Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

Covalent Polymer-RNA Conjugates for Potent Activation of the RIG-I Pathway.

RNA ligands of retinoic acid-inducible gene I (RIG-I) are a promising class of oligonucleotide therapeutics with broad potential as antiviral agents, vaccine adjuvants, and cancer immunotherapies. However, their translation has been limited by major drug delivery barriers, including poor cellular uptake, nuclease degradation, and an inability to access the cytosol where RIG-I is localized. Here this challenge is addressed by engineering nanoparticles that harness covalent conjugation of 5'-triphospate RNA (3pRNA) to endosome-destabilizing polymers. Compared to 3pRNA loaded into analogous nanoparticles via electrostatic interactions, it is found that covalent conjugation of 3pRNA improves loading efficiency, enhances immunostimulatory activity, protects against nuclease degradation, and improves serum stability. Additionally, it is found that 3pRNA could be conjugated via either a disulfide or thioether linkage, but that the latter is only permissible if conjugated distal to the 5'-triphosphate group. Finally, administration of 3pRNA-polymer conjugates to mice significantly increases type-I interferon levels relative to analogous carriers that use electrostatic 3pRNA loading. Collectively, these studies have yielded a next-generation polymeric carrier for in vivo delivery of 3pRNA, while also elucidating new chemical design principles for covalent conjugation of 3pRNA with potential to inform the further development of therapeutics and delivery technologies for pharmacological activation of RIG-I.

Photoinduced in situ generation of DNA-targeting ligands: DNA-binding and DNA-photodamaging properties of benzo[c]quinolizinium ions.

The photoreactions of selected styrylpyridine derivatives to the corresponding benzo[c]quinolizinium ions are described. It is shown that these reactions are more efficient in aqueous solution (97-44%) than in organic solvents (78-20% in MeCN). The quinolizinium derivatives bind to DNA by intercalation with binding constants of 6-11 × 104 M-1, as shown by photometric and fluorimetric titrations as well as by CD- and LD-spectroscopic analyses. These ligand-DNA complexes can also be established in situ upon irradiation of the styrylpyridines and formation of the intercalator directly in the presence of DNA. In addition to the DNA-binding properties, the tested benzo[c]quinolizinium derivatives also operate as photosensitizers, which induce DNA damage at relative low concentrations and short irradiation times, even under anaerobic conditions. Investigations of the mechanism of the DNA damage revealed the involvement of intermediate hydroxyl radicals and C-centered radicals. Under aerobic conditions, singlet oxygen only contributes to marginal extent to the DNA damage.

Optimization, characterization, and cytotoxicity studies of novel anti-tubercular agent-loaded liposomal vesicles.

The treatment of tuberculosis is still a challenging process due to the widespread of pathogen strains resistant to antibacterial drugs, as well as the undesirable effects of anti-tuberculosis therapy. Hence, the development of safe and effective new anti-antitubercular agents, in addition to suitable nanocarrier systems, has become of utmost importance and necessity. Our research aims to develop liposomal vesicles that contain newly synthesized compounds with antimycobacterial action. The compound being studied is a derivative of imidazo-tetrazine named 3-(3,5-dimethylpyrazole-1-yl)-6-(isopropylthio) imidazo [1,2-b] [1,2,4,5] tetrazine compound. Several factors that affect liposomal characteristics were studied. The maximum encapsulation efficiency was 53.62 ± 0.09. The selected liposomal formulation T8* possessed a mean particle size of about 205.3 ± 3.94 nm with PDI 0.282, and zeta potential was + 36.37 ± 0.49 mv. The results of the in vitro release study indicated that the solubility of compound I was increased by its incorporation in liposomes. The free compound and liposomal preparation showed antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC 27294) at MIC value 0.94-1.88 µg/ml. We predict that the liposomes may be a good candidate for delivering new antitubercular drugs.

Methyltransferase Set7/9 controls PARP1 expression and regulates cisplatin response of breast cancer cells.

