Drug Discovery

Determine which existing drugs or novel chemical compounds most safely and effectively disrupt the Alzheimer’s pathology generated by the highest priority genes.

Development of an APP-specific B-secretase inhibitor for Alzheimer’s disease therapy

Researchers: 
Funding year(s): 
2014
Funding to date: 
$100,000

Developments of disease-modifying therapeutics that can slow or ultimately halt disease progression are urgently needed for treating Alzheimer’s disease (AD). Till date all anti-amyloid measures to treat AD have failed. This is often construed as amyloid being the wrong target but most of the amyloid-reducing approaches including targeting the amyloid producing enzymes have side effects. An attempt to develop a specific therapy with minimum mechanism-based side effects is proposed here. AD is characterized by the deposition of the b-amyloid (Ab) peptides, the production of which is initiated by β-Secretase (β-site APP cleaving enzyme 1; BACE1). Hence it is a prime target for AD therapy. Full or partial deletion of BACE1, although prevent the development of AD-like pathologies and memory impairments in different lines of APP transgenic mice, is also associated with specific behavioral and physiological alterations in mice, which are likely to be caused by failure in the physiological processing of various substrates. Hence, general inhibition of BACE1 might be associated with mechanism-based side effects as BACE1 mediates its various physiological functions through the processing of different substrates. An APP-specific BACE1 inhibitor has the potential to specifically inhibit Aβ production without the inhibition of the processing of other substrates. Hence, studying the biology of these substrates, examination of their structural, molecular and biochemical properties is essential for designing a BACE1 modulator. In this work, we propose to study the BACE1 cleavages of three newly identified substrates, namely: Neuregulin, Neural cell adhesion molecules CHL1 and L1. Biochemical, structural and cell biological examinations of the substrates will be performed to determine the affinity, cleavage efficiency, subcellular site of their beta-cleavage and sorting determinants. Our preliminary analysis of NRG1 cleavage suggests that BACE1 binds NRG1 with higher affinity  and cleaves it with a higher catalytic efficiency  than APP. As a result, BACE1 processing of NRG1 probably occurs in the biosynthetic compartment similar to the processing of the Swedish mutant of APP. Consistently, inhibition of endocytosis did not affect Neuregulin cleavage but did that of APP. This suggests that there are two pools of BACE1 in the cell: one the endosomal pool of BACE1 responsible for APP cleavage and the other non-endosomal pool cleaving high-affinity substrates such as NRG1. Exploiting this observation, we would like to check if endosomally targeted BACE1 inhibitors or anti-BACE1 ectodomain antibodies can specifically inhibit endosomal BACE1 cleavage of APP and thus spare those of NRG1, CHL1 and L1. Such therapies will reduce amyloid burden without much side effects from BACE1 inhibition.

Roadmap: 

Elucidation of the Molecular Target of Potent γ-Secretase Modulators

Researchers: 
Funding year(s): 
2014
Funding to date: 
$250,000

A promising series of soluble γ-secretase modulators (SGSMs) has been discovered in our lab at UCSD in collaboration with MGH which inhibit the formation of the aggregation prone Aβ42 peptide in favor of shorter less pathogenic Aβ isoforms. Despite the development of numerous potent SGSMs, the molecular target and the mechanism of action remain unknown. We propose the synthesis of three distinct clickable SGSM-photoprobes for cross-linking studies to demonstrate the binding site of these ligands within the γ-secretase enzyme. Additional experiments will be conducted using novel Aβ substrates in order to evaluate the mechanism by which SGSMs affect the processivity of γ-secretase. This research will identify the critical sites of interaction between the SGSMs and their molecular target, as well as provide valuable information toward the development of more potent and selective compounds. In addition, evaluating the processivity of γ-secretase will enable a critical understanding of the mechanism by which the SGSMs selectively attenuate the production of the pathogenic Aβ42 peptide and enrich our fundamental understanding of this enigmatic enzyme. A subset of these studies will directly test the processivity model of γ-secretase activity.  Collectively, these studies serve to identify the target and mode of action of our novel SGSMs with the goal of discovering potential therapeutic agents for the treatment of Alzheimer’s disease (AD).

