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.

摘要：G-protein coupled receptors (GPCRs) constitute a large family of eukaryotic cell-surface receptors, characterized structurally by crossing the cell membrane seven times. They are involved in several important signaling pathways, and aberrant GPCR expression is associated with many diseases. The GPCR-associated sorting protein-1 (GASP-1) regulates multiple GPCRs through both ligand-dependent and ligand-independent mechanisms.Previously, researchers identified GASP-1 as a novel biomarker for breast cancer and showed that GASP-1 is overexpressed in breast cancer tumors, as well as other tumors. In this study, they proposed to determine the function of GASP-1 in breast cancer progression.In triple-negative breast cancer (TNBC) cell lines, the researchers found that anti-GASP-1 antibodies inhibited cell growth; further, transient downregulation of GASP-1 greatly inhibited cell proliferation. Using stably transfected cells designed to over- and under-express GASP-1, they showed that GASP-1 downregulation promoted cell death. GASP-1 promoted cell invasion in two different assays that mimicked cell invasion in the tumor environment. Further, the size of GASP-1 granules, formed in the cytoplasm after overexpression of GASP-1, were predictive of which tumors became invasive. The researchers conclude that GASP-1 is a breast cancer biomarker with prognostic as well as therapeutic potential.

摘要：Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases in the US. The main types of NSCLC are adenocarcinomas, squamous-cell carcinomas and—less commonly—large-cell carcinomas. One of the most common targetable genetic aberrations in NSCLC is activating mutations in the epidermal growth factor receptor (EGFR) gene; in these cases, treatment with tyrosine kinase inhibitors (TKIs) has shown clinical benefit. In particular, the third-generation TKI osimertinib has proven superior in treatment of advanced-stage NSCLC with EGFR mutations.A significant issue with TKI treatment is the eventual development of resistance, especially with first- and second-generation inhibitors. Earlier studies showed that inhibition of a specific microRNA (miRNA) could restore sensitivity to TKIs in NSCLC cells in vitro. The current study aimed to investigate the involvement of miRNAs in acquired resistance to osimertinib and to determine whether specific miRNAs could serve as markers for osimertinib resistance.miRNA expression profiling in parental and osimertinib-refractory EGFR mutant NSCLC cell lines identified several miRNAs that were differentially expressed. Inhibitory screening revealed a specific miRNA (miR-494-3p) that partially conferred resistance to osimertinib in osimertinib-refractory cells. Using a pan-cancer array, the researchers identified 18 transcripts displaying >2-fold differential expression in osimertinib-resistant cells compared with parental cells; 10 of these transcripts were predicted mIR-494-3p targets in silico. Further, analysis of plasma samples from NSCLC patients with mutated EGFR receiving osimertinib treatment showed that miR-494-3p was significantly elevated in plasma sampled at disease progression, compared to plasma sampled at treatment baseline. The researchers conclude that miR-494-3p may serve as a potential biomarker to monitor NSCLC disease progression in patients treated with osimertinib. Further study will be required to determine the therapeutic potential of miR-494-3p in vivo.