Strong Activity Research Articles

In vitro antileishmanial activities of hydro-methanolic crude extracts and solvent fractions of Clematis simensis fresen leaf, and Euphorbia abyssinica latex.

As a result of increasing drug resistance, crossover resistance development, prolonged therapy, and the absence of different agents with innovative methods for implementation, the efficacy of recent antileishmanial medications is severely declining. So, it is vital to look for other medications from botanical remedies that have antileishmanial activity. The latex of Euphorbia abyssinica (E abyssinica) and the leaves of Clematis simensis fresen (C simensis) were macerated in methanol (80%). In vitro antileishmanial activity of the preparation was tried on promastigotes of Leishmania aethiopica (L aethiopica) and Leishmania donovani (L donovani) using resazurin assay, and fluorescence intensity was measured. One percent of dimethyl sulfoxide (DMSO) and media as negative control and amphotericin B as positive control were used. Additionally, hemolytic & phytochemical tests of the preparation were done. The mean and standard errors of each extract were evaluated and interpreted for statistical significance using one-way analysis of variance. From sigmoidal dose-response curves of % inhibition, half maximal inhibitory concentration (IC50) values were determined by GraphPad Prism and Microsoft Excel; outcomes were presented as mean ± standard error of mean of triplicate trials. P < .05 was statistical significance. The phytochemical screening of C simensis and E abyssinica confirmed the existence of steroids, phenols, tannins, saponins, alkaloids, terpenoids, flavonoids and glycosides. C simensis possesses antileishmanial activity with IC50 outcomes of 46.12 ± 0.03 and 8.18 ± 0.10 µg/mL on the promastigotes of L aethiopica and L donovani, respectively. However, E abyssinica showed stronger activity with IC50 outcomes of 16.07 ± 0.05 µg/mL and 4.82 ± 0.07 µg/mL on L aethiopica and L donovani, respectively. C simensis and E abyssinica have a less hemolytic effect on human red blood cells at low concentrations. The outcomes from this investigation demonstrated that the preparation of C simensis and E abyssinica indicated significant antileishmanial activity. Therefore, further in vivo assessment of antileishmanial, cytotoxicity activity and quantitative identification of secondary metabolites are highly recommended.

Substance specific EEG patterns in mice undergoing slow anesthesia induction

The exact mechanisms and the neural circuits involved in anesthesia induced unconsciousness are still not fully understood. To elucidate them valid animal models are necessary. Since the most commonly used species in neuroscience are mice, we established a murine model for commonly used anesthetics/sedatives and evaluated the epidural electroencephalographic (EEG) patterns during slow anesthesia induction and emergence. Forty-four mice underwent surgery in which we inserted a central venous catheter and implanted nine intracranial electrodes above the prefrontal, motor, sensory, and visual cortex. After at least one week of recovery, mice were anesthetized either by inhalational sevoflurane or intravenous propofol, ketamine, or dexmedetomidine. We evaluated the loss and return of righting reflex (LORR/RORR) and recorded the electrocorticogram. For spectral analysis we focused on the prefrontal and visual cortex. In addition to analyzing the power spectral density at specific time points we evaluated the changes in the spectral power distribution longitudinally. The median time to LORR after start anesthesia ranged from 1080 [1st quartile: 960; 3rd quartile: 1080]s under sevoflurane anesthesia to 1541 [1455; 1890]s with ketamine. Around LORR sevoflurane as well as propofol induced a decrease in the theta/alpha band and an increase in the beta/gamma band. Dexmedetomidine infusion resulted in a shift towards lower frequencies with an increase in the delta range. Ketamine induced stronger activity in the higher frequencies. Our results showed substance-specific changes in EEG patterns during slow anesthesia induction. These patterns were partially identical to previous observations in humans, but also included significant differences, especially in the low frequencies. Our study emphasizes strengths and limitations of murine models in neuroscience and provides an important basis for future studies investigating complex neurophysiological mechanisms.

