Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates.

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Improved synthesis of [18F] fallypride and characterization of a Huntington's disease mouse model, zQ175DN KI, using longitudinal PET imaging of D2/D3 receptors.

PURPOSE: Dopamine receptors are involved in pathophysiology of neuropsychiatric diseases, including Huntington's disease (HD). PET imaging of dopamine D2 receptors (D2R) in HD patients has demonstrated 40% decrease in D2R binding in striatum, and D2R could be a reliable quantitative target to monitor disease progression. A D2/3R antagonist, [18F] fallypride, is a high-affinity radioligand that has been clinically used to study receptor density and occupancy in neuropsychiatric disorders. Here we report an improved synthesis method for [18F]fallypride. In addition, high molar activity of the ligand has allowed us to apply PET imaging to characterize D2/D3 receptor density in striatum of the recently developed zQ175DN knock-in (KI) mouse model of HD. METHODS: We longitudinally characterized in vivo [18F] fallypride -PET imaging of D2/D3 receptor densities in striatum of 9 and 12 month old wild type (WT) and heterozygous (HET) zQ175DN KI mouse. Furthermore, we verified the D2/D3 receptor density in striatum with [3H] fallypride autoradiography at 12 months of age. RESULTS: We implemented an improved synthesis method for [18F] fallypride to yield high molar activity (MA, 298-360 GBq/µmol) and good reproducibility. In the HET zQ175DN KI mice, we observed a significant longitudinal decrease in binding potential (BPND) (30.2%, p < 0.001, 9 months of age and 51.6%, p < 0.001, 12 months of age) compared to WT littermates. No mass effect was observed when the MA of [18F] fallypride was > 100 GBq/µmol at the time of injection. Furthermore, the decrease of D2/D3 receptor density in striatum in HET zQ175DN KI was consistent using [3H] fallypride autoradiography. CONCLUSIONS: We observed a significant decrease in D2/D3R receptor densities in the striatum of HET zQ175DN KI mice compared to WT mice at 9 and 12 months of age. These results are in line with clinical findings in HD patients, suggesting [18F] fallypride PET imaging has potential as a quantitative translational approach to monitor disease progression in preclinical studies.

MR-based spatial normalization improves [18F]MNI-659 PET regional quantification and detectability of disease effect in the Q175 mouse model of Huntington's disease.

The positron emission tomography (PET) tracer [18F]MNI-659, selective for phosphodiesterase 10A (PDE10A), is a promising tool to assess an early biomarker for Huntington's disease (HD). In this study we investigated [18F]MNI-659 uptake in the Q175 mouse model of HD. Given the focal striatal distribution of PDE10A as well as the striatal atrophy occurring in HD, the spatial normalization approach applied during the processing could sensibly affect the accuracy of the regional quantification. We compared the use of a magnetic resonance images (MRI) template based on individual MRI over a PET and CT templates for regional quantification and spatial normalization of [18F]MNI-659 PET images. We performed [18F]MNI-659 PET imaging in six months old heterozygous (HET) Q175 mice and wild-type (WT) littermates, followed by X-ray computed tomography (CT) scan. In the same week, individual T2-weighted MRI were acquired. Spatial normalization and regional quantification of the PET/CT images was performed on MRI, [18F]MNI-659 PET, or CT template and compared to binding potential (BPND) using volumes manually delineated on the individual MR images. Striatal volume was significantly reduced in HET mice (-7.7%, p<0.0001) compared to WT littermates. [18F]MNI-659 BPND in striatum of HET animals was significantly reduced (p<0.0001) when compared to WT littermates using all three templates. However, BPND values were significantly higher for HET mice using the PET template compared to the MRI and CT ones (p<0.0001), with an overestimation at lower activities. On the other hand, the CT template spatial normalization introduced larger variability reducing the effect size. The PET and CT template-based approaches resulted in a lower accuracy in BPND quantification with consequent decrease in the detectability of disease effect. This study demonstrates that for [18F]MNI-659 brain PET imaging in mice the use of an MRI-based spatial normalization is recommended to achieve accurate quantification and fully exploit the detectability of disease effect.

Noninvasive Relative Quantification of [11C]ABP688 PET Imaging in Mice Versus an Input Function Measured Over an Arteriovenous Shunt.