The protein-specific methyltransferase Set7/9 is known for its ability to add methyl groups to lysine residues on many targets, including as histones H1.4, H2A, H2B, H3, and non-histone proteins such as p53, NFκB, E2F1, pRb, Hif1α, ß-catenin, STAT3, and YY1 transcription factors. Set7/9 affects both the landscape of histone modifications and the functionality of the aforementioned TFs, and acts as an essential mediator of vital cellular functions, regulating tumor growth and the neoplastic transformation of normal cells. The number of studies demonstrating the determining role of Set7/9 in cancer is growing. Importantly, the effect of Set7/9 on tumor progression is ambivalent and cancer-type dependent. In this study we analyzed the potential participation of Set7/9 in the essential cellular processes in breast cancer cells and revealed that Set7/9 may be involved in DNA damage signaling and DNA repair processes. We further demonstrated that Set7/9 expression is downregulated in cancerous breast tissues and inversely correlated to PARP1 expression level. Using breast cancer cell lines of HER2-positive and triple negative subtypes we have shown that the attenuation of Set7/9 led to the stabilization of PARP1 on both mRNA and protein levels that in turn resulted in cisplatin resistance acquiring. Finally, we demonstrated that the combination of cisplatin with FDA approved PARP1 inhibitor niraparib (Zejula) has a synergistic effect with cisplatin and thereby allows to overcome cisplatin resistance of Set7/9 deficient breast cancer cells.

Synthesis of new acyclic chelators H4aPyta and H6aPyha and their complexes with Cu2+, Ga3+, Y3+, and Bi3.

In this article, we present the synthesis and characterization of new acyclic pyridine-containing polyaminocarboxylate ligands H4aPyta and H6aPyha, which differ in structural rigidity and the number of chelating groups. Their abilities to form complexes with Cu2+, Ga3+, Y3+, and Bi3+ cations, as well as the stability of the complexes, were evaluated by potentiometric titration method, radiolabeling with the corresponding radionuclides, in vitro studies, mass spectrometry, and HPLC. The structures of the resulting complexes were determined using NMR spectroscopy and DFT calculations. The results obtained made it possible to evaluate the influence of the structural features of the complexes on their stability. The developed chelators H4aPyta and H6aPyha were proved to be promising for further research in the field of radiopharmaceuticals.

Novel Substituted Azoloazines with Anticoagulant Activity.

Hypercytokinemia, or cytokine storm, often complicates the treatment of viral and bacterial infections, including COVID-19, leading to the risk of thrombosis. However, the use of currently available direct anticoagulants for the treatment of COVID-19 patients is limited due to safety reasons. Therefore, the development of new anticoagulants remains an urgent task for organic and medicinal chemistry. At the same time, new drugs that combine anticoagulant properties with antiviral or antidiabetic activity could be helpfull in the treatment of COVID-19 patients, especially those suffering from such concomitant diseases as arterial hypertension or diabetes. We have synthesized a number of novel substituted azoloazines, some of which have previously been identified as compounds with pronounced antiviral, antibacterial, antidiabetic, antiaggregant, and anticoagulant activity. Two compounds from the family of 1,2,4-triazolo[1,5-a]pyrimidines have demonstrated anticoagulant activity at a level exceeding or at least comparable with that of dabigatran etexilate as the reference compound. 7,5-Di(2-thienyl)-4,5-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine has shown the highest ability to prolong the thrombin time, surpassing this reference drug by 2.2 times. This compound has also exhibited anticoagulant activity associated with the inhibition of thrombin (factor IIa). Moreover, the anticoagulant effect of this substance becomes enhanced under the conditions of a systemic inflammatory reaction.

Phytochemicals Target Multiple Metabolic Pathways in Cancer.

Cancer metabolic reprogramming is a complex process that provides malignant cells with selective advantages to grow and propagate in the hostile environment created by the immune surveillance of the human organism. This process underpins cancer proliferation, invasion, antioxidant defense, and resistance to anticancer immunity and therapeutics. Perhaps not surprisingly, metabolic rewiring is considered to be one of the "Hallmarks of cancer". Notably, this process often comprises various complementary and overlapping pathways. Today, it is well known that highly selective inhibition of only one of the pathways in a tumor cell often leads to a limited response and, subsequently, to the emergence of resistance. Therefore, to increase the overall effectiveness of antitumor drugs, it is advisable to use multitarget agents that can simultaneously suppress several key processes in the tumor cell. This review is focused on a group of plant-derived natural compounds that simultaneously target different pathways of cancer-associated metabolism, including aerobic glycolysis, respiration, glutaminolysis, one-carbon metabolism, de novo lipogenesis, and ß-oxidation of fatty acids. We discuss only those compounds that display inhibitory activity against several metabolic pathways as well as a number of important signaling pathways in cancer. Information about their pharmacokinetics in animals and humans is also presented. Taken together, a number of known plant-derived compounds may target multiple metabolic and signaling pathways in various malignancies, something that bears great potential for the further improvement of antineoplastic therapy.

