Micromolar Concentrations Research Articles

Exposure to select PFAS and PFAS mixtures alters response to platinum-based chemotherapy in endometrial cancer cell lines

BackgroundExposure to per- and poly-fluoroalkyl substances (PFAS) has been associated with significant alterations in female reproductive health. These include changes in menstrual cyclicity, timing of menarche and menopause, and fertility outcomes, as well as increased risk of endometriosis, all of which may contribute to an increased risk of endometrial cancer. The effect of PFAS on endometrial cancer cells, specifically altered treatment response and biology, however, remains poorly studied. Like other gynecologic malignancies, a key contributor to lethality in endometrial cancer is resistance to chemotherapeutics, specifically to platinum-based agents that are used as the standard of care for patients with advanced-stage and/or recurrent disease.ObjectivesTo explore the effect of environmental exposures, specifically PFAS, on platinum-based chemotherapy response and mitochondrial function in endometrial cancer.MethodsHEC-1 and Ishikawa endometrial cancer cells were exposed to sub-cytotoxic nanomolar and micromolar concentrations of PFAS/PFAS mixtures and were treated with platinum-based chemotherapy. Survival fraction was measured 48-h post-chemotherapy treatment. Mitochondrial membrane potential was evaluated in both cell lines following exposure to PFAS ± chemotherapy treatment.ResultsHEC-1 and Ishikawa cells displayed differing outcomes after PFAS exposure and chemotherapy treatment. Cells exposed to PFAS appeared to be less sensitive to carboplatin, with instances of increased survival fraction, indicative of platinum resistance, observed in HEC-1 cells. In Ishikawa cells treated with cisplatin, PFAS mixture exposure significantly decreased survival fraction. In both cell lines, increases in mitochondrial membrane potential were observed post-PFAS exposure ± chemotherapy treatment.DiscussionExposure of endometrial cancer cell lines to PFAS/PFAS mixtures had varying effects on response to platinum-based chemotherapies. Increased survival fraction post-PFAS + carboplatin treatment suggests platinum resistance, while decreased survival fraction post-PFAS mixture + cisplatin exposure suggests enhanced therapeutic efficacy. Regardless of chemotherapy sensitivity status, mitochondrial membrane potential findings suggest that PFAS exposure may affect endometrial cancer cell mitochondrial functioning and should be explored further.

Kinetic Modeling Assisted Analysis of Vitamin C-Mediated Copper Redox Transformations in Aqueous Solutions.

The kinetics of oxidation of micromolar concentrations of ascorbic acid (AA) catalyzed by Cu(II) in solutions representative of biological and environmental aqueous systems has been investigated in both the presence and absence of oxygen. The results reveal that the reaction between AA and Cu(II) is a relatively complex set of redox processes whereby Cu(II) initially oxidizes AA yielding the intermediate ascorbate radical (A•-) and Cu(I). The rate constant for this reaction was determined to have a lower limit of 2.2 × 104 M-1 s-1. Oxygen was found to play a critical role in mediating the Cu(II)/Cu(I) redox cycle and the oxidation reactions of AA and its oxidized forms. Among these processes, the oxidation of the ascorbate radical by molecular oxygen was identified to play a key role in the consumption of ascorbic acid, despite being a slow reaction. The rate constant for this reaction () was determined for the first time with a calculated value of 54 ± 8 M-1 s-1. The kinetic model developed satisfactorily describes the Cu/AA/O2 system over a range of conditions including different concentrations of NaCl (0.2 and 0.7 M) and pH (7.4 and 8.1). Appropriate adjustments to the rate constant for the reaction between Cu(I) and O2 were found to account for the influence of the chloride ions and pH on the kinetics of the process. Additionally, the presence of Cu(III) as the primary oxidant resulting from the interaction between Cu(I) and H2O2 in the Cu(II)/AA system was confirmed, along with the coexistence of HO•, possibly due to an equilibrium established between Cu(III) and HO•.

