Jianing Huang

Drug discovery in the ubiquitin regulatory pathway.

The ubiquitin system has been implicated in the pathogenesis of numerous disease states, including oncogenesis, inflammation, viral infection, CNS disorders and metabolic dysfunction. Ubiquitin conjugation and deconjugation to substrate proteins is carried out by multiple families of proteins, each with a defined role in the enzymatic cascade. This conjugation-deconjugation system parallels the kinase-phosphatase system in that both alter protein function by the addition and removal of post-translational modifiers. Our understanding of ubiquitin biology and strategies to interfere pharmacologically with the ubiquitin regulatory machinery is progressing rapidly. In light of increased interest in ubiquitin pathways as drug targets, we review the ubiquitin enzymatic cascades, highlighting therapeutic opportunities and enzymatic mechanisms. We also discuss the challenges of targeting this class of enzymes with small molecules, as well as current approaches and progress in drug discovery.

Cell cycle regulatory E3 ubiquitin ligases as anticancer targets

Disregulation of the cell cycle and proliferation play key roles in cellular transformation and tumorigenesis. Such processes are intimately tied to the concentration, localization and activity of enzymes, adapters, receptors, and structural proteins in cells. Ubiquitination of these cellular regulatory proteins, governed by specific enzymes in the ubiquitin (Ub) conjugation cascade, has profound effects on their various functions, most commonly through proteasome targeting and degradation. This review will focus on a variety of E3 Ub ligases as potential oncology drug targets, with particular emphasis on the role of these molecules in the regulation of stability, localization, and activity of key proteins such as tumor suppressors and oncoproteins. E3 ubiquitin ligases that have established roles in cell cycle and apoptosis, such as the anaphase-promoting complex (APC), the Skp-1-Cul1-F-box class, and the murine double minute 2 (MDM2) protein, in addition to more recently discovered E3 ubiquitin ligases which may be similarly important in tumorigenesis, (e.g. Smurf family, CHFR, and Efp), will be discussed. We will present evidence to support E3 ligases as good biological targets in the development of anticancer therapeutics and address challenges in drug discovery for these targets.

Substrate Modification with Lysine 63-linked Ubiquitin Chains through the UBC13-UEV1A Ubiquitin-conjugating Enzyme

Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys63 of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys63-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys63-linked ubiquitin chain synthesis mechanism.

A Novel E3 Ubiquitin Ligase TRAC-1 Positively Regulates T Cell Activation

TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn2+-chelating ability of its amino-terminal RING finger domain abolished TRAC-1’s ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation.

High-throughput screening for inhibitors of the e3 ubiquitin ligase APC.

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the securin protein and mitotic cyclins, resulting in the regulation of the onset of sister-chromatid separation and mitotic exit. In an effort to identify novel therapeutic compounds that modulate cell proliferation and, therefore, have potential applications in oncology, a plate-based in vitro ubiquitination assay that uses recombinant purified E1, E2 (UbcH5c), E3 (APC11/APC2), and Flag-ubiquitin has been established and used to screen for small molecule inhibitors of APC E3 ligase activity. In this assay, APC2/APC11 is immobilized on the plate, and its E3 ligase activity (i.e., the incorporation of Flag-tagged polyubiquitin chain onto APC2/APC11 as a result of auto-ubiquitination) is detected with anti-Flag-horseradish peroxidase-conjugated antibody by monitoring the luminescence signal from the plate. Here we describe in detail the protocol for high-throughput screening of APC, including expression and purification of the individual proteins, assay development, and optimization. This assay has been validated in a 96-well plate format and successfully implemented to identify novel small molecule compounds that potently inhibit APC2/APC11 ligase activity.

R-253 Disrupts Microtubule Networks in Multiple Tumor Cell Lines

Purpose: The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments. Experimental Design: A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC50, 20 nmol/L) when examined in a spectrum of tumor cell lines. Results: R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G2-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes. Conclusion: Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.

Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction.

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines.