Effect of TiO2 Nanotubes on Biological Activity of Osteoblasts and Focal Adhesion Kinase/Osteopontin Level

Osteoblasts are important cells for bone formation and play a major part in bone diseases and bone defects. Clinically, we usually adopt bone implants for related diseases. Also, nanotechnology is important in bones and joints. This study assessed the effects of TiO2 nanotubes of different diameters on osteoblast activity, FAK and OPN levels, aiming to provide an experimental foundation for selection of clinical bone implant materials. The morphology of MG-63 human osteosarcoma cells changed with expansion of TiO2 nanotubes’ diameter. From the biological activity, the cell proliferation and adhesion were enhanced as the diameter of the TiO2 nanotube was increased and its proliferation and adhesion were highest in the 100 nm TiO2 nanotube, which is related to increased ALP activity, FAK and OPN protein and mRNA expression. ELISA detected ALP activity and found that MG-63 cells cultured with 70 nm nanotube had strongest activity. Immune blotting and PCR results showed that, FAK and OPN activities were highest in 70 nm TiO2 nanotube cells. In summary, TiO2 nanotubes increased cell proliferation and adhesion by up-regulating the activities of FAK and OPN in a concentration-dependent relationship.

Extraction Optimization, Structure Analysis and Antioxidant Activity of Polysaccharide from Sanghuangporus baumii

Introduction: In this study, we optimized the extraction process, analyzed the structure and assessed the antioxidant activity of Sanghuangporus baumii polysaccharide (SBP). The present results provide important information for the Sanghuangporus baumii polysaccharides in potential natural antioxidant effects. Background: The extraction and structural analysis of polysaccharides from Sanghuangporus has gained significant attention in the fields of chemistry, medicine, and life sciences. There is great significance in maximizing the extraction of polysaccharides from Sanghuangporus and developing their potential products in a scientific and rational manner. Objective: The study was designed to establish an efficient and practical extraction process for SBP, and then investigated the structure and the antioxidant activity. Methods: The Response Surface Methodology (RSM) based on Box-Behnken design was used to explore the ultrasound-assisted extraction of SBP, and the structure of SBP was studied by ultraviolet spectroscopy, infrared spectroscopy and other instrumental analysis methods. The total antioxidant capacity of SBP was studied by the Ferric Reducing Ability of Plasma (FRAP) method, and the scavenging capacity of ABTS+·, DPPH· and OH· was carried out as the index to investigate its antioxidant activity in vitro. Results: The statistical analysis results showed that the optimal conditions for extracting SBP were an ultrasound time of 20.74 min, ultrasound power of 268.40 W and material-liquid ratio of 1:25.71. Under optimal conditions, the experimental yield of SBP was 3.36 ± 0.01%. The RSM optimization process was applied to the experiment of complex enzyme extraction of SBP, and the yield increased to 4.72 ± 0.03%. Structural analysis showed that SBP mainly consisted of glucose, a small amount of mannose and galactose, and the molecular weight distribution was uneven, mainly concentrating in the three parts of 24.5,6.4,2.5 kDa. Moreover, SBP exhibited dose-dependent and strong reducing power and radical scavenging activity. For DPPH·, ABTS+· and OH· radical scavenging assays, IC50 values were 1.505 ± 0.050 mg/ml, 0.065 ± 0.036 mg/ml and 0.442 ± 0.007 mg/ml, respectively. Conclusion: In the present study, a β-linked heteroglucan (SBP) was extracted using the optimized process combining enzymes from the fruiting bodies of Sanghuangporus baumii. SBP exhibited effective and dose-dependent antioxidant activities. Our findings were of great value in terms of developing polysaccharides with potential natural antioxidants.

Antimicrobial and cytotoxic naphthoquinones from microbial origin: An updated review.

Naphthoquinones (NQs) are small molecules bearing two carbonyl groups. They have been the subject of much research due to their significant biological activities such as antiproliferative, antimicrobial, anti-inflammatory, antioxidant, and antimalarial effects. NQs are produced mainly by bacteria, fungi and higher plants. Among them, microorganisms are a treasure of NQs with diverse skeletons and pharmacological properties. The purpose of the present study is to provide a comprehensive update on the structural diversity and biological activities of 91 microbial naphthoquinones isolated from 2015 to 2022, with a special focus on antimicrobial and cytotoxic activities. During this period, potent cytotoxic NQs such as naphthablin B (46) and hygrocin C (30) against HeLa (IC50=0.23 µg/ml) and MDA-MB-431 (IC50=0.5 µg/ml) cell lines was reported, respectively. In addition, rubromycin CA1 (39), exhibited strong antibacterial activity against Staphylococcus aureus (MIC of 0.2 µg/ml). As importance bioactive compounds, NQs may open new horizon for treatment of cancer and drug resistant bacteria. As such, it is hoped that this review article may stimulates further research into the isolation of further NQs from microbial, and other sources as well as the screening of such compounds for biological activity and beneficial uses.

