Activity In Cell-based Assays Research Articles

Synthesis, structure of the N-(Alkyl/Arylsulfonyl) substituted 5-(Bromo/Iodo)-3-methylindazoles and bioactivity screening against some of the biochemical targets linked to type 2 diabetes mellitus

The indazole scaffold and sulfonamide moiety have evoked high interest in medicinal chemistry especially in the context of antidiabetic drug development. A series of the C-5 unsubstituted (2a–f), 5‑bromo (2g–l) and 5-iodo (2m–r) substituted 1-(alkyl/arylsulfonyl)-3-methylindazoles were prepared, characterized and then evaluated through enzymatic assay in vitro for anti-α-glucosidase activity and for potential free radical scavenging properties. Compounds 2b, 2d, 2f–h, 2k, 2p and 2q exhibited strong activity against α-glucosidase compared to acarbose (IC50 = 1.30 ± 0.02 µM) with IC50 values in the range of 0.72 ± 0.03 – 1.20 ± 0.01 µM. These compounds exhibited increased or strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) and/or nitric oxide (NO) free radical scavenging activities. The compounds were, in turn, evaluated through enzymatic assays in vitro for the potential to inhibit or activate superoxide dismutase (SOD), lipoxygenase-15 (LOX-15) and/or vascular endothelial growth factor receptor-2 (VEGFR-2) activities. An in vitro cell-based antioxidant activity assay involving lipopolysaccharide induced reactive oxygen species (ROS) production in the human breast (MCF-7) cancer cell line revealed that compounds 2d, 2f and 2h exhibit moderate activity (35.5 %, 47 % and 28 %, respectively) compared to staurosporine (24 %). Compounds 2d, 2f and 2h significantly reduced the ROS level in the human embryonic kidney (HEK293-T) cells (65.5 %, 58 % and 48 %, respectively) compared to staurosporine (34 %). The planar benzo-fused heterocyclic scaffold and the distorted tetrahedral geometry of the N-sulfonyl moiety are predicted to facilitate hydrophobic and/or hydrogen bonding interactions to stabilize protein-ligand interaction in the receptors. The ADME analysis predicted that the test compounds have drug-like properties, a safe toxicity profile in silico without any carcinogenic and mutagenicity side effect. Molecular modelling was also used to attempt to rationalize the biological activity of these compounds and to correlate the results of in vitro and in silico studies.

Inhibition of kynurenine production by N,O-substituted hydroxylamine derivatives

The inhibition of kynurenine production is considered a promising target for cancer immunotherapy. In this study, an amino acid derivative, compound 1 was discovered using a cell-based assay with our screening library. Compound 1 suppressed kynurenine production without inhibiting indoleamine 2,3-dioxygenase 1 (IDO1) activity. The activity of 1 was derived from the inhibition of IDO1 by a metabolite of 1, O-benzylhydroxylamine (OBHA, 2a). A series of N-substituted 2a derivatives that exhibit potent activity in cell-based assays may represent effective prodrugs. Therefore, we synthesized and evaluated novel N,O-substituted hydroxylamine derivatives. The structure–activity relationships revealed that N,O-substituted hydroxylamine 2c inhibits kynurenine production in a cell-based assay. We conducted an in vivo experiment with 2c, although the effectiveness of O-substituted hydroxylamine derivatives in vivo has not been previously reported. The results indicate that N,O-substituted hydroxylamine derivatives are promising IDO1 inhibitors.

Abstract 1811: Efficient identification of new small molecules targeting succinate dehydrogenase in non-small cell lung cancer

