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The research on neurodegenerative diseases is challenged by accurate disease diagnosis and evaluation of treatment response, thus there is a need for indicators at the molecular level, specifically biomarkers, that reliably provide disease diagnostic, progression, and prognostic information. The parenchyma of the brain is immersed in the interstitial fluid (ISF), which is in equilibrium with the cerebrospinal fluid (CSF) allowing for ex vivo sample analysis of potential endogenous in vivo biomarkers. Details on the kinetic parameters of these analytes may provide a more sensitive, specific and accurate measure of disease pathology, biological physiology, and pharmacodynamic response of therapeutics. To study the differential changes in the de novo biosynthesis as well as the clearance of proteinaceous/peptidic biomarkers, a mass spectrometric detectable label is introduced via stable isotope labeling in mammals (SILAM) methodology. Proteolytic processing of the proteins generates surrogate proteotypic peptides. The mass isotopomer distribution analysis (MIDA) of these proxy reporters via high resolution accurate mass spectrometry (HRMS) provides kinetic profiles of the proteins. Presented here is the conceptual methodology of applying LC-HRMS analysis to obtain MIDA data of CSF proteins which were modified by SILAM methodology utilizing D-labeled water (2H2O, D2O).

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of pathological accumulations of misfolded, phosphorylated α-synuclein (αSyn) protein. Multiple lines of evidence indicate that synucleinopathy disease progression is driven by a prion-like process of transmission of a pathologic form of αSyn. One potential therapeutic approach to prevent cell-to-cell propagation is to target this transmissible species with selective antibodies. In this study, a rodent primary neuronal culture reporter system was developed to monitor induction of detergent-insoluble, phosphorylated (pS129) aggregates of αSyn. Induction of pS129 αSyn pathology was observed with both synthetic αSyn fibrils (PFFs) and brain lysates from multiple system atrophy (MSA) patients but not αSyn monomers or human brain lysate controls. The induction-competent species in MSA lysates could be enriched by high-speed centrifugation suggesting that it is present as a high molecular weight aggregate. Furthermore, samples derived from brain lysates from Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) patients also induced pS129 αSyn pathology, but required longer incubation times. Lastly, the potential of αSyn selective antibodies to immunodeplete induction-competent forms of αSyn from both PFF and synucleinopathy brain samples is described. The results demonstrate that antibodies targeting the C-terminal of αSyn are most effective for immunodepletion of pathology-inducing forms of αSyn from samples derived from human synucleinopathy brains. Furthermore, the data support the hypothesis that antibodies that recognize a C-terminal epitope and exhibit selectivity for oligomeric forms over monomeric forms of αSyn represent a desirable target for immunotherapy for synucleinopathy patients.

The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.

A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity.

We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.

BMS-299897 is a gamma-secretase inhibitor that has the potential for treatment of Alzheimer's disease. The metabolism of [(14)C]BMS-299897 was investigated in human liver microsomes, in rat, dog, monkey and human hepatocytes and in bile duct cannulated rats. Seven metabolites (M1-M7) were identified from in vitro and in vivo studies. LC-MS/MS analysis showed that M1 and M2 were regioisomeric acylglucuronide conjugates of BMS-299897. Metabolites M3, M4 and M6 were identified as monohydroxylated metabolites of BMS-299897 and M5 was identified as the dehydrogenated product of monooxygenated BMS-299897. In vivo, 52% of the radioactive dose was excreted in bile within 0-6 h from bile duct cannulated rats following a single oral dose of 15 mg/kg of [(14)C]BMS-299897. Glucuronide conjugates, M1 and M2 accounted for 80% of the total radioactivity in rat bile. In addition to M1 and M2, M7 was observed in rat bile which was identified as a glucuronide conjugate of an oxidative metabolite M5. For structure elucidation and pharmacological activity testing of the metabolites, ten microbial cultures were screened for their ability to metabolize BMS-299897 to form these metabolites. Among them, the fungus Cunninghamella elegans produced two major oxidative metabolites M3 and M4 that had the same HPLC retention time and mass spectral properties as those found in in vitro incubations. NMR analysis indicated that M3 and M4 were stereoisomers, with the hydroxyl group on the benzylic position. However, M3 and M4 were unstable and converted to their corresponding lactones readily. Based on x-ray analysis of the synthetically prepared lactone of M3, the stereochemistry of benzylic hydroxyl group was assigned as the R configuration. Both the hydroxy metabolites (M3 and M4) and the lactone of M3 showed gamma-secretase inhibition with IC(50) values similar to that of the parent compound. This study demonstrates the usefulness of microbial systems as bioreactors to generate metabolites of BMS-299897 in large quantities for structure elucidation and activity testing. This study also demonstrates the biotransformation profile of BMS-299897 is qualitatively similar across the species including rat, dog, monkey and human which provides a basis to support rat, dog and monkey as preclinical models for toxicological testing.