Four pairs of new enantiomeric guaiane sesquiterpenoids from the rhizomes of Alisma plantago-aquatica

Four pairs of undescribed enantiomeric guaiane sesquiterpenoids, (±)-alismaenols A-D (1a/1b, 3a/3b-5a/5b), together with a pair of known ones (2a/2b) were isolated from the rhizomes of Alisma plantago-aquatica. The structures and relative configurations of the isolates were established by analysis of their 1D, 2D-NMR and HRESIMS data. Their absolute configurations were determined by comparison of their experimental CD spectra and calculated electronic circular dichroism (ECD) spectra or by single-crystal X-ray diffraction analysis. All compounds (1a/1b-5a/5b) were evaluated for their inhibitory effects on nitric oxide (NO) production in LPS-induced RAW 264.7 cells, and compound 1a exhibited stronger activity (IC50 = 12.89 μM) than indomethacin (IC50 = 14.03 μM).

Synthesis and Antibacterial Activity of Ultrasmall Silver Nanoparticles by Pulsed Laser Ablation in Deionized Water

Background: The main objective of this work is the synthesis and evaluation of silver nanoparticles (Ag NPs) by using pulsed laser ablation of a silver (Ag) target in deionized water and examining their antibacterial activity. Methods: Colloidal solutions of silver nanoparticles were prepared with different pulsed laser energies (620, 880, and 1000) mJ of wavelength 1064 nm and frequency 10 Hz. To determine their structure, optical, morphology, elemental composition, and infrared spectra, the synthesized Ag NPs were characterized using various high-throughput analytical techniques such as (UVVis) spectroscopy, transmission electron microgram (TEM), electron dispersive X-ray spectroscopy (EDX), Fourier transform infrared (FTIR) spectra, and Zeta potential. Results: The results show that the properties of synthesized Ag NPs depend much more on the laser energy. The laser energy can be used to control the properties of the prepared nanoparticles. Uniform distributions of spherical ultrasmall Ag NPs with an average size of (3) nm were obtained suspended in deionized water, which is the most effective size for antibacterial activity. However, the result indicated that the ablated Ag NPs were stable for 4 months in deionized water. The antibacterial activity of the colloidal solution of synthesized Ag NPs against Gramnegative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria was then examined using the agar-well diffusion method. Conclusion: It was found that the prepared nanoparticles exhibited strong activity against E. coli and S. aureus bacteria growth. The average zones of inhibition of Ag NPs were found to be about (26) mm for E. coli and (32) mm for S. aureus bacteria.

Revealing the spatiotemporal brain dynamics of covert speech compared with overt speech: A simultaneous EEG-fMRI study

Covert speech (CS) refers to speaking internally to oneself without producing any sound or movement. CS is involved in multiple cognitive functions and disorders. Reconstructing CS content by brain-computer interface (BCI) is also an emerging technique. However, it is still controversial whether CS is a truncated neural process of overt speech (OS) or involves independent patterns. Here, we performed a word-speaking experiment with simultaneous EEG-fMRI. It involved 32 participants, who generated words both overtly and covertly. By integrating spatial constraints from fMRI into EEG source localization, we precisely estimated the spatiotemporal dynamics of neural activity. During CS, EEG source activity was localized in three regions: the left precentral gyrus, the left supplementary motor area, and the left putamen. Although OS involved more brain regions with stronger activations, CS was characterized by an earlier event-locked activation in the left putamen (peak at 262 ms versus 1170 ms). The left putamen was also identified as the only hub node within the functional connectivity (FC) networks of both OS and CS, while showing weaker FC strength towards speech-related regions in the dominant hemisphere during CS. Path analysis revealed significant multivariate associations, indicating an indirect association between the earlier activation in the left putamen and CS, which was mediated by reduced FC towards speech-related regions. These findings revealed the specific spatiotemporal dynamics of CS, offering insights into CS mechanisms that are potentially relevant for future treatment of self-regulation deficits, speech disorders, and development of BCI speech applications.