Elongation of N6-benzyladenosine scaffold via Pd-catalyzed C–C bond formation leads to derivatives with antiflaviviral activity

Decoration of nucleoside analogues with lipophilic groups often leads to compounds with improved antiviral activity. For example, N6-benzyladenosine derivatives containing elongated lipophilic substituents in the benzyl core efficiently inhibit reproduction of tick-borne encephalitis virus (TBEV), while N6-benzyladenosine itself potently inhibits reproduction of human enterovirus A71 (EV-A71). We have extended a series of N6-benzyladenosine analogues using effective synthetic methods of CC bond formation based on Pd-catalyzed cross-coupling reactions (Sonogashira and Suzuki) in order to study the influence of bulky lipophilic substituents in the N6 position of adenosine on the antiviral activity against flaviviruses, such as TBEV, yellow fever virus (YFV) and West Nile virus (WNV), as well as a panel of enteroviruses including EV-A71, Echovirus 30 (E30), and poliovirus type 2 (PV2). Reproduction of tested flaviviruses appeared to be inhibited by the micromolar concentrations of the compounds, while cytotoxicity in most cases was beyond the detection limit. Time-of-addition studies demonstrated that the hit compounds inhibited the stage of viral RNA synthesis, but not the stages of the viral entry or protein translation. As a result, several new promising antiflaviviral leads have been identified. On the other hand, none of the synthesized compounds inhibited enterovirus reproduction, indicating a possibility of involvement of flavivirus-specific pathways in their mechanism of action.

Investigating the Cell Entry Mechanism, Disassembly, and Toxicity of the Nanocage PCC-1: Insights into Its Potential as a Drug Delivery Vehicle.

The porous coordination cage PCC-1 represents a new platform potentially useful for the cellular delivery of drugs with poor cell permeability and solubility. PCC-1 is a metal-organic polyhedron constructed from zinc metal ions and organic ligands through coordination bonds. PCC-1 possesses an internal cavity that is suitable for drug encapsulation. To better understand the biocompatibility of PCC-1 with human cells, the cell entry mechanism, disassembly, and toxicity of the nanocage were investigated. PCC-1 localizes in the nuclei and cytoplasm within minutes upon incubation with cells, independent of endocytosis and cargo, suggesting direct plasma membrane translocation of the nanocage carrying its guest in its internal cavity. Furthermore, the rates of cell entry correlate to extracellular concentrations, indicating that PCC-1 is likely diffusing passively through the membrane despite its relatively large size. Once inside cells, PCC-1 disintegrates into zinc metal ions and ligands over a period of several hours, each component being cleared from cells within 1 day. PCC-1 is relatively safe for cells at low micromolar concentrations but becomes inhibitory to cell proliferation and toxic above a concentration or incubation time threshold. However, cells surviving these conditions can return to homeostasis 3-5 days after exposure. Overall, these findings demonstrate that PCC-1 enters live cells by crossing biological membranes spontaneously. This should prove useful to deliver drugs that lack this capacity on their own, provided that the dosage and exposure time are controlled to avoid toxicity.

Anti-Tuberculosis Potential of OJT008 against Active and Multi-Drug-Resistant Mycobacterium Tuberculosis: In Silico and In Vitro Inhibition of Methionine Aminopeptidase.

Despite the recent progress in the diagnosis of tuberculosis (TB), the chemotherapeutic management of TB continues to be challenging. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is classified as the 13th leading cause of death globally. In addition, 450,000 people were reported to develop multi-drug-resistant TB globally. The current project focuses on targeting methionine aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the excision of the N-terminal methionine (NME) during protein synthesis, allowing the enzyme to be an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms, MtMetAP1a and MtMetAP1c, which are vital for Mtb viability and, hence, a promising chemotherapeutic target for Mtb therapy. In this study, we cloned and overexpressed recombinant MtMetAP1c. We investigated the in vitro inhibitory effect of the novel MetAP inhibitor, OJT008, on the cobalt ion- and nickel ion-activated MtMetAP1c, and the mechanism of action was elucidated through an in silico approach. The compound's potency against replicating and multi-drug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at 8 h with a final concentration of 1 mM Isopropyl β-D-1-thiogalactopyranoside. The average yield from 1 L of Escherichia coli culture for MtMetAP1c was 4.65 mg. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions occurred at 100 µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were 11 µM and 40 µM, respectively. The in silico study showed OJT008 strongly binds to both metal-activated MtMetAP1c, as evidenced by strong molecular interactions and a higher binding score, thereby corroborating our result. This in silico study validated the pharmacophore's metal specificity. The potency of OJT008 against both active and MDR Mtb was <0.063 µg/mL. Our study reports OJT008 as an inhibitor of MtMetAP1c, which is potent at low micromolar concentrations against both active susceptible and MDR Mtb. These results suggest OJT008 is a potential lead compound for the development of novel small molecules for the therapeutic management of TB.

