Immune evasion is a hallmark feature of tumors as they employ various strategies to suppress the immune system's ability to recognize and destroy cancer cells. Significant advances in the understanding of the mechanisms of cancer immune evasion have led to the successful development of immunotherapies that exhibit clinical efficacy against hematological as well as solid tumors. These include monoclonal antibodies, allogeneic hematopoietic stem cell transplantation, vaccination, cytokines, T cell checkpoint blockade, and adoptive transfer of engineered T cells. Due to the underlying complexity of cancer immune evasion, however, several different approaches will likely be necessary for effective therapeutic management. We are taking a dual targeting approach, focused on the ubiquitin proteasome pathway, to develop novel small molecule cancer immunotherapeutics. We have identified inhibitors of a deubiquitylase (DUB) that is highly expressed in regulatory T (Tregs) cells and plays a critical role in promoting Treg functions. In addition, we have identified inhibitors of an E3 ubiquitin ligase that negatively regulates T‐effector cell function. These inhibitors are expected to suppress Treg functions and promote T‐effector activities. A combination of such inhibitors should provide powerful anti‐cancer immunotherapy.

Despite widespread use of statins and other therapeutics, hypercholesterolemia remains a significant medical issue for a many patients unable to effectively regulate cholesterol levels. Cholesterol uptake is primarily mediated by the hepatic low density lipoprotein receptor (LDLR), which binds and internalizes plasma LDL. Elevated LDLR is associated with reduced LDL levels. Recently, the E3 ligase Idol (Inducible Degrader of LDLR) was shown to be a key regulator of LDLR levels. IDOL is a unique E3 ligase that utilizes its FERM domain to specifically target LDLR for polyubiquitylation and subsequent lysosomal degradation. Genetic ablation of IDOL raises levels of LDLR. Thus, inhibition of IDOL may be beneficial for the treatment of hypercholesterolemia. The ubiquitin‐proteasome system is a rich landscape for drug discovery. E3 ligases in particular are attractive therapeutic targets in various disorders, including metabolic disorders. Nevertheless, it has been challenging to discover and develop E3 ligase inhibitors as first in class clinical candidates. Here, we report the identification of novel IDOL inhibitors that modulate cellular cholesterol homeostasis. These compounds increased LDLR levels and increased LDL association in various cellular models. Biophysical characterization revealed direct binding of the compounds to IDOL and perturbation of IDOL:LDLR interactions. The most promising compounds were used as starting points to develop novel drug like molecules and the lead compounds are being evaluated in translational models of hypercholesterolemia. Data will be presented summarizing our progress to date targeting IDOL for the treatment of hypercholesterolemia. Work supported in part by NIH grant HL127893.

Abstract The degradation of most cellular proteins is regulated by coordinated addition and removal of ubiquitin by families of ubiquitin E3 ligases and deubiquitylating enzymes (DUBs) respectively. DUBs proteolytically cleave ubiquitin molecules from proteins resulting in modifications of protein activity, localization and function. Several DUBs are aberrantly regulated in cancer, including the best studied, USP7, selective inhibitors of which are active in cancer models. USP22, is another validated anticancer target, being one of 11 genes in the death-from-cancer gene signature, a component of the human SAGA transcriptional cofactor complex regulating myc transcription, and a regulator of the expression of p21, the histone deacetylase Sirt 1, and p53 activity. USP22 is overexpressed in oral squamous cell carcinoma, breast, non-small cell lung, colorectal, and other cancers and its expression is inversely correlated with survival. Unlike most other DUBs, USP22 exhibits robust activity only as a component of a multi-subunit complex. Initial studies reported activity solely as a member of the 2MDa SAGA complex, but more recently it has been demonstrated that USP22 exhibits similar activity in a four-protein DUB module derived from the SAGA complex. USP22 inhibitors are expected to have broad anti-cancer activities. Progenra's UbiProTM discovery platform was utilized to screen ∼200K member diversity based library of small molecules for identifying novel USP22 inhibitors. Data will be presented describing these results. Citation Format: Feng Wang, Timothy R. Stanek, Leelabati Biswas, Matthew Kodrasov, James LaRocque, Jian Wu, David Sterner, Joseph Weinstock, Michael Mattern, Steven B. McMahon, Suresh Kumar. Discovery and characterization of USP22 inhibitors as novel anti-cancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5432. doi:10.1158/1538-7445.AM2015-5432

Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates.