Effects of priming glycosyltransferase genes cps 2E and cps 4E on the exopolysaccharide biosynthesis by Lactiplantibacillus plantarum YM-4-3 strain

Microbial exopolysaccharides (EPS) have various physiological functions such as antioxidant, anti-tumor, cholesterol lowering, and immune regulation. However, improving traditional fermentation conditions to increase the production of EPS from Lactiplantibacillus plantarum (L. plantarum) is limited. In this study, we aimed to better improve EPS production and physiological functions of L. plantarum YM-4-3 strain by overexpressing and knocking out the priming glycosyltransferase genes cps 2E and cps 4E for the first time. As a result, the EPS production of the overexpression strain was 30.15 %, 26.84 % and 36.29 % higher than WT, respectively. The EPS production of the knockout strain was significantly lower than that of the WT. At the same time, transcriptome data showed that the gene expression levels of each experimental strain had changed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways found that the glycolysis/gluconeogenesis pathway had the highest gene enrichment in the metabolic pathway. The monosaccharide components of the EPS of each experimental strain were different from those of the WT and the EPS of the experimental strain showed stronger activity against oxidation. In conclusion, this study contributes to the efficient production and application of L. plantarum EPS and helps to understand the mechanism of EPS regulation in L. plantarum.

Abusive use of Zolpidem as a Result of COVID-19 and Perspectives of Continuity of the Problem in the Post-Pandemic Period.

Zolpidem is a non-benzodiazepine hypnotic drug that works as a positive modulator of Gamma-Amino Butyric Acid-A (GABA-A) receptors, with high selectivity for α1 subunits. Given this selective binding, the drug has a strong hypnotic activity. Social isolation during the SARS-CoV-2 pandemic has contributed to increased rates of anxiety, depression, and insomnia. As a result, studies have pointed to a possible increase in the indiscriminate use of drugs with sedative effects, such as Zolpidem, during the pandemic. The aim of this work was to present prospective evidence that warns of the possibility of the abusive use of Zolpidem even after the pandemic. High rates of addiction to this drug have been reported around the world after the emergence of the coronavirus. Data from the National Survey on Drug Use and Health and from Medicaid support the continuing growth in prescription and indiscriminate use of Zolpidem during the pandemic and afterward. Therefore, there is enough evidence to support the indiscriminate use of this drug since the beginning of the pandemic. Rates of indiscriminate use of sedatives may continue to increase in the post-pandemic period, especially if strict control measures are not taken by health authorities.

Two putative calcium-dependent protein kinases are involved in the regulation of sugarcane defense genes

Sugarcane is a primary sugar crop and an important source of bioenergy. Pathogens are the major factors affecting sugarcane yield and sugar content. However, the mechanisms of sugarcane defense regulation remain largely unknown, and research on prospective genetic targets for modification is scarce. As the main class of calcium sensors, calcium-dependent protein kinases (CDPKs/CPKs) play a crucial role in the immune regulatory network. Using sugarcane genomic data, we identified 229 putative ScCDPKs in primordial specie Saccharum officinarum. Searching the putative CDPKs in sugarcane cultivars from National Center for Biotechnology Information (NCBI), 12 putative ScCDPKs in cultivars were identified. Phylogenetic analysis revealed evolutionary relationships among these CDPKs in sugarcane cultivars and those in S. officinarum, Arabidopsis thaliana, and rice. Truncation mutants of ScCDPKs were introduced into the reporter system and examined for activity in inducing Sugarcane Pathogenesis Related Protein 1 (ScPR1). The results showed that truncated ScCDPK1 and ScCDPK8 induce higher expression of ScPR1 than full-length ScCDPK1 and ScCDPK8. Additionally, transient expression of truncated ScCDPK1 and ScCDPK8 exhibit stronger activity in sugarcane protoplasts for activation of ScPR1 and sugarcane Serine Protease Inhibitor (ScSPI). These results demonstrate that ScCDPK1 and ScCDPK8 possess auto-inhibitory activity. The findings of this study provide a basis for an in-depth study of the sugarcane CDPK gene family and lay the foundation for further genetic improvement.