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria.

Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (∼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. KEY POINTS: Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non-invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non-invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.

Scots pine defensins inhibit Ips acuminatus α-amylase activity

Background. Pine bark beetle Ips acuminatus (Gyllenhal, 1827) is one of the most harmful pests of pine trees as it affects the phloem of the upper part of the stem and branches, disrupting the flow of nutrients and water to the crown. I. acuminatus feeds by plant tissues rich in starch, so α-amylases must play a pivotal role in the carbohydrate metabolism of these insects. However, in conifer bark beetles, α-amylases remain poorly understood. Materials and Methods. To detect the α-amylase activity in the digestive system of I. acuminatus, we obtained extracts from larvae, pupae, and adults that were collected from naturally infested Scots pine. The α-amylase activity of crude extracts from different stages and parts of the bark beetle’s body was assessed using 1% starch agar plates. The quantitative evaluation of the α-amylase inhibitory activity of recombinant defensins PsDef1, PsDef2, and PsDef5.1 was performed using the Bernfeld method. The docking models of Scots pine defensins and Ips typographus L. α-amylase (AmyIp) complexes were predicted using the ClusPro 2.0 web server. Results and Discussion. As a result, we found the presence of α-amylase activity in the digestive systems of both larvae and adults of I. acuminatus, but not in pupae. All tested defensins, PsDef1, PsDef2, and PsDef5.1, exhibited inhibitory activity against insect α-amylase at micromolar concentrations. The IC50 values for these peptides were 4.9±0.6 μM, 4.6±0.8 μM, and 2.8±0.5 μM, respectively. In the PsDefs-AmyIt complexes, a network of hydrogen bonds, ionic bridges, and nonbonded contacts are formed between the enzyme and its inhibitor, which prevents the substrate from reaching the catalytic site. The PsDef5.1-AmyIt complex has the largest interfacial contact area, 2328 Å2, in comparison with two other defensins, which correlates well with the inhibitory activity of defensins in this study. Conclusion. Thus, we have identified α-amylase activity in I. acuminatus and demonstrated the ability of Scots pine defensins to inhibit it, sugges­ting that they play a role in pine defenses against this pest.

Photoinduced Destruction of Complex Cyanides Using Quasi-Monochromatic UVC Radiation of a KrCl Excilamp (222 nm)

The kinetic regularities of photochemical oxidation of stable complex cyanides (hexacyanoferrates) with persulfate (oxidizing system {UV/S2O}) and hydrogen peroxide (oxidizing system {UV/H2O2}) under the influence of quasi-monochromatic UVC radiation from a KrCl excilamp (222 nm) have been studied. According to the efficiency and rate of the destruction of the target compound, the oxidizing systems under study can be arranged in the following series: {UV/S2O2-8} {UV/H2O2} {UV}. The effective destruction of hexacyanoferrates at micromolar concentrations (≤47 μM) to nontoxic and biodegradable compounds in the combined {UV/S2O2-8} system is due to the high oxidizing ability of reactive oxygen species formed as a result of persulfate photolysis.

