Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.

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A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosylinsoluble assemblies of wild-type human alpha-synuclein (α-Syn) upon differentiation with retinoic acid was used to study the impact of α-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced α-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. α-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating α-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 α/β in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that α-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.

Many tumors use tryptophan (Trp)-catabolizing enzymes such as Indoleamine/Tryptophan-2,3-dioxygenases (IDO1/TDO2) to induce an immunosuppressive environment by depleting Trp, resulting in excessive generation of kynurenine (Kyn) pathway metabolites. IDO1 inhibitors in combination with immune-checkpoint inhibitors are gaining success in multiple cancer clinical trials. This, along with accruing evidences in literature, further strengthens the dual inhibition of IDO1/TDO2 as a highly effective therapeutic prospect. Herein, we describe the discovery of fused heterocycle based analogue EPL-1410 as a novel orally active dual inhibitor of IDO1/TDO2. In the biochemical and IFNγ stimulated HeLa cell based assays, EPL-1410 inhibited human IDO1 with IC(50) values of 320 nM and 23.6 nM respectively. EPL-1410 also inhibits human TDO2 with an IC(50) of 516 nM in the biochemical assay. Further studies of EPL-1410 in various ‘in vitro ADMET' screens (e.g., Metabolic stability, plasma protein binding, hCYP inhibition, hERG, Cerep Safety Screen 44) provided an optimal metabolic stability, free drug concentration fraction and safety profile necessary to qualify it as a lead candidate. The pharmacokinetic profile of EPL-1410 showed an excellent absolute oral bioavailability (%F~100%) in mice and rats. In addition, the characterization of EPL-1410 in a battery of DMPK studies including PK profile in higher species and preliminary safety data will be discussed. EPL-1410 showed a significant dose dependent pharmacological efficacy in reducing the tumor volume in mouse syngeneic cancer models of CT-26 colon carcinoma and B16F10 melanoma. A good correlation of efficacy to biomarker (Kyn/Trp) ratio was observed in plasma, tumor draining lymph node and tumor tissue with no treatment related adverse clinical signs and body weight reduction at all doses of EPL-1410 tested. The data presented will establish EPL-1410 as a novel, orally active and potent dual inhibitor of IDO1/TDO2 with desired pharmaceutical properties for further development as a potential immuno-oncology therapeutic intervention. Citation Format: Srinivas Gullapalli, Abhijit Roychowdhury, Tushar Khaladkar, Sangameshaver Sawargave, Ravindra Janrao, Vijay Kalhapure, Ganesh Urunkar, Jayanarayan Kulathingal, Ramamohan Reddy Lekkala, Sonali Bhadra, Pankaj Kumar Singh, Krishna Anand Putta, Sonali Manwatkar, Prashant Surve, Mukund Keshav Gurjar. EPL-1410, a novel fused heterocycle based orally active dual inhibitor of IDO1/TDO2, as a potential immune-oncology therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1701.