The alpha 7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 ( 3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b']-di pyrrole-1(2H)-carboxamide),a compound known as a 5-hydroxytryptamine(2B/2C) receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC20 concentration of nicotine and a corresponding EC50 of 1.5 mu M for potentiation of EC20 nicotine responses in GH4C1 cells expressing the alpha 7 receptor. SB-206553 was devoid of direct alpha 7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the alpha 7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC20 (17 mu M) and EC100 (124 mu M) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 mu M), an effect that was entirely blocked by the alpha 7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 mu M. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of alpha 7 nAChR PAMs in schizophrenia.

Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the G protein coupled and nuclear hormone receptor families. A historical limitation on ion channel targeted drug discovery in the form of the extremely low throughput nature of the gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, has been overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. These have taken advantage of various approaches to monitor ion flux or membrane potential using radioactive, non-radioactive, spectroscopic and fluorescence measurements and have significantly impacted both high-throughput screening and lead optimization efforts. In addition, major advances have been made in the development of automated electrophysiological platforms to increase capacity for cell-based screening using formats aimed at recapitulating the gold standard assay. This review addresses the options available for cell-based screening of ion channels with examples of their utility and presents case studies on the successful implementation of high-throughput screening campaigns for a ligand-gated ion channel using a fluorescent calcium indicator, and a voltage-gated ion channel using a fluorescent membrane potential sensitive dye.

Cell-based assays for identification of biologically active small molecules from chemical libraries are becoming increasingly popular for HTS aimed at a variety of drug targets. Functional assays require good coordination between several independent processes during the run. This article describes our custom designed, fully automated Thermo LAS robotic system with integrated FLIPRTETRA and several additional peripheral elements such as a BioTek ELX washer unit, a PE Evolution pipettor, and PE FlexDrop dispenser. The Thermo LAS robotic control software, Polara, ensures that each and every plate of sensitive cells in a large batch experiences the same procedure as an individual plate assayed in the hands of a scientist. Such robotic systems can process hundreds of plates a day and require large-scale automated support for cell preparation. The TAP SelecT is an automated robotic system that can plate 100– 300 plates of cells per day with defined accuracy and precision. In addition to plating cells, SelecT can also pass and expand cell lines. Here, we present a case study of a GPCR-mediated Ca-flux assay, where this robotic team enables high-throughput logistics even for an extremely sensitive cell-based assay.

The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289kDa) and a truncated TOR (132kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (Km) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8μM, respectively. Dose–response and inhibition mechanisms of several known inhibitors, the rapamycin–FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors.

In response to diverse stimuli, the transcription factor NF-κB is activated by the IKK kinase complex containing two kinases (IKKα and IKKβ) that phosphorylate IκB, an inhibitory protein of NF-κB. The phosphorylation of IκB results in ubiquitination and degradation of IκB, allowing NF-κB to translocate to the nucleus where it regulates its target genes. To elucidate the role of IKK in the NF-κB signaling pathway, we have developed and characterized two quantitative, sensitive, and nonradioactive assays for evaluating IKKβ activity: a dissociation-enhanced lanthanide fluorescence immunoassay called DELFIA and a homogeneous time-resolved fluorescence resonance energy transfer assay called LANCE. We show that the two assays have similar sensitivity and Michaelis constants (Km) for adenosine 5′-triphosphate and substrate; however, the LANCE format was far more efficient and easier to perform. Additionally, the assays were validated with the known kinase inhibitor K252a and several other kinase inhibitors, which showed that the IC50 values of the two assays were comparable. In summary, both assays are quantitative, sensitive, reproducible, and amenable to high-throughput screening with improved waste management over radioactive assays.

ABSTRACT Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.