Roadmap: 

Orbitrap Fusion Tribrid Mass Spectrometer

Researchers: 
Funding year(s): 
2014
Funding to date: 
$200,000
The proposed grant will assist in the purchase of an Orbitrap Fusion Tribrid Mass Spectrometer system to enable the development of a method to assess tau production and clearance rates in humans, animal models, and in vitro experiments. This cutting edge mass spectrometer system will provide more precise measurements with the ultra-low abundance of biomolecules of interest than current instruments can quantify. This will allow for the first time the measuring of tau kinetics enabling evaluation of tau directed therapeutics in animal models, tau kinetics in humans (e.g. AD), tau isoforms in stem cells and understanding the effects of genetic risk factors
related to tau metabolism. This system will support investigators at Washington University (CAF member Dr. Holtzman as well as other Washington University investigators) and other CAF members around the country as opportunities arise. Further, this system will enable plasma amyloid-beta kinetics to be measured. This will address central issues in amyloid-beta transport from the brain to the blood and also if peripheral kinetics canindicate brain amyloidosis.
 
The Stable Isotope Labeling Kinetics (SILK) approach has been adopted both in the academic fields of neurodegeneration and the commercial sector. The Fusion triple Mass Spectrometry system will enable the development and application of novel discoveries in amyloid-beta, tau, and related AD proteins in addition to basic discoveries in the kinetic metabolism of tau in vivo in both humans and animal models. The major limitation to the adoption and use of SILK is the technological hurdle in accurate mass spectrometry measurement of labeled biomolecules. The Fusion Tribrid MS system is the only system to demonstrate the capability to quantify very low abundant labeled biomolecules (attomole quantitation) with very low (<1%) labeling. This resource will provide accurate, high-throughput mass spectrometry analysis for tau SILK studies.
 
Compared to brain and CSF Aβ, tau and peripheral Aβ biology and pathophysiology in AD is far less understood. Importantly, tau levels in CSF correlate more closely with cognitive decline in AD patients than Aβ, and CSF tau and Aβ are critical biomarkers in precisely predicting the order and magnitude of pathologic processes in AD. Therefore, we believe that determining the tau metabolism in AD is the next critical step in the AD research field to improve future clinical trial designs and to develop an early AD detection test. Tau is
predominantly an intracellular protein but recent studies suggest that it is also released into the extracellular space, where it may be involved in spreading tau pathology to remote brain regions. Many studies have shown that tau and phosphorylated tau amounts are increased in AD, but the mechanism of tau production or clearance is not known. Elucidating tau metabolism would greatly enhance our basic knowledge of tau biology as well as our understanding of the role of tau in AD pathophysiology.

 

Roadmap: 

Molecular mechanisms of synaptic plasticity in the hippocampus: A path to novel therapies

Funding year(s): 
2014
Funding to date: 
$100,000

There is strong evidence suggesting that Alzheimer’s disease is caused in large part by the accumulation of a toxic protein termed A-beta (Aβ) in the brain. If scientists can understand in great molecular detail the very early steps of how Aβ accumulation impairs brain function, it will be possible to develop therapies that prevent these steps.  One of the earliest effects of toxic forms of Aβ is to impair the ability of the connections between nerve cells, termed synapses, to modify their own properties in response to changes in the patterns of brain activity. This synaptic plasticity, in particular in a brain region termed the hippocampus, is thought to be critical for learning and memory and thus impairments in synaptic plasticity in the hippocampus likely account for many of the early and late symptoms of Alzheimer’s.   While some of the molecular mechanisms underlying synaptic plasticity in the hippocampus have been elucidated, much is not known.  My laboratory has developed novel approaches to the study of one form of synaptic plasticity, termed long-term potentiation (LTP), which seems to be particularly important in Alzheimer’s disease in that toxic forms of Aβ inhibit the mechanisms normally responsible for this plasticity.  This impairment of LTP likely contributes to the cognitive impairment in early stages of Alzheimer’s disease as well as the eventual physical shrinkage and eventual loss of synapses. This research project will use sophisticated molecular, electrophysiological and imaging techniques to further elucidate the detailed molecular mechanisms of LTP in the hippocampus focusing on two proteins that have been genetically or biochemically associated with Alzheimer’s.  We will molecularly manipulate these different synaptic proteins in individual nerve cells and define their specific roles in LTP.  Importantly, we will express mutated versions of these proteins and determine if this prevents the detrimental effect of toxic species of Aβ on synaptic function and plasticity.  The results of these experiments will provide novel proteins and mechanisms that can be targeted for treating Alzheimer’s disease by preventing the very changes in the brain that lead to its devastating symptoms. 

