Christopher W. am Ende

γ-Secretase Modulator (GSM) Photoaffinity Probes Reveal Distinct Allosteric Binding Sites on Presenilin

Background: Potent GSMs have been identified that lower Aβ42; however, the mechanism of modulation is not well understood. Results: The photoaffinity probe E2012-BPyne specifically labels PS1-NTF at a unique site. Conclusion: Acid and imidazole GSMs bind to distinct sites on PS1-NTF and are differentially affected by L458. Significance: Our results provide evidence for multiple binding sites within γ-secretase that confer specific modulatory effects. γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

Bioorthogonal reactions, including the strain-promoted azide–alkyne cycloaddition (SPAAC) and inverse electron demand Diels–Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

Novel γ-secretase modulators: a review of patents from 2008 to 2010

Introduction: The amyloid precursor protein is first cleaved by β-secretase to generate a 99-residue membrane-bound CTF (C99 or β-CTF), which is subsequently cleaved by γ-secretase to generate amyloid β (Aβ) peptides and the APP intracellular domain. The amyloidogenic Aβ42 has attracted considerable attention because it is thought to be the most pathogenic species associated with Alzheimers disease progression. New classes of compounds, called γ-secretase modulators (GSMs), have been shown to selectively lower Aβ42 production without shutting down key γ-secretase-dependent signaling pathways. This has become an important therapeutic strategy aimed at modulating Aβ production. Areas covered: The progress on the clinical development of γ-secretase inhibitors is briefly covered in this review, followed by a discussion of the potential differentiating attributes of GSMs. Then, the patent literature covering novel GSMs is reviewed, focusing on patents from 2008 to 2010. Expert opinion: Much progress has been made in the past 2 years on developing GSMs with improved potency for lowering the production of Aβ42. However, many of these chemotypes are in a challenging chemical space and generally possess higher lipophilicity than most CNS drugs. It will be important to gain a better understanding of the specific target(s) that these GSMs interact with in order to facilitate future drug design efforts.

Design and synthesis of dihydrobenzofuran amides as orally bioavailable, centrally active γ-secretase modulators.

We report the discovery and optimization of a novel series of dihydrobenzofuran amides as γ-secretase modulators (GSMs). Strategies for aligning in vitro potency with drug-like physicochemical properties and good microsomal stability while avoiding P-gp mediated efflux are discussed. Lead compounds such as 35 and 43 have moderate to good in vitro potency and excellent selectivity against Notch. Good oral bioavailability was achieved as well as robust brain Aβ42 lowering activity at 100 mg/kg po dose.

Development of clickable active site-directed photoaffinity probes for γ-secretase

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.

Design, synthesis, and pharmacological evaluation of a novel series of pyridopyrazine-1,6-dione γ-secretase modulators.

Herein we describe the design and synthesis of a novel series of γ-secretase modulators (GSMs) that incorporates a pyridopiperazine-1,6-dione ring system. To align improved potency with favorable ADME and in vitro safety, we applied prospective physicochemical property-driven design coupled with parallel medicinal chemistry techniques to arrive at a novel series containing a conformationally restricted core. Lead compound 51 exhibited good in vitro potency and ADME, which translated into a favorable in vivo pharmacokinetic profile. Furthermore, robust reduction of brain Aβ42 was observed in guinea pig at 30 mg/kg dosed orally. Through chemical biology efforts involving the design and synthesis of a clickable photoreactive probe, we demonstrated specific labeling of the presenilin N-terminal fragment (PS1-NTF) within the γ-secretase complex, thus gaining insight into the binding site of this series of GSMs.

Synthesis of pyridopyrazine-1,6-diones from 6-hydroxypicolinic acids via a one-pot coupling/cyclization reaction.

A facile one-pot synthesis of 3,4-dihydro-1H-pyrido[1,2-a]pyrazine-1,6(2H)-diones (pyridopyrazine-1,6-diones) has been developed which employs a sequential coupling/cyclization reaction of 6-hydroxypicolinic acids and β-hydroxylamines. The transformation proceeds in good yield under mild conditions using O-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) to both carry out the amide formation and activate the hydroxyl group for intramolecular alkylation.

Discovery of indole-derived pyridopyrazine-1,6-dione γ-secretase modulators that target presenilin.

Herein we describe design strategies that led to the discovery of novel pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) incorporating an indole motif as a heterocyclic replacement for a naphthyl moiety that was present in the original lead 9. Tactics involving parallel medicinal chemistry and in situ monomer synthesis to prepare focused libraries are discussed. Optimized indole GSM 29 exhibited good alignment of in vitro potency and physicochemical properties, and moderate reduction of brain Aβ42 was achieved in a rat efficacy model when dosed orally at 30mg/kg. Labeling experiments using a clickable, indole-derived GSM photoaffinity probe demonstrated that this series binds to the presenilin N-terminal fragment (PS1-NTF) of the γ-secretase complex.

Small-molecule phosphodiesterase probes: discovery of potent and selective CNS-penetrable quinazoline inhibitors of PDE1

PDE1 is a family of calcium-activated, dual substrate phosphodiesterases expressed in both the CNS and periphery that play a role in the integration of intracellular calcium and cyclic nucleotide signaling cascades. Exploration of the potential in targeting this family of enzymes to treat neuropsychiatric disorders has been hampered by a lack of potent, selective, and brain penetrable PDE1 inhibitors. To identify such compounds we used high-throughput screening, structure-based design, and targeted synthetic chemistry to discover the 4-aminoquinazoline 7a (PF-04471141) and the 4-indanylquinazoline 27 (PF-04822163) each of which are PDE1 inhibitors that readily cross the blood brain barrier. These quinazoline-based PDE1-selective inhibitors represent valuable new tools to study the biological processes regulated by PDE1 and to begin to determine the potential therapeutic utility of such compounds to treat neuropsychiatric disorders.

Design of Pyridopyrazine-1,6-dione γ-Secretase Modulators that Align Potency, MDR Efflux Ratio, and Metabolic Stability

Herein we describe the design and synthesis of a series of pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) for Alzheimers disease (AD) that achieve good alignment of potency, metabolic stability, and low MDR efflux ratios, while also maintaining favorable physicochemical properties. Specifically, incorporation of fluorine enabled design of metabolically less liable lipophilic alkyl substituents to increase potency without compromising the sp(3)-character. The lead compound 21 (PF-06442609) displayed a favorable rodent pharmacokinetic profile, and robust reductions of brain Aβ42 and Aβ40 were observed in a guinea pig time-course experiment.