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN domain.

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5' cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here, we determine high-resolution cryoelectron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain.

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5'-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

In Silico Discovery of Group II Intron RNA Splicing Inhibitors.

Here, we describe the discovery of compounds that inhibit self-splicing in group II introns. Using docking calculations, we targeted the catalytic active site within the Oceanobacillus iheyensis group IIC intron and virtually screened a library of lead-like compounds. From this initial virtual screen, we identified three unique scaffolds that inhibit splicing in vitro. Additional tests revealed that an analog of the lead scaffold inhibits splicing in an intron-dependent manner. Furthermore, this analog exhibited activity against the group II intron from a different class: the yeast ai5γ IIB intron. The splicing inhibitors we identified could serve as chemical tools for developing group II intron-targeted antifungals, and, more broadly, our results highlight the potential of in silico techniques for identifying bioactive hits against structured and functionally complex RNAs.

Insights into Actinium Complexes with Tetraacetates─AcBATA versus AcDOTA: Thermodynamic, Structural, and Labeling Properties.

In the current research, we conducted a comparative study of the Ac3+ complex with H4DOTA and H4BATA. The stability constants of the [AcBATA]- and [AcDOTA]- complexes were studied directly by extraction methods. We discovered that the thermodynamic properties of the [AcBATA]- complex are superior to those of [AcDOTA]-. Moreover, the fast kinetics of H4BATA complexation with Ac3+ during the radiolabeling experiment was observed already at room temperature. Ac3+ was placed inside the macrocyclic cavity of the [AcBATA]- complex, preventing the release of the cation. According to DFT studies, two possible conformations were found, where two pendant arms coordinate with the metal cation on one side of the azacrown cavity and two on the other side, or three pendant arms are located on one side and one on the other. Finally, high inertness in vitro and in vivo of [AcBATA]- was discovered, making the H4BATA ligand highly preferable for application as a component of actinium-based radiopharmaceuticals.

Specific Fluorescent Probes for Imaging DNA in Cell-Free Solution and in Mitochondria in Living Cells.

New styryl dyes consisting of N-methylpyridine or N-methylquinoline scaffolds were synthesized, and their binding affinities for DNA in cell-free solution were studied. The replacement of heterocyclic residue from the pyridine to quinoline group as well as variation in the phenyl part strongly influenced their binding modes, binding affinities, and spectroscopic responses. Biological experiments showed the low toxicity of the obtained dyes and their applicability as selective dyes for mitochondria in living cells.

Silencing of PKG1 Gene Mimics Effect of Aging and Sensitizes Rat Vascular Smooth Muscle Cells to Cardiotonic Steroids: Impact on Fibrosis and Salt Sensitivity.

Background Marinobufagenin, NKA (Na/K-ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration-1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)-dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMC from old rats, due to downregulation of ANP/cGMP/PKG-dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results Cultured VSMC from the young (3-month-old) and old (24-month-old) male Sprague-Dawley rats and young VSMC with silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen-1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Conclusions Age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin-induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.

20-Hydroxyecdysone Confers Antioxidant and Antineoplastic Properties in Human Non-Small Cell Lung Cancer Cells.

20-Hydroxyecdysone (20E) is an arthropod hormone which is synthesized by some plants as part of their defense mechanism. In humans, 20E has no hormonal activity but possesses a number of beneficial pharmacological properties including anabolic, adaptogenic, hypoglycemic, and antioxidant properties, as well as cardio-, hepato-, and neuroprotective features. Recent studies have shown that 20E may also possess antineoplastic activity. In the present study, we reveal the anticancer properties of 20E in Non-Small Cell Lung Cancer (NSCLC) cell lines. 20E displayed significant antioxidant capacities and induced the expression of antioxidative stress response genes. The RNA-seq analysis of 20E-treated lung cancer cells revealed the attenuation of genes involved in different metabolic processes. Indeed, 20E suppressed several enzymes of glycolysis and one-carbon metabolism, as well as their key transcriptional regulators-c-Myc and ATF4, respectively. Accordingly, using the SeaHorse energy profiling approach, we observed the inhibition of glycolysis and respiration mediated by 20E treatment. Furthermore, 20E sensibilized lung cancer cells to metabolic inhibitors and markedly suppressed the expression of Cancer Stem Cells (CSCs) markers. Thus, in addition to the known beneficial pharmacological activities of 20E, our data uncovered novel antineoplastic properties of 20E in NSCLC cells.

Methyltransferase Set7/9 as a Multifaceted Regulator of ROS Response.

