Developing small molecule inhibitors of Pleckstrin-2 to treat thrombosis

PROJECT SUMMARY Thrombosis, often at unusual sites such as splanchnic vein and arteries, is common complication and one of the leading causes of mortality and morbidity in patients with chronic myeloproliferative neoplasms (MPNs). Developing effective therapies for thrombosis in MPNs is in its nascence. Current first-line therapy of chronic phase MPNs includes aspirin, hydroxyurea, interferon a, or anagrelide. However, these treatment approaches remain suboptimal with ongoing risks for thrombosis, hemorrhage, and impaired quality of life. Targeted molecular therapy at this stage of MPNs for thrombosis is also lacking. Aplexis, Inc is a startup company focusing on the development of new approaches to treat thrombosis in the chronic phase of MPNs, especially targeting the downstream effectors of the JAK2 pathway that is commonly activated in these disorders. One of these effectors is Pleckstrin-2 (Plek2) that is a novel target of the JAK2-STAT5 pathway. Previous studies have shown that loss of Plek2 ameliorated JAK2V617F mutant-induced myeloproliferative phenotypes, and reverted thrombosis and lethality of the JAK2V617F MPN mouse model. Importantly, Plek2 is overexpressed in the bone marrow and peripheral blood mononuclear cells from JAK2V617F positive chronic MPN patients. Given the significance of Plek2 in thrombosis pathogenesis in MPNs, the Ji laboratory at Northwestern University has identified hit compounds of Plek2 small molecule inhibitors using in silico-based high-throughput screenings and cell-based assays. Preliminary data show that the hit compounds significantly inhibit Plek2’s functions on erythroblast proliferation and lamellipodia formation in vitro. The hit compounds also dramatically reduce myeloproliferation and thrombosis in vivo in MPN mouse models. Preliminary mechanistic studies reveal that Plek2 functions as a scaffold protein to recruit PI3K effector proteins and enhances PI3K-Akt signaling. Plek2 inhibitors bind to Plek2 and disrupt this recruitment, which blocks PI3K-Akt signaling and inhibits cell hyperproliferation. Together, these findings lead us to hypothesize that Plek2 inhibitors block cell hyperproliferation in vitro and in vivo and prevent thrombosis in MPNs. The goal of this Phase I SBIR project is to establish proof-of-principle evidence for the therapeutic effects of Plek2 inhibitors in thrombosis in MPNs. In Aim 1, we propose to develop lead compounds with potent inhibitory effects of Plek2-induced cell proliferation in vitro using medicinal chemistry and cell-based assays. In Aim 2, we will examine the efficacy, pharmacokinetics, and toxicity of lead compounds in pre-clinical MPN mouse models with thrombosis. Aplexis has a strong support from Northwestern University’s Innovation and New Ventures Office (INVO) on an exclusive license for the associated patent-pending intellectual property from this technology. The collaboration with the Ji laboratory at Northwestern University will ensure the success of the proposed research, which will position Aplexis to the next step in the production of clinical candidate of Plek2 inhibitors and pre-IND investigation in Phase II.