Abstract Efficient lung cancer treatment remains challenging due to the lack of therapeutic targets. The succinate dehydrogenase (SDH) enzyme plays a critical metabolic role as an intermediate between the citric acid cycle and the electron transport chain associated with the cancer disease state. Although several existing compounds have been applied to target metabolic diseases in vitro, modulating SDH for lung cancer treatment is still elusive. By integrating the AtomNet® technology for compound identification with mitochondria- and cell-based enzyme activity assays, we identified four new small molecules from 96 predicted candidates. Cellular thermal shift assay confirmed that these small molecules bind directly to SDH subunits in lung cancer cells. Mechanistically, treatment with these small molecules increased cellular and mitochondrial reactive oxygen species, accumulated succinate, and DNA damage-induced apoptosis in lung cancer cell lines. Functionally, these small molecules reduced the growth, migration, and formation of 3D organoids in vitro in lung cancer cell lines, both in the short and long term. Our study sheds light on the function of SDH in metabolic dysfunction related to cancer and highlights the potential of modulating SDH as a viable therapeutic strategy for targeting lung cancer and other cancer types. LS, NS, and WCZ contributed equally to this work. Citation Format: Luis Silva, Nicholas Skiados, Nikitha Murugavel, Nastassja Luna, Karen Cover, Manish K. Gupta, Stephanie C. Contreras, Terrence E. O'Brien, Wen Cai Zhang. Efficient identification of new small molecules targeting succinate dehydrogenase in non-small cell lung 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 1811.

Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition

Of the targets for HIV-1 therapeutics, the capsid core is a relatively unexploited but alluring drug target due to its indispensable roles throughout virus replication. Because of this, we aimed to identify "clickable" covalent modifiers of the HIV-1 capsid protein (CA) for future functionalization. We screened a library of fluorosulfate compounds that can undergo sulfur(VI) fluoride exchange (SuFEx) reactions, and five compounds were identified as hits. These molecules were further characterized for antiviral effects. Several compounds impacted invitro capsid assembly. One compound, BBS-103, covalently bound CA via a SuFEx reaction to Tyr145 and had antiviral activity in cell-based assays by perturbing virus production, but not uncoating. The covalent binding of compounds that target the HIV-1 capsid could aid in the future design of antiretroviral drugs or chemical probes that will help study aspects of HIV-1 replication.

Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity.

Cathepsins (Cats) are proteases that mediate the successful entry of SARS-CoV-2 into host cells. We designed and synthesized a tailored series of 21 peptidomimetics and evaluated their inhibitory activity against human cathepsins L, B, and S. Structural diversity was realized by combinations of different C-terminal warhead functions and N-terminal capping groups, while a central Leu-Phe fragment was maintained. Several compounds were identified as promising cathepsin L and S inhibitors with Ki values in the low nanomolar to subnanomolar range, for example, the peptide aldehydes 9a and 9b (9a, 2.67 nM, CatL; 0.455 nM, CatS; 9b, 1.76 nM, CatL; 0.512 nM, CatS). The compounds' inhibitory activity against the main protease of SARS-CoV-2 (Mpro) was additionally investigated. Based on the results at CatL, CatS, and Mpro, selected inhibitors were subjected to investigations of their antiviral activity in cell-based assays. In particular, the peptide nitrile 11e exhibited promising antiviral activity with an EC50 value of 38.4 nM in Calu-3 cells without showing cytotoxicity. High metabolic stability and favorable pharmacokinetic properties make 11e suitable for further preclinical development.

Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50) values ranging from 1.42 to 32.7 μM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC50) values of 21.73 and 31.19 μM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.

Discovery of α-Ketoamide inhibitors of SARS-CoV-2 main protease derived from quaternized P1 groups

Although the SARS-CoV-2 pandemic has ended, multiple sporadic cases still exist, posing a request for more antivirals. The main protease (Mpro) of SARS-CoV-2, a key enzyme for viral replication, is an attractive target for drug development. Here, we report the discovery of a new potent α-ketoamide-containing Mpro inhibitor, N-((R)-1-cyclohexyl-2-(((R)-3-methoxy-1-oxo-1-((1-(2-oxo-2-((thiazol-2-ylmethyl)amino)acetyl)cyclobutyl)amino)propan-2-yl)amino)-2-oxoethyl)-4,4-difluorocyclohexane-1-carboxamide (20j). This compound presented promising enzymatic inhibitory activity against SARS-CoV-2 Mpro with an IC50 value of 19.0 nM, and an excellent antiviral activity in cell-based assay with an EC50 value of 138.1 nM. This novel covalent inhibitor may be used as a lead compound for subsequent drug discovery against SARS-CoV-2.

