Submicromolar Concentrations Research Articles

Inhibitory activity of catecholic phosphonic and phosphinic acids against Helicobacter pylori ureolysis

Catechols have been reported to be potent covalent inhibitors of ureases, and they exhibit activity by modifying cysteine residues at the entrance to enzymatic active sites. Following these principles, we designed and synthesized novel catecholic derivatives that contained carboxylate and phosphonic/phosphinic functionalities and assumed expanded specific interactions. When studying the chemical stability of the molecules, we found that their intrinsic acidity catalyzes spontaneous esterification/hydrolysis reactions in methanol or water solutions, respectively. Regarding biological activity, the most promising compound, 2-(3,4-dihydroxyphenyl)-3-phosphonopropionic acid (15), exhibited significant anti-urease potential (Ki = 2.36 μM, Sporosarcinia pasteurii urease), which was reflected in the antiureolytic effect in live Helicobacter pylori cells at a submicromolar concentration (IC50 = 0.75 μM). As illustrated by molecular modeling, this compound was bound in the active site of urease through a set of concerted electrostatic and hydrogen bond interactions. The antiureolytic activity of catecholic phosphonic acids could be specific because these compounds were chemically inert and not cytotoxic to eukaryotic cells.

A Chemical Chaperone Restores Connexin 26 Mutant Activity.

Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds 3 and 8 were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only 3 (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ∼5 μM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.

Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins.

Malignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death. The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death. CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs. CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.

De novo design of highly efficient type-I photosensitizer based on π-conjugated oligomer for photodynamic eradication of multidrug-resistant bacterial infections

Traditional photosensitizers show limited singlet oxygen generation in hypoxic infection lesions, which greatly suppress their performance in antibacterial therapy. Meanwhile, there still is lack of feasible design strategy for developing hypoxia-overcoming photosensitizers agents. Herein, radical generation of π-conjugated small molecules is efficiently manipulated by an individual selenium (Se) substituent. With this strategy, the first proof-of-concept study of a Se-anchored oligo (thienyl ethynylene) (OT-Se) with high-performance superoxide radical (O2•−) and hydroxyl radical (•OH) generation capability is present, and achieves efficient antibacterial activities towards the clinically extracted multidrug-resistant bacteria methicillin-resistant S. aureus (MRSA) and carbapenem-resistant E. coli (CREC) at sub-micromolar concentration under a low white light irradiation (30 mW/cm2). The water-dispersible OT-Se shows a good bacteria-anchoring capability, biocompatibility, and complete elimination of multidrug-resistant bacteria wound infection in vivo. This work offers a strategy to boost type-I photodynamic therapy (PDT) performance for efficient antibacterial treatments, advancing the development of antibacterial agents.

2-Hydroxyoleic Acid as a Self-Assembly Inducer for Anti-Cancer Drug-Centered Nanoparticles.

A potent nontoxic antitumor drug, 2-hydroxyoleic acid (6, 2OHOA) used for membrane lipid therapy, was selected as a self-assembly inducer due to its ability to form nanoparticles (NPs) in water. For this purpose, it was conjugated with a series of anticancer drugs through a disulfide-containing linker to enhance cell penetration and to secure drug release inside the cell. The antiproliferative evaluation of the synthesized NP formulations against three human tumor cell lines (biphasic mesothelioma MSTO-211H, colorectal adenocarcinoma HT-29, and glioblastoma LN-229) showed that nanoassemblies 16-22a,bNPs exhibit antiproliferative activity at micromolar and submicromolar concentrations. Furthermore, the ability of the disulfide-containing linker to promote cellular effects was confirmed for most nanoformulations. Finally, 17bNP induced intracellular ROS increase in glioblastoma LN-229 cells similarly to free drug 8, and such elevated production was decreased by pretreatment with the antioxidant N-acetylcysteine. Also, nanoformulations 18bNP and 21bNP confirmed the mechanism of action of the free drugs.

DNA Sequence-Specific Ligands. XIX. Synthesis, Spectral Properties, Virological and Biochemical Studies of Fluorescent Dimeric Trisbenzimidazoles DB<sub>3</sub>(<i>n</i>)

In this work, we synthesized and characterized the properties of a series of new fluorescent narrow-groove ligands DB3(n). DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), trisbenzimidazole fragments of which are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of monomeric trisbenzimidazole MB3 with α,ω-alkyldicarboxylic acids. DB3(n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20–0.30 µM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Fatty Acid Conjugated Chalcones as Tubulin Polymerization Inhibitors: Design, Synthesis, QSAR, and Apoptotic and Antiproliferative Activity.

