Protein Kinase Inhibitor Research Articles

Platelet Glycoprotein Ibα Cytoplasmic Tail Exacerbates Thrombosis During Bacterial Sepsis

Septic patients, coupling severe disseminated intravascular coagulation (DIC) and thrombocytopenia, have poor prognoses and higher mortality. The platelet glycoprotein Ibα (GPIbα) is involved in thrombosis, hemostasis, and inflammation response. However, it remains unclear whether the GPIbα cytoplasmic tail regulates sepsis-mediated platelet activation and inflammation, especially in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections. Using a mouse model of S. aureus-induced bacteremia, we found that both 10 amino acids of GPIbα C-terminal sequence deficiency and pharmacologic inhibition of protein kinase C (PKC) alleviated pathogenesis by diminishing platelet activation and aggregate formation. Furthermore, the GPIbα cytoplasmic tail promoted the phagocytosis of platelets by Kupffer cells in vivo. The genetically deficient GPIbα cytoplasmic tail also downregulated inflammatory cytokines and reduced liver damage, ultimately improving the survival rate of the septic mice. Our results illustrate that the platelet GPIbα cytoplasmic domain exacerbates excessive platelet activation and inflammation associated with sepsis through a PKC-dependent pathway. Thus, our findings provide insights for the development of effective therapeutic strategies using PKC inhibitor treatment against bacterial infection.

DNA-PK inhibition shows differential radiosensitization in orthotopic GBM PDX models based on DDR pathway deficits.

Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi's) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi's is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study we used GBM patient derived xenografts (PDXs) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300nM and above, with significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knock-down of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. While the mechanism underpinning this discordant effect in vitro vs. in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wildtype GBM lines significantly delayed growth and decreased NHEJ efficiency (but not Homologous Recombination), after peposertib exposure.

Stromal reprogramming overcomes resistance to RAS-MAPK inhibition to improve pancreas cancer responses to cytotoxic and immune therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is often resistant to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have both been implicated as drivers of resistance to therapy. Mitogen-activated protein kinase (MAPK) inhibition has not yet shown clinical efficacy, likely because of rapid acquisition of tumor-intrinsic resistance. However, the unique PDAC TME may also be a driver of resistance. We found that long-term focal adhesion kinase (FAK) inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in PDAC cells in mouse models and tissues from patients with PDAC. Concomitant inhibition of both FAK (with VS-4718) and rapidly accelerated fibrosarcoma and MAPK kinase (RAF-MEK) (with avutometinib) induced tumor growth inhibition and increased survival across multiple PDAC mouse models. In the TME, cancer-associated fibroblasts (CAFs) impaired the down-regulation of MYC by RAF-MEK inhibition in PDAC cells, resulting in resistance. By contrast, FAK inhibition reprogramed CAFs to suppress the production of FGF1, which can drive resistance to RAF-MEK inhibition. The addition of chemotherapy to combined FAK and RAF-MEK inhibition led to tumor regression, a decrease in liver metastasis, and improved survival in KRAS-driven PDAC mouse models. Combination of FAK and RAF-MEK inhibition alone improved antitumor immunity and priming of T cell responses in response to chemotherapy. These findings provided the rationale for an ongoing clinical trial evaluating the efficacy of avutometinib and defactinib in combination with gemcitabine and nab-paclitaxel in patients with PDAC and may suggest further paths for combined stromal and tumor-targeting therapies.

Tubulin-Targeted Therapy in Melanoma Increases the Cell Migration Potential by Activation of the Actomyosin Cytoskeleton─An In Vitro Study.

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines─WM35 and WM266-4─representing the less and more invasive melanomas. We investigated the mechanisms of cellular processes regulating the activation of actomyosin as an effect of colchicine treatment. Additionally, we investigated the biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632) and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy (AFM) and microconstriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers, compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased the level of cancer cell invasiveness. We emphasized that a cellular pathway of Rho-ROCK-dependent actomyosin contraction is responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy.

SESN2 Ameliorates Dihydrotestosterone-induced Human Ovarian Granulosa Cell Damage by Activating AMPK/ULK1-mediated Mitophagy.