Broad-spectrum antiviral activity of two structurally analogous CYP3A inhibitors against pathogenic human coronaviruses in vitro

Coronaviruses pose a permanent risk of outbreaks, with three highly pathogenic species and strains (SARS-CoV, MERS-CoV, SARS-CoV-2) having emerged in the last twenty years. Limited antiviral therapies are currently available and their efficacy in randomized clinical trials enrolling SARS-CoV-2 patients has not been consistent, highlighting the need for more potent treatments. We previously showed that cobicistat, a clinically approved inhibitor of Cytochrome P450-3A (CYP3A), has direct antiviral activity against early circulating SARS-CoV-2 strains in vitro and in Syrian hamsters. Cobicistat is a derivative of ritonavir, which is co-administered as pharmacoenhancer with the SARS-CoV-2 protease inhibitor nirmatrelvir, to inhibit its metabolization by CPY3A and preserve its antiviral efficacy. Here, we used automated image analysis for a screening and parallel comparison of the anti-coronavirus effects of cobicistat and ritonavir. Our data show that both drugs display antiviral activity at low micromolar concentrations against multiple SARS-CoV-2 variants in vitro, including epidemiologically relevant Omicron subvariants. Despite their close structural similarity, we found that cobicistat is more potent than ritonavir, as shown by significantly lower EC50 values in monotherapy and higher levels of viral suppression when used in combination with nirmatrelvir. Finally, we show that the antiviral activity of both cobicistat and ritonavir is maintained against other human coronaviruses, including HCoV-229E and the highly pathogenic MERS-CoV. Overall, our results demonstrate that cobicistat has more potent anti-coronavirus activity than ritonavir and suggest that dose adjustments could pave the way to the use of both drugs as broad-spectrum antivirals against highly pathogenic human coronaviruses.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

De novo design of a dual-emissive self-reporting photosensitizer with high membrane permeability via chain-engineering strategy

Photodynamics therapy (PDT) is an important and advantageous method for the selective ablation of tumor cells. However, the reported PDT sensitizers remain two critical problems: firstly, dual-emissive self-reporting photosensitizers have not been reported for the monitoring and control of light irradiation; secondly, high loading concentration (usually at micromolar concentration) of the photosensitizers was used. Herein, we have designed a dual-emissive self-reporting photosensitizer with low loading concentration (200 nM) to resolve the two knots. 4-Aminostyryl-methyl-benzothiazolium was selected as the photosensitizer, which can generate 1O2 upon light irradiation, and displayed red-shifted absorption when binding to RNA. Meanwhile different chains were modified to optimize the RNA affinity and membrane permeability concurrently. Finally, the photosensitizer with C4 chain (NPT-4) was screened out. NPT-4 targeted mitochondria in living cells to give green and red fluorescence with the excitation of 488 nm and 561 nm, respectively. During cell death, NPT-4 translocated into RNA to give dramatically decreased green emission and unchanged red fluorescence. Therefore, NPT-4 simultaneously induced and reported the damages of tumor cells upon light irradiation in dual-emissive manner. Further, NPT-4 exhibited high membrane permeability, and 200 nM NPT-4 can brightly light up the cells and efficiently induce cell damages with light irradiation. Accordingly, NPT-4 is expected to be an important self-reporting photosensitizer for PDT and relative studies.