Impairment of the metabotropic glutamate receptor 5 (mGluR5) has been implicated with various neurologic disorders. Although mGluR5 density can be quantified with the PET radiotracer [11C]ABP688, the methods for reproducible quantification of [11C]ABP688 PET imaging in mice have not been thoroughly investigated yet. Thus, this study aimed to assess and validate cerebellum as reference region for simplified reference tissue model (SRTM), investigate the feasibility of a noninvasive cardiac image-derived input function (IDIF) for relative quantification, to validate the use of a PET template instead of an MRI template for spatial normalization, and to determine the reproducibility and within-subject variability of [11C]ABP688 PET imaging in mice. Blocking with the mGluR5 antagonist MPEP resulted in a reduction of [11C]ABP688 binding of 41% in striatum (p < 0.0001), while no significant effect could be found in cerebellum (-4.8%, p > 0.99) indicating cerebellum as suitable reference region for mice. DVR-1 calculated using a noninvasive IDIF and an arteriovenous input function correlated significantly when considering the cerebellum as the reference region (striatum: DVR-1, r = 0.978, p < 0.0001). Additionally, strong correlations between binding potential calculated from SRTM (BPND) with DVR-1 based on IDIF (striatum: r = 0.980, p < 0.0001) and AV shunt (striatum: r = 0.987, p < 0.0001). BPND displayed higher discrimination power than VT values in determining differences between wild-types and heterozygous Q175 mice, an animal model of Huntington's disease. Furthermore, we showed high agreement between PET- and MRI-based spatial normalization approaches (striatum: r = 0.989, p < 0.0001). Finally, both spatial normalization approaches did not reveal any significant bias between test-retest scans, with a relative difference below 5%. This study indicates that noninvasive quantification of [11C]ABP688 PET imaging is reproducible and cerebellum can be used as reference region in mice.

Longitudinal Characterization of mGluR5 Using 11C-ABP688 PET Imaging in the Q175 Mouse Model of Huntington Disease.

Metabotropic glutamate receptor 5 (mGluR5) represents a potential therapeutic target for Huntington disease. Using 11C-ABP688 (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime), a noncompetitive and highly selective antagonist for mGluR5, we aimed to longitudinally characterize in vivo changes in mGluR5 by means of PET imaging in the Q175 mouse model of Huntington disease. Methods:11C-ABP688 PET imaging, followed by a CT scan, was performed on 18 heterozygous mice and 18 wild-type (WT) littermates at 3 different time points (6, 9, and 13 mo old). 11C-ABP688 nondisplaceable binding potential (BPND) was calculated for each time point in striatum and cortex using the cerebellum as the reference region. In addition, voxel-based statistical parametric mapping (SPM) analysis was performed on BPND images. Postmortem validation of mGluR5 level and neuronal density was performed on the mice at 6 mo old. Results: The 11C-ABP688 BPND of heterozygous animals was significantly reduced at all time points in the striatum (-13.1%, -13.5%, and -14.2% at 6, 9, and 13 mo, respectively; P < 0.001 for all) and in the cortex (-9.8%, -10.2%, and -10.6%, respectively; P < 0.01 for all), when compared with WT animals. Longitudinal changes in 11C-ABP688 BPND were also found in heterozygous mice, showing a reduction at 13 mo compared with 6 mo (-10.4%, P < 0.05). SPM analysis confirmed reduced BPND in heterozygous compared with WT mice, as well as a time-related decline in 11C-ABP688 binding in the striatum of heterozygous mice. Postmortem analysis confirmed a mGluR5 decrease in both striatum (-36.6%; P < 0.01) and cortex (-16.6%; P < 0.05) in heterozygous mice, whereas no difference in neuronal density was found. Conclusion: In vivo imaging of mGluR5 using 11C-ABP688 PET/CT revealed a marked reduction in ligand binding in the striatum and cortex of heterozygous mice, compared with WT mice, as well as a temporal decline. This study suggests that 11C-ABP688 PET imaging is a potential biomarker to monitor the progression of, and therapeutic strategies for, Huntington disease.

Quantifying autophagy using novel LC3B and p62 TR-FRET assays.

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

Longitudinal Small-Animal PET Imaging of the zQ175 Mouse Model of Huntington Disease Shows In Vivo Changes of Molecular Targets in the Striatum and Cerebral Cortex.