Roadmap: 

Elucidation of the mechanism of action of Gamma Secretase Modulators

Researchers: 
Funding year(s): 
2013
Funding to date: 
$150,000

This project focuses on ultimately defining the structure of a soluble gamma-secretase modulator (SGSM)-bound gamma-secretase enzyme complex at high resolution. Defining the structure of this complex will provide critical information towards elucidating the mechanism of action of this promising series of therapeutic molecules known as SGSMs. This structural information will enable molecular dynamics simulations and can be used to identify critical sites of interaction between the SGSMs and their molecular target which we have shown to involve the catalytic subunit of the gamma-secretase enzymatic complex. These studies should enable a critical understanding of the mechanism of how these molecules selectively attenuate only the most pathogenic of the Abeta peptides, e.g., Abeta42 and could pave the way for identifying perhaps even more potent and more selective therapeutic agents for the treatment of Alzheimer’s disease.

Roadmap: 

Effects of Inhibitors of Monoacylglycerol Lipase on Behavior and Synaptic Plasticity of Ts65Dn Mice, a Genetic Model of Down Syndrome

Funding year(s): 
2013
Funding to date: 
$100,000

Alzheimer’s Disease (AD) is caused by a complex interplay between genetic, epigenetic and environmental factors. Mutations in three genes, amyloid precursor protein (APP), presenilin (PS)‐1 and (PS)‐2 account for early onset autosomal dominant AD (Bertram and Tanzi, 2012). People with Down syndrome (DS) carry and extra copy of chromosome 21, which contains a copy of the APP gene. As a result, by the 4th decade all people with DS exhibit the AD type neuropathology and most go on to show dementia by age 60. Thus, DS can be regarded as a valid and robust model of AD. We refer to the occurrence of AD in people with DS by using the term DS/AD. Mouse genetic models of DS carry an extra copy of genes homologous to those on human chromosome 21. One of the best current genetic models of DS, Ts65Dn mice, exhibit abnormalities in brain structure and cognition similar to those observed in DS people, including the degeneration of specific neuronal populations, an effect shown to be caused by
increased gene dose for APP.

Monoacylglycerol lipase (MAGL) is an enzyme that belongs to the serine hydrolase family. It was shown that a selective antagonist of MAGL, JZL184, restored to normal the levels of pro‐inflammatory eicosanoids and inflammatory cytokines in a mouse model of AD (Piro et al., 2012); inactivation of MAGL also robustly suppressed production and accumulation of β‐amyloid (Aβ), a peptide product of APP, and improved synaptic plasticity and memory (Chen et al., 2012).

Therapeutic Hypothesis: That inhibiting MAGL will reduce AD related neuropathology, restoring to normal measures of inflammation, APP processing, synaptic plasticity and cognition.

The goal of this proposal is to validate monoacylglycerol lipase as a therapeutic target for ameliorating AD‐type neuropathology in DS, and by extension AD, thereby providing a novel approach to the treatment of these disorders.

Roadmap: 

Normalizing Abeta synaptic depression with drugs targeting PICK1

Funding year(s): 
2013
Funding to date: 
$100,000

There is general agreement that beta amyloid (Aβ) is a likely causative agent in the development of Alzheimer’s disease. There is growing evidence that early in the disease an important target of Aβ is the synapse, the site of communication between neurons. We have found that exposure of synapses to Aβ causes their weakening. In this proposal we will examine the role played by PICK1, a protein that associates with synaptic receptors and participates in the weakening of synapses by Aβ. We will test drugs that inhibit the interaction between synaptic receptors and PICK1; such drugs should act to normalize synaptic strength in the presence of elevated Aβ. These drugs may be lead compounds in the search for drugs to treat Alzheimer’s disease.

Roadmap: 

The Development of UDP Analogs for the Treatment of Alzheimer's Disease

Researchers: 
Funding year(s): 
2012
Funding to date: 
$100,000

The goal of this project is to collaborate with a medicinal chemist to design, synthesize and test the efficacy of third-generation small molecules that will activate glial receptors. The most efficacious molecules then will be tested for their ability to reverse plaque burden in mouse models of Alzheimer’s disease.