Repurposing GSK3B Small Molecule Inhibitors for Refractory Lymphoid Malignancies

Background: Small molecule GSK3B inhibitors were developed for solid tumors and neurological conditions, including Alzheimer's and Parkinson disease. Five GSK3B inhibitors achieved favorable safety and PK/PD profiles in a total of 22 Phase 1 and 2 clinical trials but no clinical response for any of these indications. GSK3B is the kinase that phosphorylates β-catenin to initiate its degradation, hence GSK3B small molecule inhibition results in rapid nuclear accumulation of β-catenin protein. Significance: Activating mutations in the Wnt/β-catenin pathway are common oncogenic drivers throughout all main types of cancer (n=64,812) but not found in B- and T-lymphoid malignancies (n=2,137). Studying tissue microarrays by β-catenin immunohistochemistry, lung, colon, breast, and melanoma specimen (n=57) showed consistently high levels of nuclear β-catenin, whereas B- and T-cell lymphomas (n=84) lacked β-catenin signal. Despite comparable mRNA levels, proteomic analyses revealed 60-400-fold lower β-catenin levels in lymphoid compared to epithelial tumors (n=1,389) suggesting that lymphoid malignancies rely on powerful mechanisms for β-catenin degradation, e.g. by GSK3B. Results: To study the effects of β-catenin activation in human cells, we induced expression of stabilized form of β-catenin in 18 lymphoid (B- and T-ALL, MCL, DLBCL, PTCL) and four myeloid leukemia cell lines. In B- and T-lymphoid cells, β-catenin accumulation compromised clonal fitness, colony formation and induced cell death, but increased competitive fitness and colony formation in myeloid leukemia cells. CEBPα-mediated reprogramming of B-ALL cells into a myeloid phenotype reversed the deleterious effects highlighting that effects of β-catenin-accumulation strictly depend on lymphoid lineage-identity. RNA-seq studies upon inducible activation of β-catenin in lymphoid cells, revealed transcriptional repression of Myc and Myc target genes as principal gene expression change. Unlike activating β-catenin:TCF7 complexes in epithelial cells, our global interactome studies in B- and T-ALL and lymphoma cells identified repressive β-catenin complexes with lymphoid-specific Ikaros zinc finger (IKZF1/2/3) proteins and several components of the NuRD complex including Mta1/2, Gatad2a/b, Chd4, HDAC1/2. Interactome and ChIP-seq analyses revealed that β-catenin together with lymphoid-specific Ikaros factors recruited repressive NuRD complex members to deacetylate H3K27 and repress MYC superenhancer (SE) regions. Genetic deletion of both IKZF1 and IKZF3 or HDRT-based mutation of Ikaros binding motifs within the Myc-SE enabled resistance to β-catenin accumulation. To leverage this previously unrecognized vulnerability of lymphoid malignancies, we examined five clinically approved GSK3B small molecule inhibitors that achieved favorable safety profiles at micromolar plasma concentrations in clinical trials for neurological disorders and solid tumors. Strikingly, LY2090314 and CHIR99021 were effective at low nanomolar concentrations in B- and T-cell malignancies that expressed lymphoid Ikaros factors. At IC50 concentrations of &amp;lt;5 nM, GSK3B-inhibitors induced massive accumulation of β-catenin, repression of MYC and acute cell death. In B-ALL cells with IKZF1-deletion, expression of the remaining IKZF3 factor was sufficient to retain full sensitivity to GSK3B-inhibition. Deletion of β-catenin, however, abolished the effects of GSK3B inhibitors and relieved suppression of MYC, demonstrating accumulation of β-catenin represents as central mechanism of action. Preclinical in vivo treatment experiments of refractory B-ALL and mantle cell lymphoma PDX validated small molecule GSK3B-inhibition as strategy to overcome conventional mechanisms of drug-resistance. Conclusions: B- and T-lymphoid malignancies not only lacked expression of β-catenin but critically depend on GSK3B for effective β-catenin degradation. Our interactome studies in lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros and NuRD complex factors. Strikingly, GSK3B small molecule inhibitors engage this new pathway at low nanomolar concentrations and demonstrated favorable safety profiles in Phase 1 and Phase 2 trials. Safety and efficacy of GSK3B-inhibitors opens up an immediate path for repurposing these agents towards refractory B-cell malignancies.

Triclocarban exhibits higher adipogenic activity than triclosan through peroxisome proliferator-activated receptors pathways

