P4-208: characterization and optimization of new chemical entity anti-protein misfolding agents

Background and Hypothesis: Both beta amyloid and tau produce neurotoxic protein misfolding co-conspirators most often implicated in the molecular causation of AD. Moreover, since the clinical presentation of AD is heterogeneous (e.g. extrapyramidal symptoms in some people with AD and dementia symptoms in some people with PD), the clinical overlap amongst neurodegenerative disorders implies that AD is more of a syndrome than a disease and that the suppression of other aberrantly misfolded protein (e.g. alpha synuclein) may afford additional therapeutic benefits. Accordingly, we are seeking to design and develop brain-penetrable small molecule new chemical entities as anti-protein misfolding agents targeting three proteins: beta amyloid 40 and 42; tau and alpha synuclein. Based upon extensive in silico modeling, a family of novel compounds has been identified. A representative compound is TRV101. Methods: Efficacy of TRV101 was measured in a variety of in vitro assays including Thioflavin T beta amyloid aggregation, Thioflavin S tau aggregation, and biotin-beta amyloid 42 oligomerization. TRV101 was incubated with full length tau and the resultant solution was examined by electron microscopy. Additionally, data was collected on blood brain penetrance, protein binding, and longevity in mouse liver microsomes. Results: TRV101 showed anti-aggregation activity against both beta amyloid and tau. TRV has drug like properties, has high brain penetrance, and is stable in various ADME tests. Conclusion: We have developed a new class of compounds capable of inhibiting aggregation of both beta amyloid and tau proteins. Our small molecules are pharmacokinetically stable and are able to reach the target tissue. Our compounds are currently being tested in animal models of both beta amyloid and tau pathologies. In Vivo PK (Mouse, p.o.)  AUC (brain) = 8692 hr*ng/ mL  T1/2 = 5.4h  Cmax = 1217 ng/mL (brain)  Bioavailability = 78.3 %  B/P = 1 MLM T1/2 > 60 min Clint = 0.008 mL/min/mg HLM T1/2 = 57 min Clint = 0.024 mL/min/mg Physical Chemistry Properties  LogD = 3.15  MW: 446 g/mol  tPSA = 60.6 Protein binding  98.9 % (mouse)  97.6 % (human) 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 A b s o rb a n c e ( 4 5 0 n m ) In vitro assays Thioflavin S aggregation assay – Thioflavin S fluoresces when in the presence of aggregated tau. The reaction is initiated by addition of heparin and the reaction is followed by fluorescence until the signal change plateaus. Test compounds are measured by change in fluorescence compared to a vehicle control. TRV101 inhibits tau-441 aggregation with an IC50 of 2.5μM Oligomer formation assay – Biotinylated amyloid-β is allowed to self-associate in the presence of compound, vehicle, and 0.1% Tween20. The reaction is stopped by addition of 0.1% Tween-20 and the sample is applied to a neutravidin ELISA plate. Oligomers are detected by addition of streptavidin-HRP, which will only detect unbound biotin label, i.e., oligomers. TRV101 inhibits biotinylated amyloidβ (1-40) oligomerization with an IC50 of 5μM Animal models APP/PS1 (amyloid-β model), n=13 The mice were dosed at 30mg/kg (p.o., qd) commencing at 6 weeks of age with behavioral testing (2d-RAWM and fear conditioning) at ~3.5 months of age followed by ex vivo LTP analysis performed at the CA3/CA1 synapse in the hippocampus.