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  • br Acknowledgments br Introduction Ubiquitin ligases limit t

    2020-01-16


    Acknowledgments
    Introduction Ubiquitin ligases limit the stability and activity of substrates by targeting them for proteasomal-dependent degradation. Siah is a two-member family of ubiquitin ligases implicated in control of key cellular processes. Among those, Siah ligases control prolyl hydroxylases, which regulate HIF1α and ATF4 stability [[1], [2], [3]]. Inhibition of Siah1/2 attenuates prostaglandin e of HIF1α, resulting in attenuated tumor development and metastasis in melanoma [4] and inhibits neuroendocrine (NE) prostate tumor development [4,5]. Through its regulation of the co-repressor NCOR1, Siah2 controls transcription of select androgen receptor-regulated genes that contribute to development of hormone refractory prostate cancers (PCa) [6]. Siah2 also determines ATF4 levels with a concomitant effect on cell death programs following stress, as occurs in ischemic conditions [3]. Moreover, Siah2 is implicated in control of mitochondrial fission, which impacts cell death under stress conditions, such as those associated with heart infarction [7]. In all cases, blocking Siah activity appears to alleviate pathological conditions – from ischemia to tumor development and metastasis – making it a high prostaglandin e value drug target. There have been numerous attempts to inhibit Siah2 ubiquitin ligase activity, including application of a meso-scale-based screening that led to identification of menadione [8]. A structure-based approach enabled identification of short peptides that covalently bind Siah2 and inhibit its ubiquitin ligase activity [9]. The crystal structure of Siah2-bound peptides has been solved, revealing a newly defined structural pocket. Siah2 inhibitory peptides were found to significantly suppress Siah2 ubiquitin ligase activity in cell culture and in vitro [9]. Yet, the utilization of such peptides has been largely limited to cultures, limiting further characterization in vivo.
    Materials and methods
    Results
    Discussion Ubiquitin ligases, which regulate signaling in numerous cell contexts, are highly desirable targets in medicine, as their deregulation results in disease-related phenotypes [30,31]. Yet, ubiquitin ligases are also challenging drug targets, given that with the exception of HECT domain family of ligases, RING finger ubiquitin ligases lack catalytic activity and their inhibition likely requires the disruption of specific protein-protein interactions. Among two members of the Siah ubiquitin ligases family, Siah2 has been linked to oncogenic activities and shown by independent laboratories to function in melanoma, breast, and prostate cancers [4,5,18,32,33]. Siah2 inhibition reportedly attenuates melanoma growth, development, and metastatic capacity and antagonizes formation of neuroendocrine prostate cancer and evolution of hormone-refractory prostate tumors, albeit by distinct mechanisms. Thus, inhibiting Siah2 is desirable in these conditions. Our earlier attempts to inhibit Siah ubiquitin ligases have included a Meso-scale based analysis (Meso Scale Discovery) that identified menadione as Siah1/2 inhibitory small molecule. Later, using a structure-based approach, we designed small peptides that effectively inhibited Siah by covalent binding, with proven effect in vitro and in cultured cells. Each inhibitor was also capable of decreasing viability of both melanoma and to a greater extent prostate cancer cultures and decreasing CFE, indicative of a positive anti-tumor effect. The most effective compound identified in the 3 campaigns was adapalene/RLS-24 and its analogue (RLS-30), both of which promoted effective cell death at nanomolar concentrations, effectively inhibited Siah2 ubiquitin ligase activity in vitro, and altered HIF1α activity in a PHD-dependent manner. At times, inhibitors were more potent in prostate cancer cells compared with melanoma, and others, melanoma better then prostate cancer cells. Among the plausible explanations are the differential activity of Siah1/2 genes in the different tumor cells, and correspondingly, different affinity of RLS\'s to Siah1 vs. Siah2. The latter is supported by the observation that certain RLS are more effective in Siah1 KO compared with Siah2 KO MEFs and vice versa, requiring further assessment of RLS to each of the Siah proteins.