Cancer is often associated with an aberrant increase in tubulin and microtubule activity required for cell migration, invasion, and metastasis. A new series of fatty acid conjugated chalcones have been designed as tubulin polymerization inhibitors and anticancer candidates. These conjugates were designed to harness the beneficial physicochemical properties, ease of synthesis, and tubulin inhibitory activity of two classes of natural components. New lipidated chalcones were synthesized from 4-aminoacetophenone via N-acylation followed by condensation with different aromatic aldehydes. All new compounds showed strong inhibition of tubulin polymerization and antiproliferative activity against breast and lung cancer cell lines (MCF-7 and A549) at low or sub-micromolar concentrations. A significant apoptotic effect was shown using a flow cytometry assay that corresponded to cytotoxicity against cancer cell lines, as indicated by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. Decanoic acid conjugates were more potent than longer lipid analogues, with the most active being more potent than the reference tubulin inhibitor, combretastatin-A4 and the anticancer drug, doxorubicin. None of the newly synthesized compounds caused any detectable cytotoxicity against the normal cell line (Wi-38) or hemolysis of red blood cells below 100 μM. It is unlikely that the new conjugates described would affect normal cells or interrupt with cell membranes due to their lipidic nature. A quantitative structure-activity relationship analysis was performed to determine the influence of 315 descriptors of the physicochemical properties of the new conjugates on their tubulin inhibitory activity. The obtained model revealed a strong correlation between the tubulin inhibitory activity of the investigated compounds and their dipole moment and degree of reactivity.

Abstract LB324: BB3008, a potent and selective small molecular HPK1 inhibitor effective in multiple syngeneic tumor models

Abstract Introduction: Hematopoietic progenitor kinase 1 (HPK1) is a serine/threonine kinase in the MAP4K family predominantly expressed in immune cells, and has been identified as a negative regulator of T cell and B cell receptor signaling and dendritic cell function. Inhibition of the kinase activity of HPK1 results in the activation of exhausted T cell in tumor microenvironment and enhanced anti-tumor immune response. Observation of HPK1 overexpression in various tumor tissues further indicates the kinase as a novel target for immuno-oncology therapy. Here we report BB3008 as a small molecular HPK1 inhibitor. Methods: BB3008 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice and dogs. Syngeneic tumor models in mice were conducted to demonstrate the in vivo efficacy of BB3008 as a single agent and in combination with mouse PD-1 antibody. Results: BB3008 shows sub-nanomolar HPK1 potency, with good selectivity against kinases in the MAP4K family and other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 by BB3008 strongly suppresses the phosphorylation at Ser376 of the downstream biomarker protein SLP-76, with IC50 at 30 nM. With the stimulation of CD3/CD28, boost of IL-2 production was observed in Jurkat cells, purified human T cells, and human peripheral blood mononuclear cells upon treatment of BB3008 at sub-micromolar concentration. BB3008 also enhanced the killing activity of T cell against EL4 lymphoma cell in vitro in a concentration-dependent manner. PK profile of BB3008 showed good bioavailability (>80%) with a nearly linear dose-dependent exposure. The compound had no significant suppression on various normal cell lines proliferation, and had no hERG liability. In preliminary safety evaluation, no abnormal clinical symptoms and serum/blood tests were observed in mice and dogs dosed up to 1000mpk and 150mpk for 14 days, respectively. BB3008 showed significant tumor-growth inhibition rates in CT26, Hepa 1-6 and 4T1 mouse models as single agent, and in MC-38 mouse model in combination with mouse PD-1 antibody. Conclusion: As a potent, selective and oral available small molecule HPK1 inhibitor, BB3008 shows good safety tolerance in preclinical animals, and promising efficacy in multiple solid tumor models, both as single agent or in combination with immune checkpoint blockade. The IND-enabling study of BB3008 is in progress. Citation Format: Gongping Duan, Min Li, Xingmin Zhang. BB3008, a potent and selective small molecular HPK1 inhibitor effective in multiple syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB324.

Reverse Screening of Boronic Acid Derivatives: Analysis of Potential Antiproliferative Effects on a Triple-Negative Breast Cancer Model In Vitro

It has been demonstrated that different organoboron compounds interact with some well-known molecular targets, including serine proteases, transcription factors, receptors, and other important molecules. Several approaches to finding the possible beneficial effects of boronic compounds include various in silico tools. This work aimed to find the most probable targets for five aromatic boronic acid derivatives. In silico servers, SuperPred, PASS-Targets, and Polypharmacology browser 2 (PPB2) suggested that the analyzed compounds have anticancer properties. Based on these results, the antiproliferative effect was evaluated using an in vitro model of triple-negative breast cancer (4T1 cells in culture). It was demonstrated that phenanthren-9-yl boronic acid and 6-hydroxynaphthalen-2-yl boronic acid have cytotoxic properties at sub-micromolar concentrations. In conclusion, using in silico approaches and in vitro analysis, we found two boronic acid derivatives with potential anticancer activity.

