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Research Highlights

Summaries of key publications that apply Promega technologies to solve research problems

Apoptosis

Soto-Gamez, A. et al. (2022) Enhanced extrinsic apoptosis of therapy-induced senescent cancer cells using a death receptor 5 (DR5) selective agonist. Cancer Lett. 525, 67–75. DOI: 10.1016/j.canlet.2021.10.038

Cellular senescence is a state of cell-cycle arrest that occurs typically in response to external stress factors. Anti-cancer therapies, including chemotherapy and radiation, are often directed toward inducing senescence or programmed cell death (apoptosis) in cancer cells. However, senescent cells are characterized by genomic instability, and they can escape from apoptosis, causing cancer relapse. These senescent cells are resistant to apoptosis-inducing factors, including cytotoxic drugs, via pathways involving so-called death receptors that recruit adapter proteins and caspases. Most death receptors belong to the tumor necrosis factor (TNF) family.

The TNF-related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via death receptors, and current anti-cancer strategies are investigating its use as a therapeutic agent. This study examined the sensitivity of senescent cells to TRAIL after exposure to genotoxic stress. Cancer cells showed variable sensitivity to TRAIL after induction of senescence, while expression of both pro- and anti-apoptotic receptors was elevated. The researchers showed that a TRAIL variant with selectivity for death receptor 5 (DR5) was more effective at inducing apoptosis than wild-type TRAIL. No apoptosis induction was observed in noncancerous cells, even at the highest concentrations tested. Therefore, the DR5-selective TRAIL variant shows promise as a novel anticancer agent to eliminate therapy-induced cancer cell senescence.

Keywords: senescence, TRAIL, apoptosis, cancer therapy, caspase

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Bioassays

Mohan, N. et al. (2022) Comparative characterization of different molecular formats of bispecific antibodies targeting EGFR and PD-L1. Pharmaceutics 14, 1381. DOI: 10.3390/pharmaceutics14071381

Programmed death-ligand 1 (PD-L1), an immune checkpoint protein, is overexpressed in triple-negative breast cancers (TNBC). TNBC is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 receptor (HER2). Bispecific antibodies that can simultaneously target two different antigens offer new therapeutic modalities to treat cancer, but they can be challenging to characterize. The researchers generated two formats of IgG1-like bispecific antibodies targeting the same antigens, EGFR and PD-L1. They used the CellTiter-Glo® assay to investigate the potency of these antibody formats in TNBC cell model systems. With the PD-1/PD-L1 Blockade Bioassay, they showed that both antibody formats could disrupt the engagement of PD-1 to its ligand PD-L1, promoting TCR signaling, transcriptional activation and cytokine production. Further, the researchers demonstrated that one of the antibody formats induced potent ADCC activity in high-EGFR-expressing cells, using the ADCC Reporter Bioassay. They conclude that selection of appropriate cell lines and assays is critically important for assay development and potency testing of bispecific antibodies.

Keywords: bispecific antibodies, EFGR, PD-L1, triple-negative breast cancer (TNBC), bioassays

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Inflammation

McBride, C. et al. (2022) Overcoming preclinical safety obstacles to discover (S)N((1,2,3,5,6,7-Hexahydrosindacen-4-yl)carbamoyl)-6-(methylamino)-6,7-dihydro5Hpyrazolo[5,1b][1,3]oxazine-3-sulfonamide (GDC-2394): A potent and selective NLRP3 inhibitor. J. Med. Chem. DOI: 10.1021/acs.jmedchem.2c01250

Inflammasomes—multi-unit protein complexes—have become a central concept in immune system research. In particular, activation of the NLRP3 inflammasome has been widely studied, and it is implicated in variety of diseases. Accordingly, small-molecule inhibitors of NLRP3 inflammasome activation are attractive therapeutic targets.

In this study, the researchers developed a series of novel NLRP3 inhibitors targeted to minimize the risk of drug-induced liver injury observed with a well-known NLRP3 inhibitor, MCC950. Using a lipophilic ligand efficiency (LLE) strategy and starting from MCC950, the researchers developed a series of novel NLRP3 inhibitor candidates. Two candidates advanced to safety studies in nonhuman primates; however, the first lead had an inadequate solubility profile. Therefore, research efforts shifted to compound GDC-2394, which did not show adverse renal or hepatic effects in nonhuman primates. In vitro and in vivo studies with GDC-2394 demonstrated a safety profile suitable for advancing the compound into clinical trials.

Keywords: inflammasome, NLRP3, caspase-1, MCC950, NLRP3 inhibitor

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Kinase Biology

Phadke, S. et al. (2022) Insights into the modular design of kinase inhibitors and application to Abl and Axl. RSC Med. Chem. 13, 64. DOI: 10.1039/d1md00296a

Scaffold hopping is a drug design strategy in which the core chemical structure of a drug candidate is replaced isosterically, which leads to structurally novel compounds. This strategy is commonly used in designing kinase inhbitors. All kinases have a conserved activation loop characterized by an Asp-Phe-Gly (DFG) motif. This motif exists in a “DFG-in” structural conformation, and a conformational change to a “DFG-out” structure that can no longer bind Mg2+ results in inactivation.

In this study, the researchers describe a strategy to generate an array of DFG-out conformation inhibitors with three different hinge-binders and two DFG-pocket groups. They systematically evaluated parts of DFG-out inactive conformation inhibitors that affect kinome-wide selectivity and identified elements that could be used as the starting point for targeting 36 distinct kinases. Next, they applied their strategy to Abl kinase to measure potency of selective inhibitors under “real-world” conditions using the NanoLuc®-Abl1 fusion expression vector and NanoBRET™ Intracellular Target Engagement Assay. They also studied inhibitor binding to Axl kinase using a radiometric assay. Based on their results, the researchers identified two selective inhibitors that display low nanomolar potency against Axl or wild-type and clinically relevant mutants of Abl.

Keywords: kinase inhibitors, kinase target engagement, Abl, Axl

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Luciferase Assays

Chen, R. et al. (2022). Engineering circular RNA for enhanced protein production. Nat. Biotechnol. DOI: 10.1038/s41587-022-01393-0

The success of mRNA vaccines has prompted interest in extending the duration of protein expression for gene therapy applications. Circular RNAs (circRNAs), in which coding RNA molecules are joined head to tail, show significant promise in this regard. In this study, the researchers report the development of a modular high-throughput platform to make and test synthetic circRNAs. To maximize circRNA translation, they optimized five elements: vector topology, 5′ and 3′ untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. To optimize translation activity, they prepared NanoLuc® luciferase reporter constructs and transfected HeLa cells. They also examined circRNA expression levels in vivo by luminescence imaging in mice. The authors conclude that their optimized circRNA synthesis platform increased circRNA protein yields by several hundred-fold and enabled potent and durable protein production in vivo.

Keywords: gene delivery, gene therapy, nucleic acid therapeutics, RNA splicing

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Adamson, H. et al. (2022) Rapid quantification of C. difficile glutamate dehydrogenase and toxin B (TcdB) with a NanoBiT split-luciferase assay. Anal. Chem. 94, 8156−8163. DOI: 10.1021/acs.analchem.1c05206

The Gram-positive bacillus Clostridioides (formerly Clostridium) difficile is responsible for most hospital-related infections, with global incidence rising over the past decade. Two large toxins, toxin A (TcdA) and toxin B (TcdB), are responsible for triggering host responses that can result in significant intestinal damage. There is an urgent need for sensitive and rapid tests to detect true C. difficile infection, as distinguished from disease-free carriers.

In this study, the researchers used NanoBiT® technology to design “split-luciferase” assays that could detect TcdB and another C. difficile biomarker, glutamate dehydrogenase (GDH). The assay development strategy used affimers (synthetic nonimmunoglobulin-binding proteins) with high binding affinity for different regions of TcdB and GDH. These affimers are more convenient than antibodies, because they are smaller, stable and easily expressed as fusions with LgBiT and SmBiT tags. Binding to the target antigen in solution reconstitutes functional NanoBiT® enzyme, resulting in a bright luminescent signal. The assay performance with fecal samples was equivalent to that of a current point-of-care (POC) test, but it had the advantages of being quantitative, requiring less user steps, and being able to distinguish clinically relevant TcdB. The researchers conclude that their assay method is suitable for a wide range of biomarkers and offers the potential to develop ultrasensitive and rapid POC tests for many infectious diseases.

Keywords: C. difficile, bioluminescence, NanoBiT, sensors, toxins

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