Abstract C086: NP1867, a potent, selective, covalent small molecule inhibitor of DNA Mismatch Repair (MMR) protein PMS2, functionally inhibits MMR in cells and elicits COSMIC mutational signatures consistent with MMR-deficient patient samples

Abstract Two complementary mechanisms safeguard genome fidelity during DNA replication: DNA polymerase proofreading and DNA mismatch repair (MMR). DNA polymerase proofreading or MMR deficiencies can occur independently or, more rarely, simultaneously; loss of function of either or both pathways leads to the accumulation of DNA mutations. Lynch Syndrome is caused by mono-allelic germline mutations in MMR genes MLH1, MSH2, MSH6 or PMS2. Gene expression from the wild-type allele elicits sufficient MMR activity until a second hit somatic mutation inactivates the wild-type allele leading to MMR deficiency (MMR-d). Constitutional mismatch repair deficiency (CMMRD) syndrome is an inherited early onset cancer predisposition syndrome resulting from biallelic germline mutations in MLH1, MSH2, MSH6 or PMS2. MMR-d cancers are commonly and typically characterized by accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences - microsatellite instability (MSI); importantly, such mutations occur at higher rates in genetically unstable tumour cells under replicative stress than in normal cells. Cancers that harbour >40% microsatellite variations (positive for two or more of five clinically tested microsatellite markers) are described as MSI-high (MSI-H). Importantly, MMR-d, MSI-H tumours are now eligible for immune checkpoint therapy, irrespective of tissue type, providing substantial patient benefit and a major advance in cancer treatment. This clinical context is consistent with the hypothesis that MMR-d elicits an immunogenic state. However, there is an unmet need for chemical tools to explore the role of therapeutic MMR inhibition alongside immune checkpoint blockade. Here, for the first time, we demonstrate that NP1867 is a potent, selective small molecule inhibitor of the Gyrase, Hsp90, Histidine Kinase, MutL (GHKL) family endonuclease PMS1 Homolog 2, Mismatch Repair System Component (PMS2). We demonstrate by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in cell-based assays of MMR-dependent DNA repair. NP1867 displays minimal off-target binding in broad biochemical screening panels including kinases and ATPases. Treatment of MMR-proficient, colorectal cancer cell lines with NP1867 increases tumour mutational burden and generates COSMIC mutational signatures consistent with MMR-d patient samples demonstrating, for the first time, that acute inhibition of PMS2 with a small molecule phenocopies the epigenetic silencing and/or genetic loss of MMR observed in patients. We characterise cell- and tissue-based target engagement, and in vivo pharmacokinetic and pharmacodynamic properties of NP1867 to inform optimal use by the research community for further exploring PMS2 and MMR function. Citation Format: Julian Blagg, Philippe Riou, Alexia Hervieu, Eleonora Piumatti, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Sam Weeks, Maria Rodriguez, Kalpesh Parmar, Pradip Patel, Adam Peall, Paige Tongue, Tess McLaren, Robert Workman, Ruzica Bago, Stuart Robjohns, Rebecca Beaumont, Mike Briggs, Gareth Langley, Charles Nichols, David Clark, Paul Winship, Benoit Rousseau, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. NP1867, a potent, selective, covalent small molecule inhibitor of DNA Mismatch Repair (MMR) protein PMS2, functionally inhibits MMR in cells and elicits COSMIC mutational signatures consistent with MMR-deficient patient samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C086.

Exploring the Fatty Acid Binding Pocket in the SARS-CoV-2 Spike Protein - Confirmed and Potential Ligands.