Since the discovery of the HTT gene in 1993, numerous animal models have been developed to study the progression of Huntington disease (HD) and to evaluate potential new therapeutics. In the present study, we used small-animal PET to characterize the expression of molecular targets in the recently reported HD animal model, the zQ175 mouse model. Methods: Male heterozygous zQ175 (Htttm1Mfc/190JChdi, CHDI-81003003) and wild-type (WT, C57BL/6J) animals were imaged with the dopamine D2 receptor radioligand 11C-raclopride, the PDE10A radioligand 18F-MNI-659, the dopamine D1 receptor radioligand 11C-NNC 112, and the 5-HT2A radioligand 11C-MDL 100907 at 6 and 9 mo of age. The outcome measure was the binding potential (BPND), using the cerebellum as the reference region. Selected regions of interest were the striatum for all radioligands and additionally the striatum, rostral cortex, caudal cortex, and hippocampus for 11C-NNC 112 and 11C-MDL 100907. Results: At 6 mo of age, the BPND in the striatum was lower in zQ175 than WT animals by 40% for 11C-raclopride, by 52% for 18F-MNI-659, by 28% for 11C-NNC, and by 11% for 11C-MDL 100907. In the rostral cortex, D1 receptor binding was 22% lower in zQ175 than WT animals. We found an overall reduction in D1 and 5-HT2A binding in the hippocampus of zQ175 compared with WT animals. The BPND of 11C-MDL 100907 in the caudal cortex was also lower in zQ175 WT animals. At 9 mo, there was a slight further reduction of D1, D2, and 5-HT2ABPND in the striatum, whereas PDE10A reached a plateau. Cortical markers were also slightly further decreased at 9 mo in zQ175 animals. Conclusion: Our study indicates a marked reduction of ligand binding to D1 and D2 and 5-HT2A receptors as well as loss of PDE10A enzyme in the striatum of zQ175 mice as compared with WT animals, in agreement with data obtained in clinical PET studies of patients with HD. The zQ175 mouse model recapitulates the expression pattern seen in humans with HD and may have value in further elucidating pathophysiologic events and therapeutic strategies.

Lead optimization toward proof-of-concept tools for Huntington's disease within a 4-(1H-pyrazol-4-yl)pyrimidine class of pan-JNK inhibitors.

Through medicinal chemistry lead optimization studies focused on calculated properties and guided by X-ray crystallography and computational modeling, potent pan-JNK inhibitors were identified that showed submicromolar activity in a cellular assay. Using in vitro ADME profiling data, 9t was identified as possessing favorable permeability and a low potential for efflux, but it was rapidly cleared in liver microsomal incubations. In a mouse pharmacokinetics study, compound 9t was brain-penetrant after oral dosing, but exposure was limited by high plasma clearance. Brain exposure at a level expected to support modulation of a pharmacodynamic marker in mouse was achieved when the compound was coadministered with the pan-cytochrome P450 inhibitor 1-aminobenzotriazole.

Development of a series of aryl pyrimidine kynurenine monooxygenase inhibitors as potential therapeutic agents for the treatment of Huntington's disease.

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

A monoclonal antibody TrkB receptor agonist as a potential therapeutic for Huntington's disease.

Huntington's disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

Discovery and structure-activity relationship of potent and selective covalent inhibitors of transglutaminase 2 for Huntington's disease.

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

SAR Development of Lysine-Based Irreversible Inhibitors of Transglutaminase 2 for Huntington's Disease.

We report a series of irreversible transglutaminase 2 inhibitors starting from a known lysine dipeptide bearing an acrylamide warhead. We established new SARs resulting in compounds demonstrating improved potency and better physical and calculated properties. Transglutaminase selectivity profiling and in vitro ADME properties of selected compounds are also reported.

Irreversible 4-Aminopiperidine Transglutaminase 2 Inhibitors for Huntington's Disease.

A new series of potent TG2 inhibitors are reported that employ a 4-aminopiperidine core bearing an acrylamide warhead. We establish the structure-activity relationship of this new series and report on the transglutaminase selectivity and in vitro ADME properties of selected compounds. We demonstrate that the compounds do not conjugate glutathione in an in vitro setting and have superior plasma stability over our previous series.

Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases.

The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp(2) character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.

A profiling platform for the characterization of transglutaminase 2 (TG2) inhibitors.