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.

The formation of a reactive intermediate was found to be responsible for CYP3A4 metabolism-dependent inhibition (MDI) observed with (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenyl-acrylamide (1). Structure-3A4 MDI relationship studies culminated in the discovery of a difluoro analogue, (S)-N-[1-(4-fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluoro-phenyl)acrylamide (2), as an orally bioavailable KCNQ2 opener free of CYP3A4 MDI.

Members of the TAM family of receptor tyrosine kinases (RTKs) play important roles in tumorigenesis and homeostatic regulation of the immune system. Two closely related ligands, Protein S (PROS1) and Growth Arrest Specific 6 (GAS6), bind and activate the TAMs, with GAS6 activating all TAMs and PROS1 activating MerTK and Tyro3. TAM RTKs are aberrantly expressed in many types of cancer where they may promote survival, metastasis, and chemoresistance. These receptors are also expressed on innate immune cells, particularly macrophages and dendritic cells, where they play important roles in clearance of apoptotic cells and negative regulation of inflammation. In tumors, activation of TAM signaling cascades appears to promote an antiinflammatory and immunosuppressive microenvironment, in turn driving tumor growth. Thus, inhibition of TAM signaling pathways through selective targeting with monoclonal antibodies could result in direct antitumor effects and promote antitumor immunity, whereas activation of TAMs could have antiinflammatory and immunosuppressive effects. We have used a variety of antibody-discovery strategies to identify potent and selective antagonists and agonists of human and rodent TAMs. Robust preclinical model systems for characterizing the mechanistic and functional effects of these antibodies have been established using engineered and nonengineered cell lines and primary cells. These include models to assess TAM activation and downstream signaling in cells and effects on cytokine secretion in LPS-stimulated U937 monocyte-derived and primary macrophages and primary dendritic cells. Effects of TAM inhibition on T-cell responses in mixed lymphocyte reactions are also being evaluated, as are in vivo mouse models. Utilizing these models, we have identified anti-TAM antibodies with antagonist or agonist activity, suggesting they may have potential in modulating the activity of these pathways. Citation Format: Diego Alvarado, Laura Vitale, Michael Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Craig Polson, James R. Storey, Jenifer Widger, Laura Mills-Chen, Karuna Sundarapandiyan, Andrea Crocker, Colleen Patterson, Biwei Zhao, Russell A. Hammond, Li-Zhen He, Venky Ramakrishna, Joel Goldstein, Lawrence Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Identification and characterization of monoclonal antibodies targeting the Tyro3, Axl and MerTK (TAM) family of receptor tyrosine kinases [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B194.

