Beyond amyloid: a diverse portfolio of novel drug discovery programs for Alzheimer's disease and related dementias

Although the molecular mechanisms underlying the pathogenesis of Alzheimer's disease and other related neurodegenerative diseases remain unclear, accumulation of misfolded proteins, neuroinflammation, mitochondrial dysfunction and perturbed calcium homeostasis have been identified as key events leading to neuronal loss during neurodegeneration. Evidence for 'druggable' targets for each of these key mechanisms was presented by the Alzheimer's Drug Discovery Foundation-funded investigators at the 12th International Conference on Alzheimer's Drug Discovery, Jersey City, NJ, 26-27 September 2011 http://www.worldeventsforum.com/addf/addrugdiscovery.


Neuroprotective strategies
Th erapies that are neuroprotective or neurotrophic and can protect against neuronal loss are attractive targets in preventing or slowing the progression of Alzheimer's disease (AD). Carmela Abraham (Boston University School of Medicine, USA) discussed the development of modulators of Klotho expression, a protein that has been shown to decrease with age and regulate maturation of myelinating oligodendrocytes. Th ree hit compounds were identifi ed from a high-throughput screen (HTS) of 150,000 compounds that inhibit methylation at CpG islands and thereby increase Klotho expression. Hits have been validated in several cell lines and are now undergoing a medicinal chemistry strategy to ensure a desirable pharmacological profi le. A second neuroprotective strategy was highlighted by Eugenia Trushina (Mayo Clinic College of Medicine, USA). Trushina and team identifi ed and characterized an orally available and blood brain barrier (BBB) permeable tricyclic pyrone, CP-2, which eliminates 80% of amyloid deposits (fi brillar and oligomers) in the 5x familial AD (FAD) transgenic model. Probing the mechanism of action revealed metabolomic changes and altered mitochondrial traffi cking and function prior to the onset of memory and neurological phenotypes, all of which were reversible with CP-2 treatment.
Other novel neuroprotective strategies targeted neuroinfl ammation through the orally available tetramethylpyrazine (TMP), derived from a Chinese medical herb (Ziqun Tan, University of California, USA). Martin Watterson (Northwestern University, USA) presented the develop ment program for MW151, a small molecule modulator of p38α mitogen-activated protein kinase (MAPK) activity in neuronal and glial cells with 'druggability' at the forefront of development. Watterson and colleagues undertook a strategic medicinal chemistry program to address the major barriers to AD therapeutics: central nervous system penetrance and undesired pharmacology. Th is program resulted in the development of MW151, a highly selective, BBB penetrable compound that eff ectively attenuated pro-infl ammatory cytokine production and reversed behavioral defi cits following oral administration in an amyloid-β-induced brain injury mouse model.
Robert Mahley (University of California, USA) presented an update on PY101, a small molecule that restores apolipoprotein E4 (ApoE4) to an ApoE3-like structure. Ten-day oral adminis tration of PY101 in the neuronspecifi c enolase ApoE4 (NSE-ApoE4) mouse model resulted in decreased production of toxic ApoE fragments and increased mito chon drial cyclooxygenase-1 levels in the hippo cam pus. Identi fi cation of this toxic gain of function for ApoE4 opens up additional targets in

Abstract
Although the molecular mechanisms underlying the pathogenesis of Alzheimer's disease and other related neurodegenerative diseases remain unclear, accumulation of misfolded proteins, neuroinfl ammation, mitochondrial dysfunction and perturbed calcium homeostasis have been identifi ed as key events leading to neuronal loss during neurodegeneration. Evidence for 'druggable' targets for each of these key mechanisms was presented by the Alzheimer's Drug Discovery  this pathway, including protease inhibitors to prevent neuron-specifi c proteolysis of ApoE4 and mitochondrial protectors to prevent ApoE4 fragment interaction with mitochondria. Finally, the most advanced of these neuroprotective strategies, in phase II/III for the orphan neuro degener ative disease progressive supranuclear palsy (PSP), was presented by Illana Gozes (Allon Th erapeutics, USA). Allon Th era peutics' drug (davunetide), a short eight amino acid frag ment of activity dependent neurotrophic factor (ADNF), met limited effi cacy endpoints in a phase II AD trial, but is now being tested in an improved formulation for PSP.

