Author Correction: Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells.

With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism.

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate Kinetics.

A growing body of pathological, biomarker, genetic, and mechanistic data suggests that amyloid accumulation, as a result of changes in production, processing, and/or clearance of brain amyloid-ß peptide (Aß) concentrations, plays a key role in the pathogenesis of Alzheimer's disease (AD). Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid-ß protein precursor (AßPP) to Aß peptides, with the soluble N terminal fragment of AßPP (sAßPPß) as a direct product, and BACE1 inhibition is an attractive target for therapeutic intervention to reduce the production of Aß. Here, we report the in vitro and in vivo pharmacological profile of AZD3293, a potent, highly permeable, orally active, blood-brain barrier (BBB) penetrating, BACE1 inhibitor with unique slow off-rate kinetics. The in vitro potency of AZD3293 was demonstrated in several cellular models, including primary cortical neurons. In vivo in mice, guinea pigs, and dogs, AZD3293 displayed significant dose- and time-dependent reductions in plasma, cerebrospinal fluid, and brain concentrations of Aß40, Aß42, and sAßPPß. The in vitro potency of AZD3293 in mouse and guinea pig primary cortical neuronal cells was correlated to the in vivo potency expressed as free AZD3293 concentrations in mouse and guinea pig brains. In mice and dogs, the slow off-rate from BACE1 may have translated into a prolongation of the observed effect beyond the turnover rate of Aß. The preclinical data strongly support the clinical development of AZD3293, and patients with AD are currently being recruited into a combined Phase 2/3 study to test the disease-modifying properties of AZD3293.

Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.

The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Population PKPD modeling of BACE1 inhibitor-induced reduction in Aß levels in vivo and correlation to in vitro potency in primary cortical neurons from mouse and guinea pig.

PURPOSE: The aims were to quantify the in vivo time-course between the oral dose, the plasma and brain exposure and the inhibitory effect on Amyloid ß (Aß) in brain and cerebrospinal fluid, and to establish the correlation between in vitro and in vivo potency of novel ß-secretase (BACE1) inhibitors. METHODS: BACE1-mediated inhibition of Aß was quantified in in vivo dose- and/or time-response studies and in vitro in SH-SY5Y cells, N2A cells, and primary cortical neurons (PCN). An indirect response model with inhibition on Aß production rate was used to estimate unbound in vivo IC 50 in a population pharmacokinetic-pharmacodynamic modeling approach. RESULTS: Estimated in vivo inhibitory potencies varied between 1 and 1,000 nM. The turnover half-life of Aß40 in brain was predicted to be 0.5 h in mouse and 1 h in guinea pig. An excellent correlation between PCN and in vivo potency was observed. Moreover, a strong correlation in potency was found between human SH-SY5Y cells and mouse PCN, being 4.5-fold larger in SH-SY5Y cells. CONCLUSION: The strong in vivo-in vitro correlation increased the confidence in using human cell lines for screening and optimization of BACE1 inhibitors. This can optimize the design and reduce the number of preclinical in vivo effect studies.

AZ-4217: a high potency BACE inhibitor displaying acute central efficacy in different in vivo models and reduced amyloid deposition in Tg2576 mice.

Aß, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimer's disease (AD). ß-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to Aß peptides. Small molecule BACE1 inhibitors are expected to decrease Aß-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pM in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of Aß production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.

Low background and high contrast PET imaging of amyloid-ß with [11C]AZD2995 and [11C]AZD2184 in Alzheimer's disease patients.