Screening and evaluation of prebiotic exopolysaccharide of Lactobacillus plantarum on treating IBD in mice

PurposeThis research aimed at ascertaining the effects of prebiotic exopolysaccharide on inflammatory bowel disease (IBD). MethodsWe studied the prebiotic activity of EPSs from 5 Lactobacillus plantarum strains using an in vitro gut microbiota model. The EPS with the strongest activity was fed to DSS-induced IBD mice. We then examined the mice's gut microbiota, serum cytokines, oxidative stress, and metabolic conditions. ResultsIn the in vitro experiment, Only EPS32 of the 5 EPSs exhibited a good prebiotic activity similar to inulin. The EPS32 is composed of rhamnose, fructose, and galactose in a ratio of 8.2:1:1.7. When feeding mice with 50 mg/kgBW/day EPS32 for 10 days, it significantly alleviated the IBD symptoms including restoring gut microbiota dysbiosis, increasing short-chain fatty acid production, lowering pro-inflammatory cytokine levels, lowering oxidative stress in the lumen, and restore the metabolic conditions. ConclusionContinuous intake of prebiotic EPS can effectively alleviate IBD symptoms.

Asymmetric Total Synthesis and Structural Revision of DAT2, an Antigenic Glycolipid from Mycobacterium tuberculosis.

DAT2 is a member of the diacyl trehalose family (DAT) of antigenic glycolipids located in the mycomembrane of Mycobacterium tuberculosis (Mtb). Recently it was shown that the molecular structure of DAT2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT2 and its methyl-branched acyl substituent mycolipanolic acid is determined. For this, four different stereoisomers of mycolipanolic acid were prepared in a stereoselective and unified manner, and incorporated into DAT2. A rigorous comparison of the four isomers to the DAT isolated from Mtb H37Rv by NMR, HPLC, GC, and mass spectrometry allowed a structural revision of mycolipanolic acid and DAT2. Activation of the macrophage inducible Ca2+-dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants.

Asymmetric Total Synthesis and Structural Revision of DAT2, an Antigenic Glycolipid from Mycobacterium tuberculosis

AbstractDAT2 is a member of the diacyl trehalose family (DAT) of antigenic glycolipids located in the mycomembrane of Mycobacterium tuberculosis (Mtb). Recently it was shown that the molecular structure of DAT2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT2 and its methyl‐branched acyl substituent mycolipanolic acid is determined. For this, four different stereoisomers of mycolipanolic acid were prepared in a stereoselective and unified manner, and incorporated into DAT2. A rigorous comparison of the four isomers to the DAT isolated from Mtb H37Rv by NMR, HPLC, GC, and mass spectrometry allowed a structural revision of mycolipanolic acid and DAT2. Activation of the macrophage inducible Ca2+‐dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants.

A Fluorous Peptide Amphiphile with Potent Antimicrobial Activity for the Treatment of MRSA-induced Sepsis and Chronic Wound Infection.

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad-spectrum and low resistance tendency. Herein, a library of fluoro-lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non-fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co-assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA-induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug-resistant bacterial infections.

From brain to body: exploring the connection between altered reward processing and physical fitness in schizophrenia

Understanding the underlying mechanisms that link psychopathology and physical comorbidities in schizophrenia is crucial since decreased physical fitness and overweight pose major risk factors for cardio-vascular diseases and decrease the patients’ life expectancies. We hypothesize that altered reward anticipation plays an important role in this. We implemented the Monetary Incentive Delay task in a MR scanner and a fitness test battery to compare schizophrenia patients (SZ, n = 43) with sex- and age-matched healthy controls (HC, n = 36) as to reward processing and their physical fitness. We found differences in reward anticipation between SZs and HCs, whereby increased activity in HCs positively correlated with overall physical condition and negatively correlated with psychopathology. On the other handy, SZs revealed stronger activity in the posterior cingulate cortex and in cerebellar regions during reward anticipation, which could be linked to decreased overall physical fitness. These findings demonstrate that a dysregulated reward system is not only responsible for the symptomatology of schizophrenia, but might also be involved in physical comorbidities which could pave the way for future lifestyle therapy interventions.