Design, synthesis and biological evaluation of pyrrolopyrimidine derivatives as novel and selective positive modulator of the small conductance Ca2+-activated K+ channels

The type 2 small conductance Ca2+-activated K+ channels (SK2) have been considered as one of the most promising therapeutic targets for spinocerebellar ataxias type 2 (SCA2) by playing a critical role in the control of normal purkinje cells (PCs) pacemaking. Herein, a novel series of pyrrolopyrimidine derivatives were designed and synthesized from the lead compound NS13001 as subtype-selective modulators of SK channels. Among them, the halogen-substituted compound 12b (EC50 = 0.34 ± 0.044 μM) was identified with a ∼5.4-fold higher potency on potentiating SK2-a channels at submicromolar concentrations as compared to NS13001 (EC50 = 1.83 ± 0.50 μM). Furthermore, compound 12b exhibited selectivity on SK2-a/SK3 subtype by displaying 93.33 ± 3.26% efficacies on SK2-a channels, and 84.54% ± 7.49% on SK3 channels. In addition, compound 12b demonstrated the potential to cross the blood-brain barrier (BBB) with suitable pharmacokinetic properties and low cytotoxicity. Molecular docking study also unveiled the binding interactions of compound 12b with SK2-CaM protein complex. Overall, the novel pyrrolopyrimidines provide an insightful guidance for future structural optimization of SK channel agonists.

Observation of Cytotoxicity of Phosphonium Derivatives Is Explained: Metabolism Inhibition and Adhesion Alteration.

The search for new antibiotics, substances that kill prokaryotic cells and do not kill eukaryotic cells, is an urgent need for modern medicine. Among the most promising are derivatives of triphenylphosphonium, which can protect the infected organs of mammals and heal damaged cells as mitochondria-targeted antioxidants. In addition to the antioxidant action, triphenylphosphonium derivatives exhibit antibacterial activity. It has recently been reported that triphenylphosphonium derivatives cause either cytotoxic effects or inhibition of cellular metabolism at submicromolar concentrations. In this work, we analyzed the MTT data using microscopy and compared them with data on changes in the luminescence of bacteria. We have shown that, at submicromolar concentrations, only metabolism is inhibited, while an increase in alkyltriphenylphosphonium (CnTPP) concentration leads to adhesion alteration. Thus, our data on eukaryotic and prokaryotic cells confirm a decrease in the metabolic activity of cells by CnTPPs but do not confirm a cytocidal effect of TPPs at submicromolar concentrations. This allows us to consider CnTPP as a non-toxic antibacterial drug at low concentrations and a relatively safe vector for delivering other antibacterial substances into bacterial cells.

Inhibiting a promiscuous GPCR: iterative discovery of bitter taste receptor ligands.

The human GPCR family comprises circa 800 members, activated by hundreds of thousands of compounds. Bitter taste receptors, TAS2Rs, constitute a large and distinct subfamily, expressed orally and extra-orally and involved in physiological and pathological conditions. TAS2R14 is the most promiscuous member, with over 150 agonists and 3 antagonists known prior to this study. Due to the scarcity of inhibitors and to the importance of chemical probes for exploring TAS2R14 functions, we aimed to discover new ligands for this receptor, with emphasis on antagonists. To cope with the lack of experimental structure of the receptor, we used a mixed experimental/computational methodology which iteratively improved the performance of the predicted structure. The increasing number of active compounds, obtained here through experimental screening of FDA-approved drug library, and through chemically synthesized flufenamic acid derivatives, enabled the refinement of the binding pocket, which in turn improved the structure-based virtual screening reliability. This mixed approach led to the identification of 10 new antagonists and 200 new agonists of TAS2R14, illustrating the untapped potential of rigorous medicinal chemistry for TAS2Rs. 9% of the ~ 1800 pharmaceutical drugs here tested activate TAS2R14, nine of them at sub-micromolar concentrations. The iterative framework suggested residues involved in the activation process, is suitable for expanding bitter and bitter-masking chemical space, and is applicable to other promiscuous GPCRs lacking experimental structures.

Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general “rocker switch” alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9.

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (-)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.

