Enzyme Fragment Complementation Research Articles

Abstract 3045: PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer

Abstract Peroxisome proliferator activated receptor alpha (PPARα) is a transcription factor and master regulator of fatty acid oxidation. PPARα ligands modulate the switch between co-activator and co-repressor transcription complexes at PPARα-controlled target genes. In addition, PPARα has been shown to transrepress NF-κB signaling and inhibit angiogenesis. Renal cell carcinoma (RCC) expresses high levels of PPAR-α and is a highly angiogenic cancer. Current frontline treatments for RCC include chemotherapy, anti-angiogenics, and immunotherapy but are limited by the immune suppressive state of the tumor microenvironment. TPST-1120 is a novel PPARα antagonist for the treatment of cancer patients that binds in the ligand binding domain, positioning the AF-2 activation helix in an inactive conformation. The inactive conformation has a lower affinity for co-activator motifs and reduces activation of PPARα regulated genes. In a CHO-based reporter cell line, TPST-1120 potently competed against the PPARα-specific agonist GW7647 (IC50 = 36 nM at 20 nM GW7647) and an endogenous PPARα agonist oleoylethanolamide (OEA; IC50 = 15 nM at 30 µM OEA). Furthermore, in an enzyme fragment complementation assay, we demonstrated that TPST-1120 inhibited agonist-induced binding of PPARα to MED1 by destabilizing the co-activator complex thereby stabilizing the inactive conformation of PPAR-α. In a murine model of renal cell adenocarcinoma (RENCA), TPST-1120 treatment for 12 days (30 mg/kg qd) reduced tumor growth by 52% as a monotherapy (p<0.0001). Consistent with these findings, TPST-1120 inhibited tumor angiogenesis and proliferation as quantified by Ki67 levels. Combination treatment with current frontline therapeutics resulted in synergistic tumor inhibition of 74% with anti-PD1 (200 ug Q3D for 12 days) and 81% with cabozantinib (15 mg/kg QD for 15 days) (p<.0001). Flow cytometry-based analysis of tumors demonstrated increased CD8+ T cell infiltration in the TME as well as decreases in M2 and elevations in M1 macrophages. Importantly, we demonstrate that TPST-1120 can reverse an immunosuppressive class of immune cells to promote anti-tumor immunity and anti-angiogenesis in kidney cancer in the absence of overt toxicity. These data support advancement of TPST-1120 into RCC. Citation Format: Michael Gillespie, Valerie Chen, Isabella Howard, Keira Smith, Dave Freund, Nathan Standifer, Dara Burdette, Thomas W. Dubensky, Sam Whiting, Dipak Panigrahy. PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3045.

Abstract 3932: Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is)

Abstract CC-99282 is a novel CELMoD® agent that was optimized for activity in non-Hodgkin lymphoma (NHL). CC-99282 potently degrades Ikaros and Aiolos, resulting in enhanced antiproliferative, apoptotic, and immune-stimulatory activity in diffuse large B-cell lymphoma (DLBCL) models, including those with acquired chemoresistance (Lopez-Girona A, et al. Hematol Oncol. 2021). In lymphocytes, Ikaros negatively regulates gene expression via histone modifications, including polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) (Oravecz A, et al. Nat Commun. 2015); in NHL, these epigenetic mechanisms are unclear. Using DLBCL models, we explored the relationship between CC-99282 activity and epigenetic status, and the mechanism of synergy of CC-99282 treatment and inhibition of EZH2, a PRC2 component. Baseline characteristics of human DLBCL lines and effects of CC-99282 ± tazemetostat (TAZ), a representative EZH2i, were evaluated in vitro and in vivo by chromatin immunoprecipitation sequencing, gene expression, flow cytometry, immunoblotting, enzyme fragment complementation assays, and/or CRISPR/Cas9 gene editing. Analysis of Ikaros/Aiolos degradation in > 20 DLBCL cell lines showed their loss is necessary, but not sufficient for CC-99282 efficacy. Evaluation of baseline histone marks showed that the sensitive cell lines exhibit aberrant, higher H3K27me3 coverage at promoter regions of expressed genes. This suggests a direct correlation between H3K27me3 status at these regions and CC-99282 sensitivity. In T cells, Oravecz et al. found that loss of Ikaros reduces H3K27me3 at specific chromatin sites due to its interaction with PRC2. We confirmed that these affected regions are enriched in genes upregulated upon CC-99282 treatment in DLBCL cell lines, suggesting a similar role for Ikaros in DLBCL. Pathway analysis following CC-99282 or TAZ treatment demonstrated high overlap between pathways altered by both agents. CC-99282 + TAZ combined demonstrated additive and/or synergistic antiproliferative and apoptotic effects in DLBCL cell lines. This combination did not alter Ikaros degradation or overall H3K27me3 status but increased downstream effects. Results were confirmed by CRISPR/Cas9 knockout competition assays with EZH2 and other PRC2 components. This effect was independent of cell of origin, EZH2 mutational status, or degree of CC-99282 sensitivity. Synergy was confirmed using the SU-DHL6 and DB xenograft models that are intrinsically resistant to EZH2is and CC-99282, respectively. Combination treatment yielded durable responses and tumor-free animals. Collectively, these data suggest that Ikaros could act as an epigenetic modulator through PRC2 recruitment and support the combination of CC-99282 with EZH2is in NHL (NCT03930953) to favor broad and durable clinical responses. Citation Format: Soraya Carrancio, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, Diana Jankeel, Adam Blattler, Preethi Janardhanan, Matthew Groza, Jim Leisten, Rama Krishna Narla, Antonia Lopez-Girona, Daniel W. Pierce. Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3932.

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms.

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a β-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of β-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4, potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.

Functionality of Melatonin Receptors: Internalization.

The main step of classical desensitization of a receptor, by mean of its disappearance from the plasma membrane, is its internalization. This is a key factor in the regulation of agonist-mediated signaling pathways, as it most of the time stops the activation of the receptor. Internalization is thus important to evaluate, as a complementary information for a natural ligand or an alternative synthetic agonist. Enzyme fragment complementation is an elegant but delicate way to measure this phenomenon, by fusing two complementary parts of an enzyme to two partners, and to measure the activity of the reconstituted enzyme upon complexation of the partners. In the present chapter, using two parts of β-galactosidase, one fused to the C-terminus of the MT1 receptor, the other to an endosomal protein, one can measure the formation of the complex; thus, the transfer of the receptor to the endosome from which MT1 will be recirculated.

Functionality of Melatonin Receptors: Recruitment of β-Arrestin at MT1.

The main process of downregulation of G protein-coupled receptors is desensitization by which the receptor is extruded from the plasma membrane and directed to the endosomal compartment for recycling. Typically, the first step of this phenomenon consists in the recruitment of the protein β-arrestin induced by the agonist. Melatonin receptors undergo the same process: melatonin leads to the recruitment of β-arrestin and is subsequently sent away from the membrane, leading to a de facto stop of the melatonin receptor-mediated G protein signaling, because the receptors are not at the membrane level to receive the message brought by melatonin. The way one can measure this recruitment is based on the elegant technique of enzyme fragment complementation by which two parts of an enzyme are fused to two partners and reform an active enzyme upon the formation of the complex between these two partners. The basic way to set up this technique is presented here.

NanoLuc-Based Methods to Measure β-Arrestin2 Recruitment to G Protein-Coupled Receptors.

Cytosolic β-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or β-arrestin recruitment makes β-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor β-arrestin2 recruitment to the human histamine H1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H1 receptor.

Pharmacological Activation of Nrf2 by Rosolic Acid Attenuates Endoplasmic Reticulum Stress in Endothelial Cells.

Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases.

Gymnema montanum improves endothelial function via inhibition of endoplasmic reticulum stress by activating Nrf2 signaling

Objective: To investigate the effects of Gymnema montanum leaf extract against endoplasmic reticulum (ER) stress-induced toxicity in endothelial cells. Methods: The immortalized endothelial hybrid cell, EA.hy926 was treated with different concentrations of Gymnema montanum leaf extract (0-100 μg/mL) and the ER stress inducer, tunicamycin. The cytotoxicity was assessed by MTT as well as lactate dehydrogenase and malondialdehyde levels were determined. The levels of ER stress markers, GRP78 and CHOP were analysed by Western blot assay. The Gymnema montanum leaf extract-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by cell-based luciferase enzyme fragment complementation assay and antioxidant responsive element driven luciferase reporter assay. The levels of phosphoproteins of the MAPK pathway were analyzed using the Bioplex system. Results: A dose-dependent cytoprotective effect of Gymnema montanum leaf extract was observed in tunicamycin-induced toxicity. Gymnema montanum leaf extract significantly reduced lactate dehydrogenase activity and malondialdehyde levels in ER stress-induced endothelial cells. It also suppressed ER stress markers dose dependently and inhibited the phosphorylation of JNK, ERK, MEK and p38 MAPK in tunicamycin-induced endothelial cells. Moreover, Gymnema montanum leaf extract increased the expression of Nrf2 and its downstream targets in endothelial cells. Conclusions: Gymnema montanum leaf extract attenuates ER stress by increasing the expression of Nrf2 and its downstream genes.

Identification of Cosalane as an Inhibitor of Human and Murine CC–Chemokine Receptor 7 Signaling via a High-Throughput Screen

CC–chemokine receptor 7 (CCR7) is a G protein–coupled receptor expressed on a variety of immune cells. CCR7 plays a critical role in the migration of lymphocytes into secondary lymphoid tissues. CCR7 expression, however, has been linked to numerous disease states. Due to its therapeutic relevance and absence of available CCR7 inhibitors, we undertook a high-throughput screen (HTS) to identify small-molecule antagonists of the receptor. Here, we describe a robust HTS approach using a commercially available β-galactosidase enzyme fragment complementation system and confirmatory transwell chemotaxis assays. This work resulted in the identification of several compounds with activity against CCR7. The most potent of these was subsequently determined to be cosalane, a cholesterol derivative previously designed as a therapeutic for human immunodeficiency virus. Cosalane inhibited both human and murine CCR7 in response to both CCL19 and CCL21 agonists at physiologic concentrations. Furthermore, cosalane produced durable inhibition of the receptor following a cellular incubation period with subsequent washout. Overall, our work describes the development of an HTS-compatible assay, completion of a large HTS campaign, and demonstration for the first time that cosalane is a validated CCR7 antagonist. These efforts could pave the way for new approaches to address CCR7-associated disease processes.

A Pharmacochaperone-Based High-Throughput Screening Assay for the Discovery of Chemical Probes of Orphan Receptors.

G-protein-coupled receptors (GPCRs) have varying and diverse physiological roles, transmitting signals from a range of stimuli, including light, chemicals, peptides, and mechanical forces. More than 130 GPCRs are orphan receptors (i.e., their endogenous ligands are unknown), representing a large untapped reservoir of potential therapeutic targets for pharmaceutical intervention in a variety of diseases. Current deorphanization approaches are slow, laborious, and usually require some in-depth knowledge about the receptor pharmacology. In this study we describe a cell-based assay to identify small molecule probes of orphan receptors that requires no a priori knowledge of receptor pharmacology. Built upon the concept of pharmacochaperones, where cell-permeable small molecules facilitate the trafficking of mutant receptors to the plasma membrane, the simple and robust technology is readily accessible by most laboratories and is amenable to high-throughput screening. The assay consists of a target harboring a synthetic point mutation that causes retention of the target in the endoplasmic reticulum. Coupled with a beta-galactosidase enzyme-fragment complementation reporter system, the assay identifies compounds that act as pharmacochaperones causing forward trafficking of the mutant GPCR. The assay can identify compounds with varying mechanisms of action including agonists and antagonists. A universal positive control compound circumvents the need for a target-specific ligand. The veracity of the approach is demonstrated using the beta-2-adrenergic receptor. Together with other existing assay technologies to validate the signaling pathways and the specificity of ligands identified, this pharmacochaperone-based approach can accelerate the identification of ligands for these potentially therapeutically useful receptors.

Effect of Biased GLP-1/Glucagon Receptor Co-agonists on Insulin Secretion

Introduction: The effect of dual GLP-1 and glucagon analogues on insulin secretion remains unclear. Here, we investigated the effect of novel GLP-1 / glucagon receptor (GLP-1R/GCGR) co-agonists on beta cell lines, finding augmented insulin release compared to reference agonist Exendin-4. This effect was related to signalling bias at the GLP-1R. Methods: GLP-1R/GCGR co-agonists were designed, based on the sequence of oxyntomodulin. INS-1 832/3 beta cells and MIN6B1 beta cells were used to perform 24 hour insulin secretion assays. Ligand-directed signal bias was determined from cyclic adenosine monophosphate (cAMP) and β arrestin-1/2 responses measured by enzyme fragment complementation in CHO-K1 cells expressing GLP-1R or GCGR. Results: Several co-agonists show enhanced maximal insulin release compared to Exendin-4 (representative co-agonist Peptide A vs. Exendin-4 fold insulin release, normalized to 11mM glucose: INS-1 823/3 cells 4.45 ± 0.24 vs. 2.55 ± 0.22, p = 0.0002; MIN6B1 cells 2.28 ± 0.22 vs. 1.57 ± 0.08, p = 0.002). At the GLP-1 receptor, co-agonists were biased towards cAMP generation, compared to the β arrestin-2 pathway (delta log (τ/KA) -0.70 ± 0.11 [Peptide A], vs. 0.29 ± 0.11 [Exendin-4], p= <0.0001). At the GCGR, no significant change in bias was seen relative to native glucagon. Conclusion: GLP-1R/GCGR co-agonists show enhanced insulin release in vitro compared to exendin-4. As canonical effects of β arrestin recruitment include desensitisation and loss of cell surface receptors via endocytosis, the observed signal bias with these peptides may be linked to their insulinotropic effect. These findings may inform the design of more potent insulinotropic compounds for therapeutic use in type 2 diabetes. Disclosure G. Farooq: None. B. Jones: None. J.S. Minnion: None. S.R. Bloom: None.

Mu Opioid Receptor Agonists with Dopamine D3 Receptor Antagonism: Toward Developing Analgesics with Less Abuse Potential

Opioid analgesics such as morphine are widely used in clinical practice for the management of moderate to severe pain. While they are the gold standard for painkillers, they are also highly addictive: approximately 10% of patients prescribed opioids for pain‐relief develop an opioid‐use disorder. Abuse of prescription opioids and heroin has become an epidemic in the United States with opioid overdose deaths quadrupling since 1999. Opioids induce their physiological effects through the activation of the mu opioid receptor (MOR). Interestingly, co‐administration of dopamine D3 (DRD3) receptor antagonists with opioids attenuates the self‐administration and rewarding effects of the drugs, but has no effect on their ability to relieve pain. This suggests that the development of compounds that both activate MOR and antagonize DRD3 may be a means to maintain the beneficial effects of opioids, while reducing their abuse potential. We have recently developed a library of small molecules with a novel scaffold that are agonists at MOR. Herein we screened our MOR agonists for antagonism at DRD3 in a DRD3/βarrestin enzyme fragment complementation assay. We identified multiple compounds that antagonize DRD3 that are as potent as Sulpiride, a known DRD3 antagonist. Further characterization of these MOR agonists with polypharmacology at DRD3 may allow for the development of opioid analgesics with reduced abuse liabilities.Support or Funding InformationFunded by NIDA grant R01 DA033073 to LMB Fellowship to NMS: NSF award 1359369. and TDB, and NSF Undergraduate ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation–based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

Homo- and Heterodimerization of Proteins in Cell Signaling: Inhibition and Drug Design.

Protein dimerization controls many physiological processes in the body. Proteins form homo-, hetero-, or oligomerization in the cellular environment to regulate the cellular processes. Any deregulation of these processes may result in a disease state. Protein-protein interactions (PPIs) can be inhibited by antibodies, small molecules, or peptides, and inhibition of PPI has therapeutic value. PPI drug discovery research has steadily increased in the last decade, and a few PPI inhibitors have already reached the pharmaceutical market. Several PPI inhibitors are in clinical trials. With advancements in structural and molecular biology methods, several methods are now available to study protein homo- and heterodimerization and their inhibition by drug-like molecules. Recently developed methods to study PPI such as proximity ligation assay and enzyme-fragment complementation assay that detect the PPI in the cellular environment are described with examples. At present, the methods used to design PPI inhibitors can be classified into three major groups: (1) structure-based drug design, (2) high-throughput screening, and (3) fragment-based drug design. In this chapter, we have described some of the experimental methods to study PPIs and their inhibition. Examples of homo- and heterodimers of proteins, their structural and functional aspects, and some of the inhibitors that have clinical importance are discussed. The design of PPI inhibitors of epidermal growth factor receptor heterodimers and CD2-CD58 is discussed in detail.

Abstract 3637: Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules

Abstract Introduction: Immunotherapy with the combination of monoclonal antibodies (mAbs) that block PD-1 (nivolumab) and CTLA-4 (ipilimumab) has shown clinical benefit beyond that observed with either mAb alone. We have developed PD-1 x CTLA-4 bispecific proteins aimed at inducing antitumor immunity through simultaneous blockade of both checkpoint molecules. Two proteins, a tetravalent PD-1 x CTLA-4 bispecific DART® protein (bivalent for both PD-1 and CTLA-4) and a trivalent PD-1 x CTLA-4 TRIDENT™ protein (bivalent for PD-1 but monovalent for CTLA-4) were engineered; the TRIDENT protein was designed to promote CTLA-4 blockade through avidity when bound to PD-1 on cells co-expressing both receptors, thus favoring PD-1 over CTLA-4 blockade in cells that do not co-express both molecules. Methods: Proteins were engineered from humanized PD-1 and CTLA-4 mAb sequences and demonstrated favorable biophysical properties. Binding assays were performed by ELISA or flow cytometry. Co-ligation of PD-1 and CTLA-4 was assessed in an enzyme-fragment complementation assay (DiscoverX). T-cell activation was tested in reporter cells, staphylococcus enterotoxin B-stimulated PBMCs or MLR assays. Results: Both DART and TRIDENT proteins showed equivalent potency in binding immobilized recombinant PD-1 or PD-1-expressing cell lines, inhibition of PD-1 interaction with PD-L1 or PD-L2 as well as reversal of PD-1/PD-L1 mediated T-cell signal inhibition. In all assays, both formats showed activities that were comparable to those of the precursor PD-1 mAb. With respect to CTLA-4, the DART protein showed a minor potency loss in binding to CTLA-4 expressing cells, inhibition of CTLA-4/B7 interaction and reversal of T-cell signal inhibition compared to the precursor mAb. The TRIDENT protein, however, showed substantial lower potency than the DART protein in all CTLA-4 assays, consistent with the monovalent nature of the CTLA-4 arm. Importantly, in cells that co-express both receptors, DART and TRIDENT proteins show comparable co-engagement of PD-1 and CTLA-4, as shown by enzyme-fragment complementation, suggesting that anchoring through PD-1 compensates for the decreased CTLA-4 avidity of the TRIDENT molecule when both target receptors are expressed. Similarly, in T-cell co-activation assays, both DART and TRIDENT proteins showed a comparable increase in IFN-gamma response that recapitulated that observed with the combination of the individual parental mAbs. Conclusion: Both PD-1 x CTLA-4 DART and TRIDENT bispecific molecules block PD-1 and CTLA-4 pathways, with the TRIDENT protein demonstrating a PD-1-biased binding preference, consistent with its design intended to reduce CTLA-4 blockade in the absence of PD-1 co-expression. Both molecules showed comparable T-cell activation activity. Further development of bispecific PD-1 x CTLA-4 molecules for cancer treatment is warranted. Citation Format: Gurunadh R. Chichili, Kalpana Shah, Ross La Motte-Mohs, Alex Berezhnoy, Daorong Liu, Jessica Hill, Leilei He, Christine Shoemaker, Jennifer Brown, Kurt Stahl, Hua Li, Jonathan Li, Sergey Gorlatov, Jim Tamura, Valentina Ciccarone, Ralph Alderson, Ezio Bonvini, Paul A. Moore, Syd Johnson. Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2017-3637

Literature Search and Review

The use of ultra-high-throughput screens is a well-accepted mechanism to identify agonists and antagonists of target receptors. We used the Path Hunter® (DiscoveRx, Fremont, CA) to screen the entire Merck compound library for glucocorticoid receptor (GR) agonists in a 2.2-μl total reaction volume assayed in a 3,456-well plate format. This single-addition, homogeneous assay, which utilizes the principle of enzyme fragment complementation (EFC) to detect nuclear translocation of GR, an initial step of receptor activation, was used to successfully screen a large library of small molecules as indicated by an average signal-to-background ratio of approximately fourfold and an average Z-factor value of 0.45. Hits from the HTS campaign were studied in a cytokine secretion assay in primary human monocytes to gain functional information regarding these compounds in a phenotypic and physiologically relevant setting. Our data indicate that using the PathHunter assay, we successfully identified compounds that showed ...

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay.

Novel Peptidomimetics for Inhibition of HER2:HER3 Heterodimerization in HER2-Positive Breast Cancer.

The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibodies or a combination of antibodies with traditional chemotherapeutic agents or kinase inhibitors. Our approach is to target clinically validated HER2 domain IV with peptidomimetics and inhibit the protein-protein interactions (PPI) of HERs. Unlike antibodies, peptidomimetics have advantages in terms of stability, modification, and molecular size. We have designed peptidomimetics (compounds 5 and 9) that bind to HER2 domain IV, inhibit protein-protein interactions, and decrease cell viability in breast cancer cells with HER2 overexpression. We have shown, using enzyme fragment complementation and proximity ligation assays, that peptidomimetics inhibit the PPI of HER2:HER3. Compounds 5 and 9 suppressed the tumor growth in a xenograft mouse model. Furthermore, we have shown that these compounds inhibit PPI of HER2:HER3 and phosphorylation of HER2 as compared to control in tissue samples derived from in vivo studies. The stability of the compounds was also investigated in mouse serum, and the compounds exhibited stability with a half-life of up to 3 h. These results suggest that the novel peptidomimetics we have developed target the extracellular domain of HER2 protein and inhibit HER2:HER3 interaction, providing a novel method to treat HER2-positive cancer.

Abstract 3742: Detection of EGFR, HER2, HER3 and HER4 homodimerization and heterodimerization using EFC-based cellular assays

Abstract Ligand-induced receptor dimerization is the first functional step in receptor signaling, representing the most proximal, functional read-out for receptor activation. Here we present a novel application of the Enzyme Fragment Complementation system to monitor receptor-receptor interactions at the surface of intact cells, applicable to diverse receptor types such as RTKs, Interleukin receptors, BMP receptors and cytokine receptors, amongst others. For the purpose of this poster, we will focus on the HER family of receptors. It is well understood that the HER family proteins can dimerize with the other members of its family leading to a complicated oncogenic signaling cascade. Surprisingly, existing cellular assays have been unable to faithfully monitor these interactions proximally in a drug discovery setting. We present EFC-based cellular assays that monitor EGFR homodimerization, EGFR-ErbB2 and ErbB2-ErbB3 heterodimerization events at the receptor. The high signal to noise ratio, serum tolerance and reproducibility make these assays ideal for a diverse range of applications for the identification and development of therapeutic small molecules and biologics, including screening, functional characterization, QC lot release assays and neutralizing antibody studies. Citation Format: Jane Lamerdin, Abhishek Saharia, Jennifer Lin-Jones, Mimi Nguyen, Hyna Fabionar, Sangeetha Gunturi, Abha Srivastava, Tom Wehrman. Detection of EGFR, HER2, HER3 and HER4 homodimerization and heterodimerization using EFC-based cellular assays. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3742. doi:10.1158/1538-7445.AM2014-3742