Sestrin 2 (SESN2) has been reported to participate in the regulation of granulosa cell function in ovarian tissues. However, the role of SESN2 in polycystic ovarian syndrome (PCOS) is still incompletely understood. Here, we investigated the functional role and mechanism of SESN2 in dihydrotestosterone (DHT)-induced granulosa cells. In this study, DHT was utilized to induce PCOS cell model and the AMP-activated protein kinase (AMPK) inhibitor Compound C (CC) was utilized to inhibit the AMPK pathway. qRT-PCR was performed to detect the expression of SESN2 in HGLS cells. Cell apoptosis was evaluated by flow cytometry. Oxidative stress was detected by DCFH-DA staining, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) kits. The expression of SESN2, cell apoptosis, oxidative stress, mitophagy and AMPK/ULK1 signaling-related proteins were measured by western blot. The results showed that SESN2 was downregulated in DHT-induced granulosa cells. Overexpression of SESN2 inhibited the DHT-induced apoptosis and oxidative stress of HGLS cells. DHT induction aggravated HGLS cell apoptosis and oxidative stress. SESN2 overexpression inhibited the DHT-induced apoptosis and oxidative stress of HGLS cells. In addition, overexpression of SESN2 activated the AMPK/ULK1 signaling pathway and promoted mitophagy. Treatment of CC reversed the regulatory effect of SESN2 on mitophagy. CC also reversed the influences of SESN2 overexpression on apoptosis and oxidative stress in DHT-induced HGLS cells. Overall, SESN2 suppressed DHT-induced apoptosis and oxidative stress in PCOS through AMPK/ULK1-mediated mitophagy.

Recurrent melanoma 25 years after initial diagnosis, presenting as metastatic disease early after heart transplantation

BackgroundCancer survivors (CS) comprise a particularly high-risk group for both de-novo and recurrent malignancies after solid organ transplantation.Case presentationWe report a case of relapsed melanoma, presented as metastatic disease seven months after heart transplantation in a patient who had an early-stage melanoma resected 25 years prior. Treatment with a combination of dabrafenib, a BRAF inhibitor, and trametinib, a mitogen-activated protein kinase (MEK) inhibitor resulted in a near-complete metabolic response, without major adverse effects.ConclusionThis case demonstrates the increased risk of recurrence in CS with melanoma, which can persist decades after cancer diagnosis. These patients may be amenable to treatment using modern treatment modalities in oncology.

Impact of protein kinase CK2 downregulation and inhibition on oncomir clusters 17 ~ 92 and 106b ~ 25 in prostate, breast, and head and neck cancers

BackgroundProtein kinase CK2 is a ubiquitous and highly conserved protein Ser/Thr kinase with diverse cell functions. CK2 is upregulated in various cancers and affects numerous aspects of their underlying pathobiology. The important role of microRNAs (miRNAs) referred to as oncomirs is also recognized in various cancers. Elevation of both CK2 and altered miRNA expression in cancers raised the question whether there was a connection between CK2 function and oncomirs in cancer.MethodsPCR array analysis was used to examine the effects of CK2 siRNA-mediated downregulation on miRNA levels in C4-2 prostate cancer cells. We employed prostate cancer, breast cancer, and head and neck squamous cell carcinoma (HNSCC) cells as well as a prostate cancer xenograft orthotopic tumor model to examine the effects of CK2 siRNA-mediated downregulation or chemical inhibition on oncomir cluster miR-17 ~ 92 and miR-106b ~ 25 constituent miRNAs by quantitative reverse-transcriptase stem-loop PCR. Pri-miRNAs were measured in cancer cell lines by quantitative reverse-transcriptase PCR. Protein levels were assessed by western blot. PC3-LN4 prostate cancer orthotopic xenograft tumors and blood were collected from nude mice following repeated treatments with tenfibgen ligand nanocapsules containing RNAi-CK2 or RNAi-Control cargoes.ResultsPCR array analysis demonstrated effect on a subset of miRNAs following CK2 downregulation; we focused our investigation on CK2 regulation of miR-17 ~ 92 and 106b ~ 25 oncomir clusters. Chemical inhibition or molecular downregulation of CK2 greatly reduced expression of miR-17 ~ 92 and 106b ~ 25 in prostate, breast and head and neck cancer cells in vitro. CK2α and CK2α´ protein levels were significantly correlated with many of the miR-17 ~ 92 and some of the miR-106b ~ 25 constituent members in prostate cancer cells. Decreased pri-miRNA levels for the miR-17 ~ 92 gene cluster transcript were observed for 5 of 6 cancer cell lines tested following CK2 downregulation. Nanocapsule-mediated delivery of RNAi-CK2 reduced CK2 protein expression in orthotopic prostate xenograft tumors and decreased intra-tumoral and serum levels of the oncomirs.ConclusionsTargeting CK2 for the development of new cancer therapies is under active investigation in many laboratories and pharmaceutical companies. Our data suggest a new role for CK2 in cell signaling and survival in multiple cancer types through maintenance of miR-17 ~ 92 and 106b ~ 25 biogenesis.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

Advances in Cancer Therapy: A Comprehensive Review of CDK and EGFR Inhibitors.