A general highly efficient synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy

The development of live-cell fluorescence nanoscopy is powered by the availability of suitable fluorescent probes. Rhodamines are among the best fluorophores for labeling intracellular structures. Isomeric tuning is a powerful method for optimizing the biocompatibility of rhodamine-containing probes without affecting their spectral properties. An efficient synthesis pathway for 4-carboxyrhodamines is still lacking. We present a facile protecting-group-free 4-carboxyrhodamines’ synthesis based on the nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps, expands the achievable structural diversity, increases overall yields and permits gram-scale synthesis of the dyes. We synthesize a wide range of symmetrical and unsymmetrical 4-carboxyrhodamines covering the whole visible spectrum and target them to multiple structures in living cells – microtubules, DNA, actin, mitochondria, lysosomes, Halo-tagged and SNAP-tagged proteins. The enhanced permeability fluorescent probes operate at submicromolar concentrations, allowing high-contrast STED and confocal microscopy of living cells and tissues.

Pyrazole-Enriched Cationic Nanoparticles Induced Early- and Late-Stage Apoptosis in Neuroblastoma Cells at Sub-Micromolar Concentrations.

Neuroblastoma (NB) is a severe form of tumor occurring mainly in young children and originating from nerve cells found in the abdomen or next to the spine. NB needs more effective and safer treatments, as the chance of survival against the aggressive form of this disease are very small. Moreover, when current treatments are successful, they are often responsible for unpleasant health problems which compromise the future and life of surviving children. As reported, cationic macromolecules have previously been found to be active against bacteria as membrane disruptors by interacting with the negative constituents of the surface of cancer cells, analogously inducing depolarization and permeabilization, provoking lethal damage to the cytoplasmic membrane, and cause loss of cytoplasmic content and consequently, cell death. Here, aiming to develop new curative options for counteracting NB cells, pyrazole-loaded cationic nanoparticles (NPs) (BBB4-G4K and CB1H-P7 NPs), recently reported as antibacterial agents, were assayed against IMR 32 and SHSY 5Y NB cell lines. Particularly, while BBB4-G4K NPs demonstrated low cytotoxicity against both NB cell lines, CB1H-P7 NPs were remarkably cytotoxic against both IMR 32 and SHSY 5Y cells (IC50 = 0.43-0.54 µM), causing both early-stage (66-85%) and late-stage apoptosis (52-65%). Interestingly, in the nano-formulation of CB1H using P7 NPs, the anticancer effects of CB1H and P7 were increased by 54-57 and 2.5-4-times, respectively against IMR 32 cells, and by 53-61 and 1.3-2 times against SHSY 5Y cells. Additionally, based on the IC50 values, CB1H-P7 was also 1-12-fold more potent than fenretinide, an experimental retinoid derivative in a phase III clinical trial, with remarkable antineoplastic and chemopreventive properties. Collectively, due to these results and their good selectivity for cancer cells (selectivity indices = 2.8-3.3), CB1H-P7 NPs represent an excellent template material for developing new treatment options against NB.

Ketamine reduces electrophysiological network activity in cortical neuron cultures already at sub-micromolar concentrations – Impact on TrkB-ERK1/2 signaling

The dissociative anesthetic ketamine regulates cortical activity in a dose-dependent manner. Subanesthetic-dose ketamine has paradoxical excitatory effects which is proposed to facilitate brain-derived neurotrophic factor (BDNF) (a ligand of tropomyosin receptor kinase B, TrkB) signaling, and activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Previous data suggests that ketamine, at sub-micromolar concentrations, induces glutamatergic activity, BDNF release, and activation of ERK1/2 also on primary cortical neurons. We combined western blot analysis with multiwell-microelectrode array (mw-MEA) measurements to examine ketamine's concentration-dependent effects on network-level electrophysiological responses and TrkB-ERK1/2 phosphorylation in rat cortical cultures at 14 days in vitro. Ketamine did not cause an increase in neuronal network activity at sub-micromolar concentrations, but instead a decrease in spiking that was evident already at 500 nM concentration. TrkB phosphorylation was unaffected by the low concentrations, although BDNF elicited prominent phosphorylation response. High concentration of ketamine (10 μM) strongly reduced spiking, bursting and burst duration, which was accompanied with decreased phosphorylation of ERK1/2 but not TrkB. Notably, robust increases in spiking and bursting activity could be produced with carbachol, while it did not affect phosphorylation of TrkB or ERK1/2. Diazepam abolished neuronal activity, which was accompanied by reduced ERK1/2 phosphorylation without change on TrkB. In conclusion, sub-micromolar ketamine concentrations did not cause an increase in neuronal network activity or TrkB-ERK1/2 phosphorylation in cortical neuron cultures that readily respond to exogenously applied BDNF. Instead, pharmacological inhibition of network activity can be readily observed with high concentration of ketamine and it is associated with reduced ERK1/2 phosphorylation.This article is part of the Special Issue on "Ketamine and its Metabolites".