Design, synthesis, and molecular docking of new phenothiazine incorporated N-Mannich bases as promising antimicrobial agents

The present work aims to synthesize four series of phenothiazine incorporation Mannich bases. Therefore, 10-methyl-10H-phenothiazine-3-sulfonamide (4) which was subjected to react with some secondary amines and formaldehyde to give the Mannich bases 5a-f, and 6–13. Compound 13 was then subjected to react with some secondary amines and formaldehyde to give the corresponding Mannich bases 14a-f. In total, twenty-two new compounds were synthesized and evaluated for in vitro growth inhibition activity against P. aeruginosa, E. coli, and S. aureus. Among the tested compounds, compounds 3, 5a, 5c, 6, 12, 13, 14d, and 14e exhibited good activity with a MIC value (12.5 μg/mL), compounds 5b, 10, 11, 14a, and 14c exhibited strong activity against the growth of S. aureus with a MIC value (6.25 μg/mL), and compound 14b superior against S. aureus with a MIC value (3.125 μg/mL) compared to drug reference ciprofloxacin with MIC value (2 μg/mL). The molecular docking investigation revealed the presence of many derivatives with high binding affinities and distinct interaction patterns with the target protein. Derivatives 14a-e emerged as the most promising possibilities, displaying the greatest binding energies and a varied variety of interaction types, including hydrogen bonding and pi interactions, over different distances, with derivative 14b exhibiting the highest binding energy at S = −8.3093 kcal/mol. These derivatives displayed superior binding affinities and various interaction mechanisms with the target protein, suggesting that they have great promise as lead compounds for future development into therapeutic medicines.

Abstract 6536: Enhanced membrane stability of mRNA nanoparticle derived TNFSF14 results in superior T-cell and NK cell stimulation

Abstract Resulting from intracellular translation of nanoparticle delivered mRNA, mRNA therapeutics offers an unparalleled opportunity to direct the expression of proteins to specific subcellular compartments. Many cytokines, especially the immunomodulatory Tumor Necrosis Factor (TNF) superfamily members, exist as both membrane and soluble proteins. Trimeric members of the TNF superfamily are known to be susceptible to protease cleavage resulting in their release from the membrane. Membrane and soluble versions of the immunomodulators may have different activity. Several studies have shown that membrane expressed TNFSF members more efficiently cross-link TNFSF receptors resulting in a stronger activation or inhibition signal. Moreover, the activity of membrane expressed proteins is confined to the cells that express the protein and the cells that interact with the protein expressing cell. Thus, directing the expression of immunostimulatory proteins to the membrane could be a transformative way to enhance the activity while mitigating the toxicity by limiting systemic exposure. In this study we utilized Nutcracker Therapeutics Inc. unique mRNA platform to develop a membrane stabilized version of tumor necrosis factor super family member 14, also known as LIGHT, with superior co-stimulatory activity and stability. LIGHT is an immunomodulatory cytokine that primarily acts on lymphocytes and innate immune cells through the cis or trans binding of HVEM or lymphotoxin beta receptor (LtbR). LIGHT has been implicated in multiple inflammatory diseases and is currently being evaluated in the development of immunotherapies against cancer. LIGHT stimulatory activity depends on the displacement of the inhibitory BTLA that is normally bound to HVEM and LtbR. Studies have shown that membrane bound LIGHT, but not soluble LIGHT, can effectively displace BTLA. In this study, we hypothesized that the advantageous activities of mRNA derived membrane expressed LIGHT could be further enhanced with an engineered LIGHT molecule that is less susceptible to membrane shedding. We made multiple variants of Engineered LIGHT where different regions of the putative protease sensitive sites were replaced with either a flexible or rigid linker. In short, we found that i) the soluble version of LIGHT can inhibit immune stimulatory capability of membrane expressed LIGHT; ii) membrane bound LIGHT enhances co-stimulation of antigen-specific T cells; iii) engineered LIGHT enhances membrane expression and increases stability; and iv) a design using a novel rigid linker to replace the putative protease sensitive region of LIGHT showed best overall activity. Here, we demonstrate the capability of our integrated platform to design and deliver an enhanced, membrane stabilized LIGHT molecule. This engineered transmembrane molecule represents a new class of drug uniquely enabled by nanoparticle delivered mRNA. Citation Format: Adrienne Sallets, Weiqun Liu, Meredith L. Leong, Ruben Prins, Diane M. Da Silva, Daniel J. Fernandez, Colin J. McKinlay, Edward E. Lemmens, Chris S. Rae, Sudha Adusumilli, Ray Low, Srinivasa Bandi, Guna Kannan, W. Martin Kast, Samuel Deutsch, Ole A. Haabeth. Enhanced membrane stability of mRNA nanoparticle derived TNFSF14 results in superior T-cell and NK cell stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6536.