Severe Acute Respiratory syndrome 2 (SARS-CoV-2) is a respiratory virus responsible for coronavirus disease 19 (COVID-19) and the still ongoing and unprecedented global pandemic. The key viral protein for cell infection is the spike glycoprotein, a surface-exposed fusion protein that both recognizes and mediates entry into host cells. Within the spike glycoprotein, a fatty acid binding pocket (FABP) was confirmed, with the crystallization of linoleic acid (LA) occupying a well-defined site. Importantly, when the pocket is occupied by a fatty acid, an inactive conformation is stabilized, and cell recognition is hindered. In this review, we discuss ligands reported so far for this site, correlating their activity predicted through in silico studies with antispike experimental activity, assessed by either binding assays or cell-infection assays. LA was the first confirmed ligand, cocrystallized in a cryo-EM structure of the spike protein, resulting in increased stability of the inactive conformation of the spike protein. The next identified ligand, lifitegrast, was also experimentally confirmed as a ligand with antiviral activity, suggesting the potential for diverse chemical scaffolds to bind this site. Finally, SPC-14 was also confirmed as a ligand, although no inhibition assays were performed. In this review, we identified 20 studies describing small-molecule compounds predicted to bind the pocket in in silico studies and with confirmed binding or in vitro activity, either inhibitory activity against the spike-ACE2 interaction or antiviral activity in cell-based assays. When considering all ligands confirmed with in vitro assays, a good overall occupation of the pocket should be complemented with the ability to make direct interactions, both hydrophilic and hydrophobic, with key amino acid residues defining the pocket surface. Among the active compounds, long flexible carbon chains are recurrent, with retinoids capable of binding the FABP, although bulkier systems are also capable of affecting viral fitness. Compounds able to bind this site with high affinity have the potential to stabilize the inactive conformation of the SARS-CoV-2 spike protein and therefore reduce the virus's ability to infect new cells. Since this pocket is conserved in highly pathogenic human coronaviruses, including MERS-CoV and SARS-CoV, this effect could be exploited for the development of new antiviral agents, with broad-spectrum anticoronavirus activity.

Supercritical fluid-assisted extraction of hop cone residue from craft breweries

This study aimed to use supercritical fluid-assisted extraction in hop cone residue from craft breweries and define the best extraction parameters. Yield, compounds extracted, in vitro chemical and cell-based antioxidant activities, and antimicrobial and cytotoxic potentials were evaluated. The variables studied were temperature (40–60 °C), pressure (10–20 MPa), and ethanol percentage as co-solvent (5–10%) using a Taguchi experimental design (11 runs). The extracts showed inhibitory action towards Gram-positive and Gram-negative bacteria, mainly Proteus mirabilis and Enterococcus faecalis (MICs = 5–10 mg/mL). Furthermore, up to the maximum tested concentration (400 μg/mL), the extracts showed no hepatotoxicity on a healthy porcine liver cell line (PLP2), indicating safety for food and pharmacological applications. Lower temperatures (40 °C), pressures (10 MPa), and ethanol percentages (5%) resulted in higher concentrations of some compounds (alpha-copaene, γ-elemene, γ-muurolene, α-muurolene, caryophyllene oxide) and higher antioxidant activity in cell-based assays, but lower yields. At the same time, lower temperatures (40 °C) and pressures (10 MPa) but higher ethanol percentages (10%) resulted in higher concentrations of other compounds (nerolidyl acetate, linalyl isobutyrate, α-acorenol, and geranyl isovalerate) and increased antioxidant activity in chemical systems. Our results support that the hop cone residue can be used to obtain supercritical fluid-assisted extracts with biological potentialities that might virtually be upcycled into multifunctional ingredients for the food and pharmacological industries. Using 40 °C, 10 MPa, and 5 or 10% ethanol percentage as process parameters is advisable.

Abstract 3111: The Sygnature CHARMD platform - combinatorial high-throughput assembly and review of molecular degraders

Abstract Recent interest the ability to degrade oncogenic proteins has grown exponentially, and the advent of Protein-Targeting Chimeras (PROTACs) has allowed many previously undruggable targets to be specifically targeted. However, the successful development of these bioavailable heterobifunctional degraders relies on the identification of a combination of ligase recruiter, linker and exit vector that allow formation of a competent ternary complex and target degradation. To this end, conventional single-compound synthesis is time-consuming and poses challenges for selection of degrader components with predictive SAR and ADME properties of the assembled bifunctional compound. Here, we demonstrate a high-throughput integrated platform for generation and assessment of bifunctional degraders, incorporating combinatorial chemistry using both commercial and bespoke linkers, cell-based activity assays, and assessment of in vitro DMPK properties (lipophilicity, metabolic stability and experimental polar surface area). Alongside this, we have in place computational methods for ternary complex modelling, linker design and PROTAC prioritisation. Altogether, our approach facilitates rapid screening and identification of bifunctional lead compounds for challenging oncology targets with the potential to deliver significant patient benefit. Our platform approach has been exemplified through application to oncology targets of significant current interests and our results in this area will be highlighted. Citation Format: Roland Hjerpe, Louise Birch, Alex Brien, Lola Cusin, Lorna Duffy, Benoit Gourdet, Rachel Lawrence, Philip MacFaul, Hannah Mortlock, Hannah Neal, Christopher Pearce, Erica Pitti, Gonzalo Robles, Edith Rodrigues, Catriona Scott, Rajesh Singh, Stuart Thomson, Yujia Zhang, Allan Jordan. The Sygnature CHARMD platform - combinatorial high-throughput assembly and review of molecular degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3111.