Calcium homeostasis and plasticity
Histone deacetylase (HDAC)2 has been previously implicated in memory formation and synaptic plasticity; however, the development of HDAC2-specifi c inhibitors has been challenging. To reduce the toxicity associated with this class of compounds, Pavel Petukhov (University of Illinois, USA) screened compounds using Binding Ensemble Profi ling with Photoaffi nity Labeling to specifi cally identify compounds that bind in an orientation that prevents chelation of the zinc group. Th is screen identifi ed a lead compound with an improved pharmacokinetic and toxicity profi le.
Altered calcium homeostasis, specifi cally increased endoplasmic reticulum calcium release, has been linked to a number of sporadic AD risk factors and is thought to underlie altered synaptic transmission and plasticity. Grace Stutzmann (Rosalind Franklin University School of Medicine, USA) and colleagues were the fi rst to observe that the endoplasmic reticulum-resident ryanodine recep tor (RyR) diff erentially regulates synaptic transmission and plasticity in non-transgenic and AD transgenic mice. Chronic treatment of 3x FAD transgenic mice with the RyR antagonist dantrolene (injection) normalized calcium signaling by preventing increased endoplasmic reticulum RyR-mediated calcium release, validating RyR as a potential target for further screening.
Increased intracellular calcium levels aff ect numerous cellular functional processes via calcium-sensitive proteins. Visinin-like protein 1 (VILIP1), a neuronal calcium sensor protein discovered by Karl-Heinz Braunewell (Southern Research Institute, USA) regulates the cell surface localization of α4β2 nicotinic acetylcholine receptors. VILIP1 receptors are decreased in the hippocampus, amygdala and cortical areas of AD patients. A phenotypic and biochemical HTS of 2,000 compounds identifi ed 2 lead compounds that will now be optimized for the desired pharmacological properties.

Protein folding and degradation
Heat shock proteins (HSPs) promote the correct folding and refolding of misfolded proteins, including tau, amyloid-β, and α-synuclein, and are therefore attractive targets for a number of neurodegenerative diseases. Several approaches to target HSP70 and HSP90 were presented. Allen Reitz (ALS Biopharma, USA) identifi ed hit com pounds that lowered tau levels by inducing HSP70 expression in human glioblastoma cell lines. Under standing the mechanism of action of a target is vitally important during drug design. Th is point was highlighted by Chad Dickey (University of South Florida, USA), who discovered that inhibition of HSP70 ATPase activity could also be eff ective at stabilizing disorganized tau. Inhibitors from a HTS based on rhodacyanine dyes have now been validated for their ability to decrease tau accumulation and rescue long-term potentiation in hippocampal slice cultures from Tg4510 tau mice. A second member of the heat shock family, HSP90, was the focus of Gabriela Chiosis's (Memorial Sloan Kettering Cancer Center, USA) drug discovery program. Chiosis identifi ed two populations of HSP90 in pathogenic or stressed tissue (tumors and dystrophic neurons); a good housekeeping HSP90 complex and an HSP90 complex that selectively binds to pathogenic protein. Hsp90 inhibitors developed by this team specifi cally bind the pathogenic HSP90 complex and reduce total tau and phospho-tau following acute treatment in the 3x FAD transgenic mouse model. Chronic studies will be required to assess behavioral outcomes.
Dysfunctional protein degradation pathways contribute to neurodegeneration. Karen Duff (Columbia University, USA) provided target validation for autophagy and proteasome targets demonstrating that mice with progres sive tauopathy show a decline in proteasome function concomitant with an induction of autophagy and accumulation of fi brillar tau aggregates. In vivo induction of autophagy following acute oral administration of trehalose, a non-reducing dissacharide, eff ectively decreased tau pathology in the P301L tau mouse model. Duff noted that the decreased proteosomal function seen during the onset of pathology could be reversed pharmacologically, highlighting both autophagy and proteasome activation as two potential drug targets in stimulating clearance of protein aggregates that present in numerous neurodegenerative diseases.
Lysosomal function plays a critical role in protein clearance. Lysosomal storage diseases, such as Gaucher disease, Fabry disease and Sandhoff disease, show evidence of accumulated intraneuronal amyloid-β in addition to decreased lysosome enzyme function. Brandon Wustman (Amicus Th erapeutics, USA) and team have developed a set of novel pharmacological chaperones that enhance the activity of critical lysosomal enzymes, are orally available, cross the BBB, and are currently in phase III for Fabry disease. Now, Wustman and team are testing a novel, potent pharmacological chaperone for its eff ects in clearing intraneuronal amyloid-β in an animal model of cerebral amyloid angiopathy, an orphan indication with relevance to AD.
Additional strategies at the proof-of-concept stage targeted protein aggregates through immunotherapy. Rakez Kayed (University of Texas, USA) has developed an oligomer immunotherapy agent, TOMA (tau oligomer monoclonal antibody), that eff ectively reduced tau pathology following intracerebroventricular injection in the P301L tau mouse model. In the plenary session, Ryan Watts (Genentech, USA) demonstrated that β-site APP cleaving enzyme (BACE) monoclonal antibodies engineered to cross the BBB via the transferrin receptor eff ectively reduced BACE activity and amyloid-β levels without aff ecting total BACE expression in an AD mouse model.

Novel biomarkers
Th e development of novel biomarkers to accurately diagnose AD prior to clinical manifestation and to predict conversion from mild cognitive impairment to AD is essential for the development of new therapeutics and understanding mechanisms of disease progression. Currently, the only defi nitive diagnostic tool for AD is at autopsy. Th e most developmentally advanced imaging Each program presented at the 12th International conference on Alzheimer's Drug Discovery is highlighted in terms of position in the drug development lifecycle at the time of the meeting. *Allon Theraputics is currently in phase II/III for progressive supranuclear palsy, following limited effi cacy in a phase II trial of davunetide for Alzheimer's disease. NB: the purpose of this fi gure is to highlight the stage of development for each program that has benefi ted from Alzheimer's Drug Discovery Foundation funding, and is not indicative of Alzheimer's Drug Discovery Foundation funding throughout the entire program lifecycle. Multiple funders have contributed to many of these programs. Aβ, amyloid-β; ADME, absorption, distribution, metabolism and excretion; ADNF, activity dependent neurotrophic factor; AMPK, AMP activated protein kinase; APOE, apolipoprotein E; BACE, β-site APP cleaving enzyme; FTLD, frontotemporal lobar degeneration; HDAC, histone deacetylase; HSP, heat shock protein; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; MSKCC, Memorial Sloan Kettering Cancer Center; PDI, protein disulphide isomerase; PoC, proof of concept; RyR, ryanodine receptor; TfR, transferrin receptor; TMP, tetramethylpyrazine; TOMA, tau oligomer monoclonal antibody; TOX, toxicity; VILIP, Visinin-like protein.
biomarker, AV-45 (fl orbetapir), is currently up for approval by the US Food and Drug Administration (FDA). Daniel Skov ronsky (Avid Radiopharmaceuticals, Inc., USA), funded by the Alzheimer's Drug Discovery Foundation (ADDF) for the initial stages of development of fl orbetapir-positron emission tomography (PET), provided the opening plenary address. Florbetapir 18 F is a PET imaging agent with high specifi city for amyoid-β binding. Skovronksy presented data from longitudinal analysis of normal older people with positive fl orbetapir-PET scans and identifi ed a correlation between scan positivity and cognitive decline in normal older people, providing further evidence that amyloid-β load correlates with cognitive function.
Diagnostic tests that correctly diagnose diff erent causes of dementia are clinically challenging and remain a critical unmet need. Biomarkers that effi ciently distin guish between frontotemporal lobar degeneration (FTLD) with TDP43 (TAR DNA-binding protein 43) or tau pathology are necessary for the development of disease-modifying treatments and to identify patients for inclusion/ exclusion from clinical trials. William Hu (Emory University School of Medicine, USA) provided an update on the program to identify relevant biomarker(s) using a targeted proteomics approach. Hu and colleagues identifi ed a panel of analytes from cerebrospinal fl uid samples that potentially distinguish between FTLD with TDP43 pathology and FTLD with tau pathology. Th e team is currently recruiting patients for a multi-center study to validate this panel. Jeff Kuret (Ohio State University, USA) presented data on a HTS to identify selective radiolabeled compounds with high selectivity for tau by utilizing amino acid side chains of cross-βsheet aggre gates to increase binding selectivity. Th e team has identifi ed potent lead candidates currently under optimization.
Allen Roses (Duke University, USA) highlighted the utility of biomarkers to enrich patient populations in clinical trials. Roses and team have identifi ed a region on chromosome 19 next to APOE that encodes the mitochondrial protein TOMM40 (translocase of outer mitochon drial membrane 40 homolog) and can help explain the age-of-onset variability within the APOE genotype. Th rough an algorithm-based approach, Roses and team are testing the prognostic value of the TOMM40 genotype, APOE variants and age for patient inclusion in a trial of pioglitazone in normal subjects. To close the program, José Luchsinger (Columbia University, USA) presented the use of [ 18 ]F-labeled 2-deoxy-2-fl uoro-Dglucose (FDG) PET with magnetic resonance imaging as a secondary endpoint for an ongoing phase II clinical trial for the anti-diabetic metformin in patients with mild cognitive impairment.
Th e ADDF drug discovery portfolio encompasses a wide spectrum of novel targets in the generation of therapeutics for the treatment of AD and related dementias. Th e work presented highlights the innovative approaches required during preclinical and clinical development. Th e ADDF will continue to support novel targets in order to achieve our mission to conquer AD through drug discovery. Please join us for our 13th annual International Conference on Alzheimer's Drug Discovery in the Fall of 2012 in Jersey City, NJ, USA.