234.23 S : A Novel Therapeutic Target for Alzheimer’s Disease Bitna Yi1, Andrew K. Evans1, Leslie J. Holsinger2, Robert Booth2, Mehrdad Shamloo1 Neurosurgery, School of Medicine; Stanford University, Palo Alto, CA, USA1 Virobay Inc, Menlo Park, CA, USA2 Background Results Chronic inflammation in response to neuronal damage is is implicated Cathepsin S is overexpressed in Inhibition of Cathepsin S activity thought to accelerate and potentially underlie disease in AD pathology mouse model of AD reduces neuroimmune response progression in AD (AD). Here, we assessed the Microglial Wild-type Transgenic 20 therapeutic potential of Cathepsin S as a novel molecular Death of Activation RS Inflammatory target for the treatment of AD. Neurons CatS 15 TriggersAb, Tau precursor Substance P, LPS Cathepsin S is a cysteine exhibiting both extra- Aβ, Tau Cathepsin S 10 and intracellular activities whose expression is largely Recruitment of Release Mature Inflammatory Cells Membrane Bound CatS confined to . By regulating a step in MHC class 5 Release of Fractalkine II , cathpesin S play an important TNF, IL-1, ROS Relative levels of CatS 0 role in modulating immunoregulatory processes. In WT Tg Figure 2. concentrations of the mature form of cathepsin S addition to the roles as inflammatory mediators, inhibition (24 kDa band) are increased in cortex in transgenic mice WT APP APP WT of cathepsin S could also potentially affect a number of Release of Soluble Dendritic remodeling expressing familial alzheimer’s related (5XFAD model, Tg) Vehicle Vehicle VBY50365 VBY50365 cellular processes involved in protein proteolysis and Fractalkine Synaptic degeneration compared to the wild-type. lysosomal function, which have implication in AD RS DG pathology such as amyloid and tau. Thus, modulating CNS-permeable Cathepsin S inhibitor as Pharmacological Tool 12 12 activity of cathepsin S might provide therapeutic benefit Selectivity of the cathepsin S inhibitor VBY50365 10 10 to treat or slow the disease progression through 8 8 Protease Class IC50 (nM) Fold selectivity (vs S) 6 6

modulation of specific element of inflammation and Cathepsin S cysteine 0.26 1 (% area) (% 4 area) (% 4 protein proteolysis. cysteine 140 538 2 2 With an aim of assessing the therapeutic potential of cysteine 590 2269

IBA-1 immunoreactivity IBA-1 0

IBA-1 immunoreactivity IBA-1 0 cathepsin S, we administered the cathepsin S inhibitor cysteine 65 250 Lond/Swe+3 BACE1 aspartic >10,000 >38,000 VBY50365 in the Thy1-hAPP mouse model of WT vehicle APP vehicle WT vehicle APP vehicle TACE (ADAM17) metallo >10,000 >38,000 WT VBY50365 WT VBY50365 AD. Chronic administration of VBY50365 lead to APP VBY50365 APP VBY50365 Cathepsin E aspartic >10,000 >38,000 Figure 5. A quantitative analysis of the microglia marker iba-1 staining revealed that improvement in cognitive deficits and pathology related VBY50365 reduces microglia staining (iba1) in cortex (RS) and hippocampus (DG). to AD. This data suggest modulation of cathepsin S 3 cysteine-aspartic acid >10,000 >38,000 activity might be a novel therapeutic strategy for the Chymotrypsin, Figure 3. Once a day dosing of mice with the Work in Progress Pancreatic , cathepsin S inhibitor VBY50365 (100 mg/kg) treatment of AD. Neutrophil elastase, serine >10,000 >38,000 yields concentrations of drug in plasma and Thrombin, Trypsin, CNS sufficient to sustain inhibition of Cathepsin S and synaptic degeneration Method Tryptase cathepsin S. Effects of In vitro Enzyme Potency and Selectivity Inhibition of Cathepsin S activity restores cognitive deficits Cathepsin S activity Potency and selectivity of VBY50365 was evaluated on a panel of inhibiton on synaptic listed in in vitro enzyme inhibition assays. All were A Y-Maze C Fear Conditioning human in origin except pancreatic elastase which was porcine. 80 80 Training day degeneration Enzyme reactions were run in conditions where substrate was equal to ** 70 * or less than the Michaelis constant, Km, and the resulting dose 60 response curves were used to determine IC of enzyme inhibition. 60 Testing Day 50 (Contextual FC) ?

In vivo transgenic AD mouse model study 50 40 % % Alteration The effects of the CatS inhibitor were evaluated in the Thy1- 40 Lond/Swe+3 Lond/Swe+ hAPP mouse model of AD. Using a 2x2 factor design, 6 month Freezing (%) 20 Figure 6. Thy1-hAPP mice have old male transgenic mice and their non-transgenic littermates received 30 synaptic degeneration in CA3 as Restoration of 100mg/kg/day of the cathepsin S inhibitor VBY50365 or vehicle daily 0 measured by quantitative until 7.5 months of age. Functional outcomes of VBY50365 treatment synaptophysin immunoreactivity. cognitive deficits WT vehicle APP vehicle WT VBY50365 on behavioral deficits were assessed at multiple time points post APP VBY50365 Min1 Min2 Min3 Min4 Min5 dosing through a battery of cognitive behavioral assay including 70 activity chamber, Y-maze, passive avoidance, and fear conditioning. B Passive Avoidance 60 300 Conclusion After behavioral testing, mice were sacrificed and brain tissue were WT Vehicle 50 harvested and used to assess the effects of VBY50365 on biochemical APP Vehicle 40 • Cathepsin S is strongly implicated in AD pathology. assessments. APP VBY50365 Administration of VBY50365 (100 mg/kg, SC) 200 30 WT VBY50365 • Selective inhibition of cathepsin S activity lead to

(3 min-5min) (3 20 Freezing (%) 10 restoration of cognitive deficits associated with AD and Day 1 Day 7 Day 14 Day 21 Day 28 Days 35-37 Days 43-45 time(S) 100 Activity Activity Activity Activity Passive Fear 0 reduced microglial activation in the CNS. Chamber Chamber Chamber Chamber Avoidance Conditioning Test 1 Test 2 Test 3 Test 4 • Modulation of cathepsin S activity may provide

0 WT Vehicle APP Vehicle therapeutic benefit in AD. Day 24 Habituation Training Testing WT VBY50365 Tissue APP VBY50365 Y-MAZE collection Figure 4. VBY50365 reverses spatial memory deficits observed in Thy1-hAPPLond/Swe+ mice in the Y-maze (A) (*p<0.05, **p<0.01 Acknowledgements by t-test) with similar trends for restoration of memory deficits observed in passive avoidance (B) and fear conditioning (C) Figure 1. In vivo transgenic AD mouse study Design memory test. This study was supported by Virobay Inc.