Previous epidemiological and animal studies have showed the lipid metabolic disruption of antimicrobial triclocarban (TCC) and triclosan (TCS). However, the present in vivo researches were mainly devoted to the hepatic lipid metabolism, while the evidence about the impacts of TCC/TCS on the adipose tissue is very limited and the potential mechanism is unclear, especially the molecular initiation events. Moreover, little is known about the toxic difference between TCC and TCS. This study aimed to demonstrate the differential adipogenic activity of TCC/TCS as well as the potential molecular mechanism via peroxisome proliferator-activated receptors (PPARα/β/γ). The in vitro experiment based on 3T3-L1 cells showed that TCC/TCS promoted the differentiation of preadipocytes into mature adipocytes at nanomolar to micromolar concentrations, which was approach to their human exposure levels. We revealed for the first time by reporter gene assay that TCC could activate three PPARs signaling pathways in a concentration-dependent manner, while TCS only activate PPARβ. The molecular docking strategy was applied to simulate the interactions of TCC/TCS with PPARs, which explained well the different PPARs activities between TCC and TCS. TCC up-regulated the mRNA expression of three PPARs, but TCS only up-regulated PPARβ and PPARγ significantly. Meanwhile, TCC/TCS also promoted the expression of adipogenic genes targeted by PPARs to different extent. The cellular and simulating studies demonstrated that TCC exerted higher adipogenic effects and PPARs activities than TCS. Our mice in vivo experiment showed that TCC could lead to adipocyte size increase, adipocyte lipid accumulation growing, fat weight and body weight gain at human-related exposure levels, and high fat diet exacerbated these effects. Moreover, male mice tended to be more susceptible to TCC induced obesogenic effect than female mice. This work highlights the potential obesogenic risks of TCC/TCS via PPARs signaling pathways, and TCC deserves more concerns for its higher activity.

Effect of Low Concentration of Nitroxides on SH-SY5Y Cells Transfected with the Tau Protein

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 μM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2′-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS•) decolorization assay and reduced Fe3+, inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters.

Unraveling viral drug targets: a deep learning-based approach for the identification of potential binding sites.

The coronavirus disease 2019 (COVID-19) pandemic has spurred a wide range of approaches to control and combat the disease. However, selecting an effective antiviral drug target remains a time-consuming challenge. Computational methods offer a promising solution by efficiently reducing the number of candidates. In this study, we propose a structure- and deep learning-based approach that identifies vulnerable regions in viral proteins corresponding to drug binding sites. Our approach takes into account the protein dynamics, accessibility and mutability of the binding site and the putative mechanism of action of the drug. We applied this technique to validate drug targeting toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein S. Our findings reveal a conformation- and oligomer-specific glycan-free binding site proximal to the receptor binding domain. This site comprises topologically important amino acid residues. Molecular dynamics simulations of Spike in complex with candidate drug molecules bound to the potential binding sites indicate an equilibrium shifted toward the inactive conformation compared with drug-free simulations. Small molecules targeting this binding site have the potential to prevent the closed-to-open conformational transition of Spike, thereby allosterically inhibiting its interaction with human angiotensin-converting enzyme 2 receptor. Using a pseudotyped virus-based assay with a SARS-CoV-2 neutralizing antibody, we identified a set of hit compounds that exhibited inhibition at micromolar concentrations.

High-throughput screening of compounds targeting RNA editing in Trypanosoma brucei: Novel molecular scaffolds with broad trypanocidal effects

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.

Crustacean cardioactive peptide signaling system in the gastropod mollusk Pacific abalone