The brain is composed of two classes of cells, electrically active neurons and electrically silent glia. Over the past 20 years, Dr. Philip Haydon’s lab has focused its research efforts on understanding the role of glia in brain function. As a consequence, the scientists made a breakthrough discovery that these often-neglected cells offer new therapeutic opportunities for the treatment of disorders of the brain. In particular, they demonstrated that the activation of a glial receptor leads to the clearance of amyloid plaques and restores learning and memory in Alzheimer’s mouse models.

The goal of this project is to collaborate with a medicinal chemist to design, synthesize and test the efficacy of third-generation small molecules that will activate these glial receptors. The most efficacious molecules then will be tested for their ability to reverse plaque burden in mouse models of Alzheimer’s disease. Success in this project will allow Dr. Haydon and associates to leverage private and federal funds to develop a small biotech spin-off focused on glial cells, and will prepare this study for IND-enabling studies as well as Phase I clinical trials of compounds developed in this project.

Roadmap: 

Novel Soluble Gamma-Secretase Modulators for the Treatment of Alzheimer’s Disease Identification of the Molecular Target of Potent Gamma-Secretase Modulators

Researchers: 
Funding year(s): 
2011 to 2012
Funding to date: 
$300,000

The goal of this project is to identify a series of highly potent gamma-secretase modulators able to lower Abeta42 and Abeta40 production while concomitantly increasing the less toxic production of Abeta38 without measurably affecting gamma-secretase-mediated processing of the Notch 1 receptor (which is very important in a variety of cellular processes for cell-to-cell communication).

Dr. Steven Wagner and his fellow researchers recently discovered two structurally related series of gamma-secretase modulators (AGSMs and SGSMs) with potencies more than a thousandfold superior to tarenflurbil and many of the NSAID-like carboxylic acid-containing GSMs. The first series of these aryl 2-aminothiazole GSMs (AGSMs) are small molecules that bind directly to gamma-secretase, decreasing Abeta42 and Abeta40 levels while concomitantly increasing Abeta38 and Abeta37 levels without affecting gamma-secretase-mediated enzymatic processing of other known substrates, such as Notch-1.

AGSMs were shown to be efficacious in vivo for lowering the levels of Abeta42 and Abeta40 in both the plasma and brain of APP transgenic mice. Chronic efficacy studies revealed that one AGSM (compound 4) dramatically attenuated AD-like pathology in the Tg2576 APP transgenic mouse model. In addition, unlike the GSls, the AGSMs, by virtue of the fact they do not inhibit gamma-secretase, do not show Notch-related side effects that invariably appear in rodents and mice when treated chronically with GSls (e.g., no evidence of intestinal goblet cell hyperplasia). However, the very poor aqueous solubility of these AGSMs (<0.1 micromolar at neutral pH) may significantly compromise their further preclinical development due to the difficulties in achieving the escalated supraefficacious exposures necessary for safety and toxicity studies required for advanced preclinical development with such poorly soluble compounds.

More recently, the researchers discovered a second series of highly potent GSMs that have significantly improved physicochemical properties (e.g., aqueous solubilities at neutral pH) compared to the previously described AGSM series. These two structurally related series, as may be expected, behave similarly with respect to their effects on APP processing in steady- state cell-based assays. Both GSM series are able to lower Abeta42 and Abeta40 production while concomitantly increasing Abeta38 production without measurably affecting gamma-secretase- mediated processing of another known gamma-secretase substrate, namely, the Notch 1 receptor.

Roadmap: 

Molecular Tweezers—Novel Inhibitors of Amyloidogenic Proteins and Promising Drug Candidates for Alzheimer’s Disease

Researchers: 
Funding year(s): 
2011
Funding to date: 
$100,000

The goal of this project is to plan expanded in vivo characterization of the efficacy of “molecular tweezers” toward development of disease-modifying therapy for AD and related diseases.

This project addresses Alzheimer’s disease (AD) in the larger context of diseases caused by aberrant protein folding and self-assembly, which leads to formation of toxic oligomers and aggregates. In the last several years, Dr. Gal Bitan’s lab has been studying novel compounds called “molecular tweezers,” which modulate the aberrant assembly process using a previously unexplored “process-specific” mechanism. Their current lead compound effectively prevents formation of toxic aggregates of several disease-related proteins, including those involved in AD, Abeta and Tau. Initial in vivo experiments show peripheral administration of low doses of this compound lead to significant reduction of Abeta and Tau in the brain of transgenic mice.

In view of these promising data, they are poised to explore further the mechanism of action of the molecular tweezers and answer critical questions about their pharmacokinetics and safety.

Roadmap: