摘要：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.

摘要：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.

摘要：Human T-lymphotropic virus type I (HTLV-I) is a retrovirus that infects CD4+ T cells and is transmitted by bodily fluids. Although infection may not result in symptoms in many cases, integration of viral RNA with the T-cell genome can later lead to adult T-cell leukemia/lymphoma (ATL). Progression of ATL results in a poor prognosis, and there is currently no effective treatment.Recent studies have implicated the viral HTLV-I bZIP factor (HBZ) in oncogenesis. The current study examined a potential therapeutic strategy, using a series of zinc-finger protein (ZFP) repressors targeting a region of the HTLV-I promoter that controls HBZ expression. To select the lead candidate, the researchers screened a series of ZFP repressors, using cotransfection assays with a bi-directional expression vector. This vector contained the HTLV-LTR driving firefly and Renilla luciferase in the sense and antisense direction, respectively. Further experiments in patient-derived TL-Om1 cells identified a ZFP repressor that significantly inhibited cell proliferation, and the effect was specific to HTLV-I. In addition, the ZFP repressors induced cell cycle arrest and apoptosis in TL-Om1 cells. The researchers conclude that ZFP repressors specifically targeting HBZ gene expression could constitute an effective therapeutic approach against HTLV-I-associated malignancies.

摘要：Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that continues to infect humans globally, causing coronavirus disease 2019 (COVID-19) in the population. Members of the Numb-associated kinase (NAK) family have been reported to control cellular trafficking, both during entry as well as assembly and egress, of several RNA viruses. Earlier research showed NAK activity was inhibited by known or novel small molecules, suggesting targeting this family of serine/threonine kinases could be a useful antiviral target.This study investigated if NAK family members adaptor-associated kinase 1 (AAK1), BMP-2 inducible kinase (BIKE/BMP2K), cyclin G-associated kinase (GAK) and serine/threonine kinase 16 (STK16) could play a role in SARS-CoV-2 infection and be potential antiviral candidates. Using a human lung epithelial cell line, all four NAKs were depleted from the cells, reducing both SARS-CoV-2 infection by 89–96%, depending on the NAK target, and viral titer. When known NAK inhibitors were applied to cells prior to SARS-CoV-2 infection, the results showed various levels of viral suppression. Combining two compounds that targeted three different NAKs (BIKE, AAK1 and GAK) increased the SARS-CoV-2 inhibition, suggesting that NAKs are a viable antiviral target. Other novel compounds that target NAKs and had previously demonstrated activity against other viruses were also effective at reducing SARS-CoV-2 infection.To examine when NAK inhibitors are effective antivirals, the potent AAK1/BIKE inhibitor RMC-76 treated cells various times after infection and the effects assessed at various intervals up to 10 hours, which is one cycle of SARS-CoV-2 infection. This experiment revealed RMC-76 inhibited SARS-CoV-2 both in the early viral entry stage and the later viral assemble/exit stage. Finally, each individual NAK was depleted from cells before wild-type SARS-CoV-2 infected the cells, resulting in intracellular viral RNA reduced by 70–93%. The researchers state that their experiments not only show NAKs play a role in SARS-CoV-2 infection but are also antiviral treatment candidates.

摘要：ST3Gal4 is a mammalian sialyltransferase that uses the substrate CMP-sialic acid (CMP-Sia) to attach terminal sugars (sialic acids) to N-glycans via α2,3 linkage. The multitransmembrane transporter SLC35A1 delivers CMP-Sia from the nucleus to the Golgi lumen and has been shown to associate as homodimers. Mutations in the SLC35A1 gene are associated with a subtype of a congenital disorder of glycosylation (CDG). Earlier research showed that two CDG-causing mutations in the SLC35A1 gene (T156R and E196K) reduced or eliminated CMP-Sia transport.The goal of this study was to determine if ST3Gal4 interacts with the wild-type SLC35A1. This interaction was tested using a two-subunit NanoBiT^? luciferase fused in various orientations with ST3Gal4 and wild-type SLC35A1 then assessed in a luminescent protein interaction assay. The data showed that ST3Gal4 and wild-type SLC35A1 associated in HEK293T cells. To test if the CDG mutations in SLC35A1 could restore N-glycan sialylation, HPLC analysis of wild-type and SLC35A1 knockout HEK293T cells were compared with results from the knockout cells expressing T156R or E196K SLC35A1 mutations. Both mutations restored sialylation with the T156R mutant providing greater correction, nearly matching the wild type cells. To examine if the T156R and E196K SLC35A1 mutations also interacted with the ST3Gal4 sialyltransferase, the same two-subunit luciferase complementation assay used for the wild-type SLC35A1 was repeated with the CDG-causing variants. Results show that the T156R mutant interacted with STEGal4, generating luminescence similarly to wild-type SLC35A1. In contrast, the E196K mutation did not associate with STEGal4.The researchers noted that this study improved understanding about the interactions between nucleotide sugar transporters and sialyltransferases, and the role the association may play in CDG.

摘要：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.

摘要：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.

摘要：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 inhibitors. 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.

摘要：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.

摘要：Rotenone, a broad-spectrum insecticide and piscicide, is often used in animal models to induce pathological characteristics of Parkinson’s disease (PD). Previously, the antidiabetic drug metformin has shown neuroprotective effects against rotenone-induced dopaminergic neuronal loss in PD models. Multiple mechanisms of action have been studied for these effects; however, little is known about metformin-induced attenuation of oxidative stress in neurons.This study examined the antioxidative and mitochondrion-protective properties of metformin. The researchers treated human neuroblastoma cells (SH-SY5Y) with various concentrations of rotenone to determine the optimal cytotoxic dose. To assess the neuroprotective effect of metformin, they pretreated cells with varying concentrations of metformin. Metformin showed neuroprotective activity and significantly reduced rotenone-induced caspase 3/7 activation. Further, this activity of metformin appeared to be dependent on the 5′ adenosine monophosphate-activated protein kinase (AMPK) pathway.The researchers also showed that metformin aided in restoration of mitochondrial function after rotenone-induced toxicity, as well as reducing cytosolic and mitochondrial levels of reactive oxygen species (ROS). They linked the restorative effects of metformin to upregulation of transcription factor Nrf2, which is responsible for regulation of various antioxidant enzyme systems and protection of mitochondria from ROS-induced damage.