PURPOSE: The aim of this study was to evaluate AZD2995 side by side with AZD2184 as novel PET radioligands for imaging of amyloid-ß in Alzheimer's disease (AD). METHODS: In vitro binding of tritium-labelled AZD2995 and AZD2184 was studied and compared with that of the established amyloid-ß PET radioligand PIB. Subsequently, a first-in-human in vivo PET study was performed using [(11)C]AZD2995 and [(11)C]AZD2184 in three healthy control subjects and seven AD patients. RESULTS: AZD2995, AZD2184 and PIB were found to share the same binding site to amyloid-ß. [(3)H]AZD2995 had the highest signal-to-background ratio in brain tissue from patients with AD as well as in transgenic mice. However, [(11)C]AZD2184 had superior imaging properties in PET, as shown by larger effect sizes comparing binding potential values in cortical regions of AD patients and healthy controls. Nevertheless, probably due to a lower amount of nonspecific binding, the group separation of the distribution volume ratio values of [(11)C]AZD2995 was greater in areas with lower amyloid-ß load, e.g. the hippocampus. CONCLUSION: Both AZD2995 and AZD2184 detect amyloid-ß with high affinity and specificity and also display a lower degree of nonspecific binding than that reported for PIB. Overall [(11)C]AZD2184 seems to be an amyloid-ß radioligand with higher uptake and better group separation when compared to [(11)C]AZD2995. However, the very low nonspecific binding of [(11)C]AZD2995 makes this radioligand potentially interesting as a tool to study minute levels of amyloid-ß. This sensitivity may be important in investigating, for example, early prodromal stages of AD or in the longitudinal study of a disease modifying therapy.

Core refinement toward permeable ß-secretase (BACE-1) inhibitors with low hERG activity.

By use of iterative design aided by predictive models for target affinity, brain permeability, and hERG activity, novel and diverse compounds based on cyclic amidine and guanidine cores were synthesized with the goal of finding BACE-1 inhibitors as a treatment for Alzheimer's disease. Since synthesis feasibility had low priority in the design of the cores, an extensive synthesis effort was needed to make the relevant compounds. Syntheses of these compounds are reported, together with physicochemical properties and structure-activity relationships based on in vitro data. Four crystal structures of diverse amidines binding in the active site are deposited and discussed. Inhibitors of BACE-1 with 3 µM to 32 nM potencies in cells are shown, together with data on in vivo brain exposure levels for four compounds. The results presented show the importance of the core structure for the profile of the final compounds.

Discovery of AZD3839, a potent and selective BACE1 inhibitor clinical candidate for the treatment of Alzheimer disease.

ß-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid ß peptide (Aß) species. Because cerebral deposition of Aß species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, Aß and sAPPß release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aß levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of Aß in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.

New aminoimidazoles as ß-secretase (BACE-1) inhibitors showing amyloid-ß (Aß) lowering in brain.

Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimer's disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aß40 and Aß42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aß was 40-50%, 1.5 h after oral dosing (100 µmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.

Design and synthesis of ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of ß-amyloid peptides.

The evaluation of a series of aminoisoindoles as ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer's disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aß40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 µM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of ß-amyloid peptides in mouse brain following oral dosing.

Synthesis and evaluation of pyridylbenzofuran, pyridylbenzothiazole and pyridylbenzoxazole derivatives as ¹8F-PET imaging agents for ß-amyloid plaques.

The synthesis and SAR of new ß-amyloid binding agents are reported. Evaluation of important properties for achieving good signal-to-background ratio is described. Compounds 27, 33, and 36 displayed desirable lipophilic and pharmacokinetic properties. Compound 27 was further evaluated with autoradiographic studies in vitro on human brain tissue and in vivo in Tg2576 mice. Compound 27 showed an increased signal-to-background ratio compared to flutemetamol 4, indicating its suitability as PET ligand for ß-amyloid deposits in AD patients. The preparation of the corresponding (18)F-labeled PET radioligand of compound 27 is presented.

BACE1 inhibition induces a specific cerebrospinal fluid ß-amyloid pattern that identifies drug effects in the central nervous system.

BACE1 is a key enzyme for amyloid-ß (Aß) production, and an attractive therapeutic target in Alzheimer's disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aß metabolism, inducing a unique pattern of secreted Aß peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aß1-40 and Aß1-42, treatment also changed the relative levels of several other Aß isoforms. In particular Aß1-34 decreased, while Aß5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aß1-40 and Aß1-42. The effects on Aß5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aß pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.

Characterization of AZD4694, a novel fluorinated Abeta plaque neuroimaging PET radioligand.