Hormetic Effects of Cerium, Lanthanum and Their Combination at Sub-micromolar Concentrations in Sea Urchin Sperm

Rare earth elements (REEs) cerium (Ce) and lanthanum (La) and their combination were tested across a concentration range, from toxic (10−4 to 10−5 M) to lower concentrations (10−6 to 10−8 M) for their effects on sea urchin (Sphaerechinus granularis) sperm. A significantly decreased fertilization rate (FR) was found for sperm exposed to 10−5 M Ce, La and their combination, opposed to a significant increase of FR following 10−7 and 10−8 M REE sperm exposure. The offspring of REE-exposed sperm showed significantly increased developmental defects following sperm exposure to 10−5 M REEs vs. untreated controls, while exposure to 10−7 and 10−8 M REEs resulted in significantly decreased rates of developmental defects. Both of observed effects–on sperm fertilization success and on offspring quality–were closely exerted by Ce or La or their combination.

Abstract P4-08-21: Development of novel inhibitors of the serum/glucocorticoid induced kinase (SGK) family to address limitations of AKT/PI3K/mTOR inhibitors in breast cancer

Abstract Activation of the AKT/PI3K/mTOR signaling through genetic and epigenetic mechanisms is a frequent event in all subtypes of breast cancer, and it is associated with resistance to various therapies. Development of inhibitors targeting this pathway has been met with limited success so far, mainly due to various feedback and crosstalk mechanisms amongst different members of the pathway. Several therapies that target the PI3K pathway have also been associated with dose limiting toxicities such as hyperglycemia, hyperinsulinemia, skin rashes and infections, limiting their use. The SGK family of kinases has recently been implicated in the resistance to AKT and PI3K inhibitors by promoting AKT-independent signaling to maintain the activation of the AKT/PI3K/mTOR pathway. The SGK and AKT families share similar domain structures as well as upstream activators such as PDK1 and mTORC2, and downstream effectors such as TSC2, FOXO3, and GSK3B, providing a strong rationale for the development of inhibitors targeting the SGK family. Here we present the development of novel small molecule ATP-competitive inhibitors of the SGK family. Several compounds displayed low nanomolar affinity towards SGK1 in whole cell assays, with IC50s ranging between 10-100 nM, which is 30-300 folds and 80-1000 folds higher than the previously characterized SGK1 inhibitors GSK650394 and EMD638683, respectively, as well as good selectivity against related AGC family kinases. Experiments in cells have shown a strong dose-dependent decrease in the phosphorylation of its downstream target NDRG1 at sub-micromolar concentrations, confirming selective targeting of SGK1 in whole cells. Single agent activity of SGK1 inhibition was modest in different cancer cell lines, with IC50 values for inhibition of their proliferation ranging from 1-10 uM, in agreement with the role of the SGK family in regulation of stress-induced signaling and not normal cell proliferation. However, combinations of SGK with AKT inhibitors displayed strong synergies in different breast cancer cell lines, especially in cells with resistance to AKT/PI3K inhibitors. For example, strong Bliss synergy index scores > 18 were obtained with different SGK1 inhibitors when combined with either AKT inhibitors MK-2206, ipatasertib, or capivasertib, in the JIMT-1 breast cancer cell line, which is a model of resistance to AKT, PI3K, and HER2-directed therapies. These results suggest that the combination of SGK with AKT inhibitors could be an effective therapeutic strategy to alleviate toxicities associated with AKT/PI3K/mTOR inhibitors by lowering their required dose, while maintaining anti-tumor activity and delaying the development of resistance. Citation Format: Delphine Labit, Sabindra Pradhananga, Maroua Khalifa, Emma Blackburn, Naheed Sajid, Marc Vidal, Debra Odink, Eric Campeau. Development of novel inhibitors of the serum/glucocorticoid induced kinase (SGK) family to address limitations of AKT/PI3K/mTOR inhibitors in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-21.

High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin.

The Burkholderia cepacia complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. We constructed a library of over 90 000 ubonodin variants with 2 amino acid substitutions and used a high-throughput screen and next-generation sequencing to examine the fitness of the entire library, generating the most comprehensive data set on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a submicromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.