Reactive oxygen species (ROS) induce multiple signaling cascades in the cell and hence play an important role in the regulation of the cell's fate. ROS can cause irreversible damage to DNA and proteins resulting in cell death. Therefore, finely tuned regulatory mechanisms exist in evolutionarily diverse organisms that are aimed at the neutralization of ROS and its consequences with respect to cellular damage. The SET domain-containing lysine methyltransferase Set7/9 (KMT7, SETD7, SET7, SET9) post-translationally modifies several histones and non-histone proteins via monomethylation of the target lysines in a sequence-specific manner. In cellulo, the Set7/9-directed covalent modification of its substrates affects gene expression, cell cycle, energy metabolism, apoptosis, ROS, and DNA damage response. However, the in vivo role of Set7/9 remains enigmatic. In this review, we summarize the currently available information regarding the role of methyltransferase Set7/9 in the regulation of ROS-inducible molecular cascades in response to oxidative stress. We also highlight the in vivo importance of Set7/9 in ROS-related diseases.

One-Pot Radiosynthesis of [18F]Anle138b-5-(3-Bromophenyl)-3-(6-[18F]fluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrazole-A Potential PET Radiotracer Targeting α-Synuclein Aggregates.

Availability of PET imaging radiotracers targeting α-synuclein aggregates is important for early diagnosis of Parkinson's disease and related α-synucleinopathies, as well as for the development of new therapeutics. Derived from a pyrazole backbone, 11C-labelled derivatives of anle138b (3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole)-an inhibitor of α-synuclein and prion protein oligomerization-are currently in active development as the candidates for PET imaging α-syn aggregates. This work outlines the synthesis of a radiotracer based on the original structure of anle138b, labelled with fluorine-18 isotope, eminently suitable for PET imaging due to half-life and decay energy characteristics (97% ß+ decay, 109.7 min half-life, and 635 keV positron energy). A three-step radiosynthesis was developed starting from 6-[18F]fluoropiperonal (6-[18F]FP) that was prepared using (piperonyl)(phenyl)iodonium bromide as a labelling precursor. The obtained 6-[18F]FP was used directly in the condensation reaction with tosylhydrazide followed by 1,3-cycloaddition of the intermediate with 3'-bromophenylacetylene eliminating any midway without any intermediate purifications. This one-pot approach allowed the complete synthesis of [18F]anle138b within 105 min with RCY of 15 ± 3% (n = 3) and Am in the range of 32-78 GBq/µmol. The [18F]fluoride processing and synthesis were performed in a custom-built semi-automated module, but the method can be implemented in all the modern automated platforms. While there is definitely space for further optimization, the procedure developed is well suited for preclinical studies of this novel radiotracer in animal models and/or cell cultures.

A protocol for capturing RNA-sensing innate immune receptors in multiple conformations by single-particle cryo-EM.

Capturing different conformations of receptor proteins that are complexed with ligands by single-particle cryo-EM facilitates our understanding toward the mechanisms of ligand recognition and receptor activation cascades. Here, we present a protocol for capturing RNA-sensing innate immune receptors, such as RIG-I, in multiple conformations by single-particle cryo-EM. We describe steps for protein-ligand sample preparation, data acquisition, and image processing covering focused three-dimensional classification. This protocol can be adapted to capture the dynamic behavior of other receptors that can be stabilized. For complete details on the use and execution of this protocol, please refer to Wang and Pyle (2022).1.

Novel candidate theranostic radiopharmaceutical based on strontium hexaferrite nanoparticles conjugated with azacrown ligand.

In this article, we report to the best of our knowledge the first modification of NPs with ligands for combined radiopharmaceuticals. Nanoparticles with suitable magnetic properties can be used both for diagnostics as a contrast for MRI and for therapy, including the insufficiently studied magneto-mechanical therapy. Strontium hexaferrite is one of the few hard-magnetic materials for which stable biocompatible colloidal solutions can be obtained. Strontium hexaferrite nanoparticles coated with silicon dioxide (SHF@SiO2) were modified with an amino silane coupling agent (3-aminopropyl)triethoxysilane and azacrown ether derivatives with six heteroatoms in rings were covalently linked to the amine group through the carboxyl group. The hard magnetic nanoparticles were then radiolabeled with 207Bi with a labelling yield of up to 99.8%. In vitro experiments showed that the complex SHF@SiO2-APTES-L2-207Bi is stable enough to be a potential theranostic radiopharmaceutical.