Protein kinases have essential responsibilities in controlling several cellular processes, and their abnormal regulation is strongly related to the development of cancer. The implementation of protein kinase inhibitors has significantly transformed cancer therapy by modifying treatment strategies. These inhibitors have received substantial FDA clearance in recent decades. Protein kinases have emerged as primary objectives for therapeutic interventions, particularly in the context of cancer treatment. At present, 69 therapeutics have been approved by the FDA that target approximately 24 protein kinases, which are specifically prescribed for the treatment of neoplastic illnesses. These novel agents specifically inhibit certain protein kinases, such as receptor protein-tyrosine kinases, protein-serine/threonine kinases, dual-specificity kinases, nonreceptor protein-tyrosine kinases, and receptor protein-tyrosine kinases. This review presents a comprehensive overview of novel targets of kinase inhibitors, with a specific focus on cyclin-dependent kinases (CDKs) and epidermal growth factor receptor (EGFR). The majority of the reviewed studies commenced with an assessment of cancer cell lines and concluded with a comprehensive biological evaluation of individual kinase targets. The reviewed articles provide detailed information on the structural features of potent anticancer agents and their specific activity, which refers to their ability to selectively inhibit cancer-promoting kinases including CDKs and EGFR. Additionally, the latest FDA-approved anticancer agents targeting these enzymes were highlighted accordingly.

Reversible covalent c-Jun N-terminal kinase inhibitors targeting a specific cysteine by precision-guided Michael-acceptor warheads

There has been a surge of interest in covalent inhibitors for protein kinases in recent years. Despite success in oncology, the off-target reactivity of these molecules is still hampering the use of covalent warhead-based strategies. Herein, we disclose the development of precision-guided warheads to mitigate the off-target challenge. These reversible warheads have a complex and cyclic structure with optional chirality center and tailored steric and electronic properties. To validate our proof-of-concept, we modified acrylamide-based covalent inhibitors of c-Jun N-terminal kinases (JNKs). We show that the cyclic warheads have high resilience against off-target thiols. Additionally, the binding affinity, residence time, and even JNK isoform specificity can be fine-tuned by adjusting the substitution pattern or using divergent and orthogonal synthetic elaboration of the warhead. Taken together, the cyclic warheads presented in this study will be a useful tool for medicinal chemists for the deliberate design of safer and functionally fine-tuned covalent inhibitors.

154 (PB142): The discovery of imidazo[4,5-c]pyridine-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitisers

154 (PB142): The discovery of imidazo[4,5-c]pyridine-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitisers

EP.03F.04 Treatment of Non-Small Cell Lung Carcinoma (NSCLC) Cells with Tumor Treating Fields (TTFields) And DNA-Dependent Protein Kinase (PK) Inhibitors

EP.03F.04 Treatment of Non-Small Cell Lung Carcinoma (NSCLC) Cells with Tumor Treating Fields (TTFields) And DNA-Dependent Protein Kinase (PK) Inhibitors

Elevated serum GDF15 level as an early indicator of proximal tubular cell injury in acute kidney injury

Acute kidney injury (AKI) is a high-burden medical condition, and current diagnostic criteria can only assess AKI after full manifestation. Stress marker growth differentiation factor 15 (GDF15) was reported to have a role in kidney injury of critical patients. Herein, we evaluated dynamic changes in GDF15 across diverse AKI scenarios and explored the underlying mechanisms of its induction. Serum parameters and renal lesions were analyzed in mouse models of unilateral ischemia-reperfusion injury (uni-IRI) and unilateral ureteral obstruction (UUO). The human proximal tubular (HK−2) cell line was stimulated with various conditions, and induction of GDF15 expression was determined. Serum GDF15 levels were rapidly induced within hours after injury in both animal models and declined thereafter. Renal GDF15 expression exhibited a temporary and early increased induction and was mainly located in aquaporin 1-positive proximal tubules in both unilateral AKI model tissues. In cell experiments, rapid GDF15 production was highly induced by t-BHP and CoCl2. Treatment with either an antioxidant or mitogen-activated protein kinase inhibitors abolished t-BHP- and CoCl2-mediated GDF15 expression. In addition, silencing nuclear factor erythroid 2-related factor 2 expression also reduced the basal and t-BHP- or CoCl2-mediated GDF15 expression level in HK-2 cells. Our data showed that elevated serum GDF15 levels could be detected early in unilateral AKI models without notable alterations in kidney function parameters. GDF15 expression was associated with oxidative stress- and hypoxia-mediated proximal tubular cell injury. These data document that elevated serum GDF15 can possibly serve as an early biomarker for proximal tubular cell injury in AKI.