A Homogeneous FRET Assay System for Multiubiquitin Chain Assembly and Disassembly

Ubiquitin (Ub, 76aa) is a small highly conserved protein present universally in eukaryotic cells. Covalent attachment of (Ub)(n) to target proteins is a well-known posttranslational modification that has been implicated in a wide array of cellular processes including cell biogenesis. Ubiquitin polymerization by the Ub activation-conjugation-ligation cascade and the reverse disassembly process catalyzed by Ub isopeptidases largely regulate substrate protein targeting to the 26S proteasome. Ub chains of four or more subunits attached by K48 isopeptide linkages have been shown to be necessary for the 26S proteasome association and subsequent degradation of protein molecules. To better understand this protein degradation event, it is important to develop Ub polymerization and depolymerization assays that monitor every reaction step involved in Ub attachment to, or detachment from, substrate protein molecules. In this chapter, we describe homogeneous, easy-to-use, nonradioactive, complementary continuous fluorescence assays capable of monitoring the kinetics of Ub chain formation by E3 Ub ligases, and their hydrolysis by isopeptidases, which rely on mixing a 1:1 population of fluorophore-labeled Ub molecules containing a FRET pair. The proximity of fluorescein (donor) and tetramethylrhodamine (acceptor) in Ub polymers results in fluorescein quenching on ligase-induced Ub chain assembly. Conversely, a dramatic enhancement of fluorescein emission was observed on Ub chain disassembly because of isopeptidase activity. These assays thus provide a valuable tool for monitoring Ub ligase and isopeptidase activities using authentic Ub monomers and polymers as substrates. Screening of a large number of small molecule compound libraries in a high-throughput fashion is achievable, warranting further optimization of these assays.

Abstract 3021: Development of small molecule direct AMPK activators for the treatment of cancer

5’-AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and is critical for maintaining energy homeostasis under conditions of nutrient stress. During cellular transformation, metabolic reprogramming enables the aberrant growth and proliferation of tumor cells. Both positive and negative roles for AMPK in tumor cell proliferation and survival have been reported. However, only a limited number of studies addressed this question with potent direct AMPK activators. AMPK exists as heterotrimers composed of the catalytic subunit α and reguratory subunits β and γ. We expressed the full length of all three human AMPK subunits in insect cells, purified the heterotrimer complexes, and used them for biochemical screening and characterization of AMPK activators. The purified complexes displayed basal activity, which was further enhanced by AMP. The compounds we identified potently activated the complexes in vitro at AC(2X)s (the concentration that gives a twofold activation) of 0.001-0.3 μM. Importantly, the compounds up-regulated substrate phosphorylation (pS79 Acetyl-CoA Carboxylase) and/or auto-phosphorylation (pT172 AMPKα) in multiple cancer cell lines including HepG2 hepatoma cells, A549 liver kinase B1 (LKB1) null lung cancer cells, and MOLM14 myeloid leukemia cells, indicating activation was irrespective of functional status of LKB1, which is a key AMPK-activation kinase. Activation of AMPK by the compounds was also confirmed using native AMPK isolated from normal tissues and tumor cells. We further investigated anti-proliferative effects of the compounds and found that up-regulation of AMPK kinase activity was correlated with anti-proliferative effects in A549 and MOLM14, but not in HepG2, suggesting that positive effects of direct AMPK activators could be cell-type dependent. Interestingly, we identified compounds that display comparable AMPK activation in HepG2 and A549 yet possessed divergent activities on proliferation across a panel of tumor lines. Analysis of cellular signaling across several of these tumor lines with this set of the compounds revealed dose-dependent effects on mTORC1 substrates, feedback signaling to PI3K and mTORC2, and inhibition of kinases downstream of RAF. Direct activation of AMPK could be a good therapeutic strategy for the treatment of subsets of cancers. Citation Format: Yasumichi Hitoshi, Yonchu Jenkins, Yingwu Li, Elmer Sampang, Xiang Xu, Guodong Dong, Jianing Huang, Nan Lin, Dane Goff, Simon Shaw, Luke Boralsky, Rajinder Singh, Sarkiz D. Issakani, Donald G. Payan. Development of small molecule direct AMPK activators for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3021.