Positron emission tomography (PET) radioligands that bind selectively to beta-amyloid plaques (Abeta) are promising imaging tools aimed at supporting the diagnosis of Alzheimer's disease and the evaluation of new drugs aiming to modify amyloid plaque load. For extended clinical use, there is a particular need for PET tracers labeled with fluorine-18, a radionuclide with 110 min half-life allowing for central synthesis followed by wide distribution. The development of fluorinated radioligands is, however, challenging because of the lipophilic nature of aromatic fluorine, rendering fluorinated ligands more prone to have high non-specific white matter binding. We have here developed the new benzofuran-derived radioligand containing fluorine, AZD4694 that shows high affinity for beta-amyloid fibrils in vitro (K(d) = 2.3 +/- 0.3 nM). In cortical sections from human Alzheimer's disease brain [(3)H]AZD4694 selectively labeled beta-amyloid deposits in gray matter, whereas there was a lower level of non-displaceable binding in plaque devoid white matter. Administration of unlabeled AZD4694 to rat showed that it has a pharmacokinetic profile consistent with good PET radioligands, i.e., it quickly entered and rapidly cleared from normal rat brain tissue. Ex vivo binding data in aged Tg2576 mice after intravenous administration of [(3)H]AZD4694 showed selective binding to beta-amyloid deposits in a reversible manner. In Tg2576 mice, plaque bound [(3)H]AZD4694 could still be detected 80 min after i.v. administration. Taken together, the preclinical profile of AZD4694 suggests that fluorine-18 labeled AZD4694 may have potential for PET-visualization of cerebral beta-amyloid deposits in the living human brain.

Synthesis and evaluation of 2-pyridylbenzothiazole, 2-pyridylbenzoxazole and 2-pyridylbenzofuran derivatives as 11C-PET imaging agents for beta-amyloid plaques.

The syntheses and SAR of new series of beta-amyloid binding agents are reported. The effort to optimize signal-to-background ratios for these ligands are described. Compounds 8, 21 and 30 displayed desirable lipophilicity and pharmacokinetic properties. Compounds 8 and 21 were evaluated with in vitro autoradiographic studies and in vivo in APP/PS1 transgenic mice. It is shown that it was possible to increase the signal-to-background ratios compared to PIB 1, as demonstrated by compounds 8 and 21.

AZD2184: a radioligand for sensitive detection of beta-amyloid deposits.

The presence of beta-amyloid plaques in brain is a hallmark of Alzheimer's disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Positron emission tomography (PET) radioligands such as Pittsburgh compound B ([(11)C]-2-(3-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol) (PIB) binds selectively to beta-amyloid and are promising new tools supporting the clinical diagnoses of AD. In addition, such methodology may be useful for evaluation of new drugs aiming at reduction of amyloid plaque load. The objective of this study is to develop a new amyloid selective PET radioligand with higher signal-to-background ratio when compared with existing amyloid PET ligands. The lead compound, AZD2184, (2-[6-(methylamino)pyridin-3-yl]-1,3-benzothiazol-6-ol) was found to have high affinity for amyloid fibrils in vitro (K(d): 8.4 +/- 1.0 nM). Two minutes after i.v. administration in rats, about 1% of the dose was in brain. In vitro autoradiography on cortical brain sections from amyloid-beta precursor protein/presenilin 1 (APP/PS1) mice and AD patients showed that while [(3)H]AZD2184 and [(3)H]PIB are mutually displaceable, [(3)H]AZD2184 displays a higher signal-to-background ratio primarily by virtue of lower background binding levels. The ratio of binding ability in prefrontal cortex (high plaque load) to subcortical white matter (background) was 4.5 for [(3)H]AZD2184 and 0.8 for [(3)H]PIB at 1 nM. In adjacent cortical sections from APP/PS1 mouse as well as from AD cortical tissue, [(3)H]AZD2184 and antibodies to human beta-amyloid labeled identical structures. In vivo administration of [(3)H]AZD2184 to APP/PS1 mice further showed that [(3)H]AZD2184 labels amyloid deposits with low non-specific background binding. Taken together, the pre-clinical profile of AZD2184 in relation to the reference ligand PIB, suggests that (11)C-labeled AZD2184 is a potential radioligand for PET-visualization of beta-amyloid deposits in the living human brain.