Discovery of SB-497115, a Small-Molecule Thrombopoietin (TPO) Receptor Agonist for the Treatment of Thrombocytopenia.
Authors: Luengo Juan I. , Duffy Kevin J. , Shaw Anthony N. , Delorme Evelyne , Wiggall Kenneth J. , Giampa Leslie , Liu Nannan , Smith Heather , Tian Shin-Shay , Miller Stephen G. , Keenan Richard M. , Rosen Jon , Dillon Susan B. , Lamb Peter , Erickson-Miller Connie L. 
Published: April 2020

Thrombopoietin (TPO) is a 332 amino acid cytokine that plays a key role in the regulation of megakaryopoiesis and platelet production. Over the past 10 years, recombinant forms of TPO have shown various degrees of effectiveness in the treatment of thrombocytopenias associated with chemotherapy or various disease states, although therapy with these agents may be associated with a risk of producing neutralizing antibodies. A small-molecule nonpeptidyl TPO receptor agonist is expected to offer a safer alternative to these protein agents and also offer advantages in terms of lower cost of production and an easier oral route of administration. The mode of action of TPO involves oligomerization of its specific cell-surface receptor followed by activation of multiple signaling proteins such as the JAK1/JAK2 kinases and the STAT transcription factors. Using a high-throughput reporter-gene assay based on activation of STATs in BAF-3/TPO-R cells, screening of a library of 260,000 small-molecule compounds resulted in the identification of diazo naphthalenesulfonic acids as a novel series of TPO-R agonists. Modification of the core structure and adjustment of unwanted functionality resulted in the development of hydrazino naphthalenesulfonates which displayed efficacies equivalent to those of TPO in several cell-based assays, such as luciferase reporter gene and proliferation in a TPO receptor-dependent way (i.e., no effect on TPO-insensitive cell lines). Furthermore, these compounds elicited signal-transduction responses, such as JAK2 and STAT-5 activation, in TPO-receptor-expressing cells similar to those in TPO itself, and promoted megakaryocyte differentiation in cultures of human bone marrow cells. Potencies for the best compounds in the series were quite high for such small molecules, with EC50 values in the 10–100 nM range. However, the compounds were devoid of oral bioavailability, presumably due to the highly polar sulfonic acid functionality. Molecular modeling studies of three different series of agonists suggested a number of potential replacements of the sulfonate group, which resulted in the development of the biphenyl carboxylates, a series of compounds that showed excellent pharmacokinetic parameters, including oral bioavailability. Out of a total of over two hundred analogs, SB-497115 was selected as the candidate for clinical studies due to its optimum biological and PK properties: the compound showed full maximal efficacy of TPO both in the proliferation of BAF-3/TPO-R cells (EC50 = 30 nM) as well as in the increase of the number of CD41+ cells, a marker of megakaryocyte differentiation, in cultures of human bone marrow cells (EC50 = 100 nM). Oral bioavailabity of SB-497115 was determined to be 26%, 83% and 89%, for rat, dog and monkey, respectively. The identification of SB-497115 (MW of 442) as a TPO mimetic provides proof of principle for drug discovery using JAK/STAT based assays, and shows for the first time that a small non-peptidyl molecule can trigger the selective activation of a cytokine receptor which is confirmed by the elevation in platelet numbers in human volunteers, as demonstrated in Phase I clinical studies. Evaluation of the potential of SB-497115 as an agent for the treatment of thrombocytopenia in humans is ongoing.