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

Alzheimer’s disease is the most prevalent cause of dementia and is associated with accumulation of amyloid-βpeptide (Aβ), particularly the 42-amino acid Aβ1-42, in the brain. Aβ1-42 levels can be decreased byγ-secretase modulators (GSM), which are small molecules that modulateγ-secretase, an enzyme essential for Aβproduction. BMS-869780 is a potent GSM that decreased Aβ1-42 and Aβ1-40 and increased Aβ1-37 and Aβ1-38, without inhibiting overall levels of Aβpeptides or other APP processing intermediates. BMS-869780 also did not inhibit Notch processing byγ-secretase and lowered brain Aβ1-42 without evidence of Notch-related side effects in rats. Human pharmacokinetic (PK) parameters were predicted through allometric scaling of PK in rat, dog, and monkey and were combined with the rat pharmacodynamic (PD) parameters to predict the relationship between BMS-869780 dose, exposure and Aβ1-42 levels in human. Off-target and safety margins were then based on comparisons to the predicted exposure required for robust Aβ1-42 lowering. Because of insufficient safety predictions and the relatively high predicted human daily dose of 700 mg, further evaluation of BMS-869780 as a potential clinical candidate was discontinued. Nevertheless, BMS-869780 demonstrates the potential of the GSM approach for robust lowering of brain Aβ1-42 without Notch-related side effects.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

An inhibitor of the epidermal growth factor (EGF) receptor was isolated from an extract of the Micronesian sponge Callyspongia fibrosa. The structure of the EGF-active compound was first proposed, on the basis of ion-spray mass spectrometry, to be the cyclic dimer 1 (n= 2) but comparison of a sample produced by an efficient synthetic route revealed that hypothesis to be incorrect. The EGF-active constituent must therefore be a large oligomer or a polymer of the same repeating subunit 1

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) 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, beta-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 Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, 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 Abeta1-40 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 Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

The synthesis and gamma-secretase inhibition data for a series of carbamate-appended N-alkylsulfonamides are described. Carbamate 54 was found to significantly reduce brain Abeta in transgenic mice. 54 was also found to possess markedly improved brain levels in transgenic mice compared to previously disclosed 1 and 2.

Targeting the metabolism of amyloid beta peptides (Abeta) is currently the leading experimental approach to treatment of Alzheimer's disease (AD). Described here is an immunoprecipitation-liquid chromatography/mass spectrometry (ip-LC/MS) assay to simultaneously characterize and quantitate different forms of Abeta in biological samples. The 4G8 antibody, specific for the 17-24 amino acid epitope of Abeta was employed to selectively isolate Abeta from in vitro samples for subsequent LC-MS analysis. A high resolution accurate mass hybrid linear ion trap-Orbitrap, LTQ-Orbitrap mass spectrometer was used to identify forms of 12 Abeta in H4-APP751 swe cell extracts based on ab initio calculations, accurate mass measurements, isotopic modeling and by de novo peptide sequencing using tandem mass spectrometry. The quantitative LC-MS analysis was performed on a linear ion trap mass spectrometer, LTQ, in full scan mode, this mode of operation enables sensitive detection levels and post-acquisition data mining for different forms of Abeta for quantitative assessment. Dosing studies with three known inhibitors of Abeta production, sulindac sulfide (SSide), BMS-299897 ('897) and compound W (CW) are reported to demonstrate the utility and analytical characteristics of the assay. This assay has the potential to provide insight into the formation of Abeta; increase understanding of drug mechanisms; and to contribute to drug efficacy studies.

The synthesis and gamma-secretase inhibition data for a series of nitrogen-appended N-alkylsulfonamides (11-47) are described. Inhibition of brain Abeta in transgenic mice was demonstrated by two of these compounds (23 and 44).

A series of amino-caprolactam sulfonamides were developed from a screening hit. Compounds with good in vitro and in vivo gamma-secretase activity are reported.

2,3-Benzodiazepin-1,4-diones were designed as peptidomimetics at the carboxy terminus of hydroxyamides. Inhibition of brain Abeta production was improved by one of the compounds containing constrained modification.

Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.

Inhibitors of amyloid-beta (Abeta) protein production have been widely pursued as a potential treatment for Alzheimer's disease. Following the identification of a 5 microM screening hit, SAR was initiated using solid-phase synthetic techniques. Two series of alpha-hydroxy esters and ketones which are sub-micromolar inhibitors of Abeta production were identified. The most potent alpha-hydroxyketone identified is approximately 30-fold more potent than the initial lead.