel targets for therapeutic intervention.
BACE1 is a key protease controlling the formation of amyloid β, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimer's disease (AD). Therefore, the development of potent ...and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academia and industry. Herein, we report the nonclinical and early clinical development of LY2886721, a BACE1 active site inhibitor that reached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translates in vitro activity into robust in vivo amyloid β lowering in nonclinical animal models. Similar potent and persistent amyloid β lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.
According to the amyloid cascade hypothesis, cerebral deposition of amyloid-β peptide (Aβ) is critical for Alzheimer's disease (AD) pathogenesis. Aβ generation is initiated when β-secretase (BACE1) ...cleaves the amyloid precursor protein. For more than a decade, BACE1 has been a prime target for designing drugs to prevent or treat AD. However, development of such agents has turned out to be extremely challenging, with major hurdles in cell penetration, oral bioavailability/metabolic clearance, and brain access. Using a fragment-based chemistry strategy, we have generated LY2811376 (S)-4-(2,4-difluoro-5-pyrimidin-5-yl-phenyl)-4-methyl-5,6-dihydro-4H-1,3thiazin-2-ylamine, the first orally available non-peptidic BACE1 inhibitor that produces profound Aβ-lowering effects in animals. The biomarker changes obtained in preclinical animal models translate into man at doses of LY2811376 that were safe and well tolerated in healthy volunteers. Prominent and long-lasting Aβ reductions in lumbar CSF were measured after oral dosing of 30 or 90 mg of LY2811376. This represents the first translation of BACE1-driven biomarker changes in CNS from preclinical animal models to man. Because of toxicology findings identified in longer-term preclinical studies, this compound is no longer progressing in clinical development. However, BACE1 remains a viable target because the adverse effects reported here were recapitulated in LY2811376-treated BACE1 KO mice and thus are unrelated to BACE1 inhibition. The magnitude and duration of central Aβ reduction obtainable with BACE1 inhibition positions this protease as a tractable small-molecule target through which to test the amyloid hypothesis in man.
The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer’s disease drug target owing to its critical role in the production of amyloid-beta. We have previously ...reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity. LY2886721 advanced to Phase II, but development was halted due to abnormally elevated liver enzymes. Herein, we report the discovery and clinical development of LY3202626, a highly potent, CNS-penetrant, and low-dose BACE inhibitor, which successfully addressed these key development challenges.
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Inhibition of BACE1 has become an important strategy in the quest for disease modifying agents to slow the progression of Alzheimer’s disease. We previously reported the ...fragment-based discovery of LY2811376, the first BACE1 inhibitor reported to demonstrate robust reduction of human CSF Aβ in a Phase I clinical trial. We also reported on the discovery of LY2886721, a potent BACE1 inhibitor that reached phase 2 clinical trials. Herein we describe the preparation and structure activity relationships (SAR) of a series of BACE1 inhibitors utilizing trans-cyclopropyl moieties as conformational constraints. The design, details of the stereochemically complex organic synthesis, and biological activity of these BACE1 inhibitors is described.
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The BACE1 enzyme is a key target for Alzheimer’s disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against ...BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6–8 is described.
The development of beta-site amyloid-beta precursor protein cleaving enzyme (BACE) 1 inhibitors for the treatment of Alzheimer's disease requires optimization of inhibitor potency, selectivity, and ...brain penetration. Moreover, there is a need for low-dose compounds since liver toxicity was found with some BACE inhibitors.
To determine whether the high
potency and robust pharmacodynamic effect of the BACE inhibitor LY3202626 observed in nonclinical species translated to humans.
The effect of LY3202626 versus vehicle on amyloid-β (Aβ) levels was evaluated in a series of
assays, as well as in
and multi-part clinical pharmacology studies. Aβ levels were measured using analytical biochemistry assays in brain, plasma, and cerebrospinal fluid (CSF) of mice, dogs and humans. Nonclinical data were analyzed using an ANOVA followed by Tukey's
test and clinical data used summary statistics.
LY3202626 exhibited significant human BACE1 inhibition, with an IC
of 0.615±0.101 nM in a fluorescence resonance energy transfer assay and an EC
of 0.275±0.176 nM for lowering Aβ
and 0.228±0.244 nM for Aβ
in PDAPP neuronal cultures. In dogs, CSF Aβ
concentrations were significantly reduced by ∼80% at 9 hours following a 1.5 mg/kg dose. In humans, CSF Aβ
was reduced by 73.1±7.96 % following administration of 6 mg QD. LY3202626 was found to freely cross the blood-brain barrier in dogs and humans.
LY3202626 is a potent BACE1 inhibitor with high blood-brain barrier permeability. The favorable safety and pharmacokinetic/pharmacodynamic profile of LY3202626 supports further clinical development.
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