Crustacean cardioactive peptide (CCAP) signaling systems have been characterized in a diverse range of protostomes, representatively in arthropods. The cyclic CX5C-type CCAP regulates various biological activities through CCAP receptors (CCAPRs), which are orthologous to neuropeptide S receptors (NPSRs) in deuterostomes. However, the CCAPRs of the lophotrochozoa remain poorly characterized; therefore, the relationship between the CCAP, NPS, and CX4C-type oxytocin/vasopressin (OT/VP) signaling systems is unclear. In this study, we identified a CCAP precursor and two CCAPR isoforms in the Pacific abalone (Haliotis discus hannai; Hdh). The Hdh-CCAP precursor was found to harbor three CX5C-type and one CX4C-type CCAPs. The Hdh-CCAPRs displayed homology with protostome CCAPRs and deuterostome NPSRs, having characteristics of the rhodopsin-type G protein-coupled receptors. Phylogenetic analysis showed that lophotrochozoan CCAPRs, including Hdh-CCAPRs, form a monophyletic group distinct from arthropod CCAPRs. Reporter assays demonstrated that all examined Hdh-CCAPs and insect CCAP-induced intracellular Ca2+ mobilization and cAMP accumulation in Hdh-CCAPR-expressing HEK293 cells, whereas none of the CCAP peptides inhibited the forskolin-stimulated cAMP signaling pathway even at micromolar concentrations. In silico ligand-receptor docking models showed that the N-terminal FCN motifs of Hdh-CCAPs are deeply inserted inside the binding pocket of Hdh-CCAPR, forming extensive hydrophobic interactions. In mature Pacific abalone, the transcripts for Hdh-CCAP precursor and Hdh-CCAPR were highly expressed in the neural ganglia compared to the peripheral tissues. Collectively, this study characterized the first CCAP signaling system linked to both Ca2+/PKC and cAMP/PKA signal transduction pathways in gastropod mollusks and gives insights into the evolutional origins of deuterostomian NPS and OT/VP signaling systems.

Investigating the Insertion Mechanism of Cell-Penetrating Peptide Penetratin into Cell Membranes: Implications for Targeted Drug Delivery

The cell-penetrating peptide (CPP) penetratin (PEN) has garnered attention for its potential to enter tumor cells. However, its translocation mechanism and lack of selectivity remain debated. This study investigated PEN’s insertion into healthy cells (H-) and cancer cells (C-) using micromolar concentrations and various techniques. Raman spectroscopy was used to determine PEN’s location in the lipid bilayer at different lipid-to-peptide ratios. Dynamic light scattering (DLS) and zeta potential analysis were used to measure the lipid–PEN complex’s size and charge. The results showed helical PEN particles directly inserted into C- membranes at a ratio of 110, while aggregated particles stayed on H- surfaces. Raman spectroscopy and scanning electron microscopy confirmed PEN insertion in C- membranes. Zeta potential studies revealed highly negative charges for PEN–C- complexes and neutral charges for PEN–H- complexes at pH 6.8. C- integrity remained unchanged at a ratio of 110. Specific lipid-to-peptide ratios with dipalmitoylphosphatidylserine (DPPS) were crucial for direct insertion. These results provide valuable insights into CPP efficacy for targeted drug delivery in cancer cells, considering membrane composition and lipid-to-peptide ratios.

Analogs of FTY720 inhibit TRPM7 but not S1PRs and exert multimodal anti-inflammatory effects.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.

The apocarotenoid β-ionone regulates the transcriptome of Arabidopsis thaliana and increases its resistance against Botrytis cinerea.

Carotenoids are isoprenoid pigments indispensable for photosynthesis. Moreover, they are the precursor of apocarotenoids, which include the phytohormones abscisic acid (ABA) and strigolactones (SLs) as well as retrograde signaling molecules and growth regulators, such as β-cyclocitral and zaxinone. Here, we show that the application of the volatile apocarotenoid β-ionone (β-I) to Arabidopsis plants at micromolar concentrations caused a global reprogramming of gene expression, affecting thousands of transcripts involved in stress tolerance, growth, hormone metabolism, pathogen defense, and photosynthesis. This transcriptional reprogramming changes, along with induced changes in the level of the phytohormones ABA, jasmonic acid, and salicylic acid, led to enhanced Arabidopsis resistance to the widespread necrotrophic fungus Botrytis cinerea (B.c.) that causes the gray mold disease in many crop species and spoilage of harvested fruits. Pre-treatment of tobacco and tomato plants with β-I followed by inoculation with B.c. confirmed the effect of β-I in increasing the resistance to this pathogen in crop plants. Moreover, we observed reduced susceptibility to B.c. in fruits of transgenic tomato plants overexpressing LYCOPENE β-CYCLASE, which contains elevated levels of endogenous β-I, providing a further evidence for its effect on B.c. infestation. Our work unraveled β-I as a further carotenoid-derived regulatory metabolite and indicates the possibility of establishing this natural volatile as an environmentally friendly bio-fungicide to control B.c.