Structure-based design, synthesis and evaluation of a novel family of PEX5-PEX14 interaction inhibitors against Trypanosoma

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates.Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

Protein-protein interaction-based high throughput screening for adenylyl cyclase 1 inhibitors: Design, implementation, and discovery of a novel chemotype.

Genetic and preclinical studies have implicated adenylyl cyclase 1 (AC1) as a potential target for the treatment of chronic inflammatory pain. AC1 activity is increased following inflammatory pain stimuli and AC1 knockout mice show a marked reduction in responses to inflammatory pain. Previous drug discovery efforts have centered around the inhibition of AC1 activity in cell-based assays. In the present study, we used an in vitro approach focused on inhibition of the protein-protein interaction (PPI) between Ca2+/calmodulin (CaM) and AC1, an interaction that is required for activation of AC1. We developed a novel fluorescence polarization (FP) assay focused on the PPI between an AC1 peptide and CaM and used this assay to screen over 23,000 compounds for inhibitors of the AC1-CaM PPI. Next, we used a cellular NanoBiT assay to validate 21 FP hits for inhibition of the AC1-CaM PPI in a cellular context with full-length proteins. Based on efficacy, potency, and selectivity for AC1, hits 12, 13, 15, 18, 20, and 21 were prioritized. We then tested these compounds for inhibition of AC1 activity in cyclic AMP (cAMP) accumulation assays, using HEK293 cells stably expressing AC1. Hit 15 contained a dithiophene scaffold and was of particular interest because it shared structural similarities with our recently reported benzamide series of AC1 inhibitors. We next tested a small set of 13 compounds containing the dithiophene scaffold for structure-activity relationship studies. Although many compounds were non-selective, we observed trends for tuning AC1/AC8 selectivity based on heterocycle type and substituents. Having an ethyl on the central thiophene caused the scaffold to be more selective for AC8. Cyclization of the alkyl substituent fused to the thiophene significantly reduced activity and also shifted selectivity toward AC8. Notably, combining the fused cyclohexane-thiophene ring system with a morpholine heterocycle significantly increased potency at both AC1 and AC8. Through designing a novel FP screen and NanoBiT assay, and evaluating hits in cAMP accumulation assays, we have discovered a novel, potent, dithiophene scaffold for inhibition of the AC1- and AC8-CaM PPI. We also report the most potent fully efficacious inhibitor of AC8 activity known to-date.

Corazonin signaling modulates the synthetic activity of male accessory gland in Grapholita molesta

Although neuropeptide corazonin (Crz) has been identified in numerous insect species, the research about its function in regulation of reproduction is still in its infancy. Herein, we characterized the Crz (GmolCrz) and its receptor (GmolCrzR) to investigate their reproductive function in Grapholita molesta. Both molecular docking result and cell-based receptor activity assay showed that GmolCrz could interact with GmolCrzR. Additionally, spatial expression patterns of GmolCrz and GmolCrzR in males were evaluated. Knockdown of GmolCrz or GmolCrzR significantly lengthened copulation duration and decreased fertility in males. In these males, we found that the production of sperm was normal, while the content of accessory gland proteins (Acps) in the accessory gland (AG) was strongly diminished. Furthermore, knockdown of GmolCrz or GmolCrzR in males had no effect on sperm and Acps transfer to females. RNA-seq and gene expression analyses further confirmed that genes involved in serine-type endopeptidase activity were significantly downregulated in the AG upon GmolCrzR knockdown. Finally, sperm activation assays demonstrated that this process was disrupted in the spermatophore of females mated with GmolCrz or GmolCrzR knockdown males, which may cause the decreased fertility in males. Our findings provide new insights into the functions of Crz signaling in a Lepidopteran insect.