Abstract 6758: Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa

Abstract Activation of the stimulator of interferon genes (STING) pathway causes the release of factors that trigger the immune response in the tumor microenvironment (TME). Transient strong activation leads to immune activation and tumor suppression, while prolonged weak activation leads to immuno-suppression promoting tumor development. Reprogramming of M2-type tumor-associated macrophages (M2-TAMs) toward an M1 phenotype, suppression of cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (mMDSCs), as well as activation of dendritic cells (DCs) to train T-cells are advantageous, while the activation of STING pathway in T-cells is leads to their apoptosis. Thus, targeted delivery of STINGa to specific cells with TME to induce strong transient activation may be a key to therapy. We have developed a delivery approach that uses one or two pH Low Insertion Peptides (pHLIPs) that collaborate in the targeted intracellular delivery of a STINGa. STINGa were conjugated with pHLIPs via S-S cleavable self-immolating linkers. Biophysical studies were performed to ensure proper interactions of pHLIP-STINGa agents with membrane lipid bilayers. pHLIPs extend the lifetime of STINGa in the blood and target them to acidic cancer and stromal cells (CAFs), as well as M2-TAMs, mMDSCs and DCs within TME. The targeting results in selective cytokine activation in tumors with no systemic activation, which triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after one or two doses of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. No tumors developed in mice re-challenged by an additional injection of cancer cells in a 90-day experiment. Thus, targeted delivery of STINGa to cancer cells, tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T- and NK-cells, which gain access to the tumor core. The cytotoxic activity of T- and NK-cells is not impaired by the acidic environment and immune memory is developed. The pHLIP technology may allow transformation of immuno-activating agents into more potent therapeutics, since pHLIP can target and deliver these agents to cancer cells, tumor stroma and myeloid cells. Citation Format: Michael DuPont, Anna Moshnikova, Marissa Iraca, Craig Klumpp, Hannah Visca, Dana Allababidi, Phoebe Pelzer, Donald Engelman, Oleg Andreev, Yana Reshetnyak. Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6758.

Abstract 945: In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models

Abstract Background: The standard treatment for high grade ovarian carcinoma (HGOC) is cytoreductive surgery followed by a platinum based therapy. Despite an initial high rate of complete remission, most of the patients relapse with a platinum resistant disease. Patients with resistant tumors have few options, including monotherapy with gemcitabine, and new effective therapeutic alternatives are needed. The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and recent evidences suggest its role in interfering with several DNA repair mechanisms. We investigated the effect of onvansertib (ONVA), a highly selective ATP-competitor PLK1 inhibitor, in combination with carboplatin (CARBO) and gemcitabine (GEM) in platinum-resistant HGOC patient-derived xenografts (PDXs). Methods: The combination of ONVA with either CARBO or GEM was investigated in two platinum-resistant HGOC PDXs, one intrinsically resistant to platinum (MNHOC#315), the other made resistant after repeated in vivo treatments with cisplatin (MNHOC#266R). Tumors were subcutaneously (s.c.; MNHOC#315) or orthotopically (i.p.; MNHOC#266R) transplanted in nude mice, mice were treated for 4 weeks with vehicle, single agents or drug combinations and followed for tumor growth (s.c. model) and survival. Results: CARBO- and GEM-ONVA combinations were well tolerated, with a maximum body weight loss of 6.6%. Both combinations showed strong anti-tumor activities in the two tested models. In MNHOC#315 PDX, CARBO- and GEM-ONVA combinations caused a tumor stabilization that extended beyond the dosing period and resulted in a higher tumor growth inhibition as compared to vehicle and single agents treated mice. In i.p. MNHOC#266R PDX, single agents were inactive, while a statistically significant increase in survival was observed in CARBO/ONVA and in GEM/ONVA treated mice as compared to vehicle treated mice, with a 6.6- and 7-fold increase in mean survival time, respectively. Pharmacodynamic studies are ongoing to decipher the mechanisms underlying the synergic effects of both combinations. Conclusions: The combinations of ONVA with either CARBO or GEM showed potent anti-tumor activities in two platinum-resistant HGOC PDXs. Additional studies in other HGOC platinum-resistant models, both in vivo and in vitro, as well pharmacodynamic studies are ongoing to validate these results and to get insights in the mechanisms underlying their efficacy. Citation Format: Michela Chiappa, Federica Guffanti, Ilaria Mengoli, Maya Ridinger, Giovanna Damia. In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 945.