Huntington's disease (HD) is associated with increased expression levels and activity of tissue transglutaminase (TG2), an enzyme primarily known for its cross-linking of proteins. To validate TG2 as a therapeutic target for HD in transgenic models and for eventual clinical development, a selective and brain-permeable inhibitor is required. Here, a comprehensive profiling platform of biochemical and cellular assays is presented which has been established to evaluate the potency, cellular efficacy, subtype selectivity and the mechanism-of-action of known and novel TG2 inhibitors. Several classes of inhibitors have been characterized including: the commonly used pseudo-substrate inhibitors, cystamine and putrescine (which are generally nonspecific for TG2 and therefore not practical for drug development), the various peptidic inhibitors that target the active site cysteine residue (which display excellent selectivity but in general have poor cellular activity), and the allosteric reversible small-molecule hydrazides (which show poor selectivity and a lack of cellular activity and could not be improved despite considerable medicinal chemistry efforts). In addition, a set of inhibitors identified from a collection of pharmacologically active compounds was found to be unselective for TG2. Moreover, inhibition at the guanosine triphosphate binding site has been examined, but apart from guanine nucleotides, no such inhibitors have been identified. In addition, the promising pharmacological profile of a TG2 inhibitor is presented which is currently in lead optimization to be developed as a tool compound.

Novel bisaryl substituted thiazoles and oxazoles as highly potent and selective peroxisome proliferator-activated receptor delta agonists.

The discovery, synthesis, and optimization of compound 1 from a high-throughput screening hit to highly potent and selective peroxisome proliferator-activated receptor delta (PPARdelta) agonists are reported. The synthesis and structure-activity relationship in this series are described in detail. On the basis of a general schematic PPAR pharmacophore model, scaffold 1 was divided into headgroup, linker, and tailgroup and successively optimized for PPAR activation using in vitro PPAR transactivation assays. A (2-methylphenoxy)acetic acid headgroup, a flexible linker, and a five-membered heteroaromatic center ring with two hydrophobic aryl substituents were required for efficient and selective PPARdelta activation. The fine-tuning of these aryl substituents led to an array of highly potent and selective compounds such as compound 38c, displaying an excellent pharmacokinetic profile in mouse. In an in vivo acute dosing model, selected members of this array were shown to induce the expression of pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3), genes that are known to be involved in energy homeostasis and regulated by PPARdelta in skeletal muscle.

N-Acylthiadiazolines, a new class of liver X receptor agonists with selectivity for LXRbeta.

We have identified a novel liver X receptor (LXR) agonist (2) that activates the LXRbeta subtype with selectivity over LXRalpha. LXRbeta selectivity was confirmed using macrophages derived from LXR mutant mice. Despite its selectivity and modest potency, the compound can induce APO-AI-dependent cholesterol efflux from macrophages with full efficacy. Our results indicate that it is possible to achieve significant LXRbeta selectivity in a small molecule while maintaining functional LXR activity.

2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor.

2-aminothiazole (1) was discovered as a novel Src family kinase inhibitor template through screening of our internal compound collection. Optimization through successive structure-activity relationship iterations identified analogs 2 (Dasatinib, BMS-354825) and 12m as pan-Src inhibitors with nanomolar to subnanomolar potencies in biochemical and cellular assays. Molecular modeling was used to construct a putative binding model for Lck inhibition by this class of compounds. The framework of key hydrogen-bond interactions proposed by this model was in agreement with the subsequent, published crystal structure of 2 bound to structurally similar Abl kinase. The oral efficacy of this class of inhibitors was demonstrated with 12m in inhibiting the proinflammatory cytokine IL-2 ex vivo in mice (ED50 approximately 5 mg/kg) and in reducing TNF levels in an acute murine model of inflammation (90% inhibition in LPS-induced TNFalpha production when dosed orally at 60 mg/kg, 2 h prior to LPS administration). The oral efficacy of 12m was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally at 0.3 and 3 mg/kg twice daily. Dasatinib (2) is currently in clinical trials for the treatment of chronic myelogenous leukemia.

3,4,5-Trisubstituted isoxazoles as novel PPARdelta agonists. Part 2.

A series of PPARdelta-selective agonists was investigated and optimized for a favorable in vivo pharmacokinetic profile. Isoxazole LCI765 (17d) was found to be a potent and selective PPARdelta agonist with good in vivo PK properties in mouse (C(max)=5.1 microM, t(1/2)=3.1 h). LCI765 regulated expression of genes involved in energy homeostasis in relevant tissues when dosed orally in C57BL6 mice. A co-crystal structure of compound LCI765 and the LBD of PPARdelta is discussed.