An Active Learning Framework Improves Tumor Variant Interpretation.

A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.

Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2.

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ± 0.75 μM) than PAC (25.90 ± 0.81 μM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25–26 μM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ± 1.089 μM) than PAC (21.022 ± 1.202 μM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

Development of Potent Immune Modulators Targeting Stimulator of Interferon Genes Receptor.

Stimulator of interferon genes (STING) is an endoplasmic reticulum-membrane protein that plays important roles in cancer immunotherapy by activating innate immune responses. We designed and synthesized STING modulators and characterized compounds 4a and 4c that share a crucial amidobenzimidazole moiety. In vitro STING binding and cell-based activity assays demonstrated the potency and efficacy of the compounds that function as direct STING agonists by stimulating STING downstream signaling and promoting type I interferon immune responses. In vitro metabolic studies and the pharmacokinetic properties of the compounds led us to investigate their anticancer activity in an in vivo syngeneic model. Intravenous injection of compounds 4a and 4c significantly decreased tumor volume in a CT26 murine colorectal carcinoma model, and the immunological memory-derived cancer inhibition was observed in 4c-treated mouse models. The present results suggest the therapeutic potential of the compounds for cancer immunotherapy via STING-mediated immune activation.

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21.

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product.

Application of Acoustic Ejection MS System to High-Throughput Screening for SARS-CoV-2 3CL Protease Inhibitors.

MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.

17R/S-Benzo-RvD1, a synthetic resolvin D1 analogue, attenuates neointimal hyperplasia in a rat model of acute vascular injury.

Persistent inflammation following vascular injury drives neointimal hyperplasia (NIH). Specialized lipid mediators (SPM) mediate resolution which attenuates inflammation and downstream NIH. We investigated the effects of a synthetic analogue of resolvin D1 (RvD1) on vascular cells and in a model of rat carotid angioplasty. Human venous VSMC and endothelial cells (EC) were employed in migration, cell shape, toxicity, proliferation and p65 nuclear translocation assays. Murine RAW 264.7 cells were utilized to test the effect of pro-resolving compounds on phagocytic activity. A model of rat carotid angioplasty was used to evaluate the effects of 17R/S-benzo-RvD1 (benzo-RvD1) and 17R-RvD1 applied to the adventitia via 25% Pluronic gel. Immunostaining was utilized to examine Ki67 expression and leukocyte recruitment. Morphometric analysis was performed on arteries harvested 14 days after injury. Exposure to benzo-RvD1 attenuated PDGF- stimulated VSMC migration across a range of concentrations (0.1-100 nM), similar to that observed with 17R-RvD1. Pre-treatment with either Benzo-RvD1 or 17R-RvD1 (10, 100nM) attenuated PDGF-BB-induced VSMC cytoskeletal changes to nearly baseline dimensions. Benzo-RvD1 demonstrated modest anti-proliferative activity on VSMC and EC at various concentrations, without significant cytotoxicity. Benzo-RvD1 (10nM) inhibited p65 nuclear translocation in cytokine-stimulated EC by 21% (p<0.05), similar to 17R-RvD1. Consistent with pro-resolving activities of other SPM, both 17R-RvD1 and benzo-RvD1 increased the phagocytic activity of RAW 264.7 cells against S. Aureus and Zymosan particles. There were no significant differences in Ki-67 or CD45 staining observed on day 3 after angioplasty. Periadventitial treatment with benzo-RvD1 reduced carotid neointimal area at 14 days compared to control (0.08 mm2 v. 0.18 mm2; p<0.05), with similar efficacy to 17R-RvD1. 17R/S-benzo-RvD1 and 17R-RvD1 exhibit similar pro-resolving and anti-migratory activity in cell-based assays, and both compounds attenuated NIH following acute arterial injury in rats. Further studies of the mechanisms of resolution following vascular injury, and the translational potential of SPM analogues, are indicated.