The AKT inhibitor AZD5363 elicits synthetic lethality in ARID1A-deficient gastric cancer cells via induction of pyroptosis.

Gastric cancer (GC) is a deadly disease with poor overall survival and limited therapeutic options. Genetic alterations such as mutations and/or deletions in chromatin remodeling gene AT-rich interactive domain 1 A (ARID1A) occur frequently in GC. Although ARID1A mutations/deletions are not a druggable target for conventional treatments, novel therapeutic strategies based on a synthetic lethal approach may be effective for the treatment of ARID1A-deficient cancers. A kinase inhibitor library containing 551 compounds was screened in ARID1A isogenic GC cells for the ability to induce synthetic lethality effect. Selected hits' activity was validated, and the mechanism of the most potent candidate drug, AKT inhibitor AD5363 (capivasertib), on induction of the synthetic lethality with ARID1A deficiency was investigated. After robust vulnerability screening of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A-/- cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the Caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment.

Green synthesized AgNPs of the Anchusa arvensis aqueous extract resulting in impressive protein kinase, antioxidant, antibacterial, and antifungal activities.

This study focused on analyzing the pharmacological activities of AgNPs synthesized from an aqueous plant extract of Anchusa arvensis. The effectiveness of AgNPs was evaluated for protein kinase inhibition, antioxidant, antibacterial, and antifungal activities. The AgNPs and plant were used to regulate the protein kinase activity using the liquid TSB and ISP4 medium protein kinase inhibition study demonstrated that nanoparticles exhibited a larger zone of inhibition (9.1 ± 0.8) compared to the plant extract (8.1 ± 0.6). The antioxidant activity was assessed using DPPH reagent, and the results indicated that AgNPs displayed potent free radical scavenging properties. In terms of antibacterial activity, AgNPs showed higher efficacy against Enterobacter aerogens (20.1 ± 0.9), Bordetella bronchiseptaca (19.1 ± 0.9), and Salmonella typhimurium (17.2 ± 0.8) at 4 mg/mL. The antifungal activity of AgNPs was prominent against Aspergillus fumagatus (14.1 ± 0.9), Mucor species (19.2 ± 0.8), and Fusarium solani (11.2 ± 0.8) at 20 mg/mL. These findings suggest that AgNPs possess multiple beneficial properties, including bactericidal/fungicidal effects, protein kinase inhibition, and potential free radical scavenging abilities. Therefore, AgNPs have potential applications in various fields, such as biomedicine and industry, due to their ability to counteract the harmful effects of free radicals.

α- Triazolylboronic acids: a novel scaffold to target FLT3 in AML.

The treatment of acute myeloid leukemia (AML) presents a challenge to current therapies because of the development of drug resistance. Genetic mutation of FMS-like tyrosine kinase-3 (FLT3) is a target of interest for AML treatment, but the use of FLT3-targeting agents on AML patients has so far resulted in poor overall clinical outcomes.1 The incorporation of the boronic group in a drug scaffold could enhance the bioavailability and pharmacokinetic profile of conventional anticancer chemotypes. Boronic acids represent an intriguing and unexplored class of compounds in the context of AML, and they are only scantly reported as inhibitors of protein kinases. We identified a-triazolylboronic acids as a novel chemotype for targeting FLT3 by screening a library of structurally heterogeneous in-house boronic acids. Selected compounds show low micromolar activities on enzymatic and cellular assays, selectivity against control cell lines and a recurring binding mode in in-silico studies. Furthermore, control analogues synthesized ad hoc and lacking the boronic acid are inactive, confirming that this group is essential for the activity of the series. All together, these results suggest α-triazolylboronic acids could be a promising novel chemotype for FLT3 inhibition, laying the ground for the design of further compounds.

Development of Nitric Oxide-Donating Netarsudil Derivatives as a Synergistic Therapy for Glaucoma with Reduced Ocular Irritation.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a series of NO-donating Netarsudil derivatives were designed, synthesized, and their activities in vitro and in vivo were evaluated. Among them, (S)-10e released an appropriate amount of NO in aqueous humor in vitro and displayed potent ROCK inhibition. Topical administration of (S)-10e significantly lowered intraocular pressure in an acute ocular hypertension rabbit model and protected retinal ganglion cells in a magnetic microbead occlusion mouse model. A metabolism investigation revealed that (S)-10e released 7a, a metabolite after NO releasing, and 13, an active metabolite of (S)-Netarsudil, in rabbit eyes. Notably, introducing an NO donor moiety attenuated ROCK inhibition-induced ocular irritation in an sGC-independent manner, suggesting that the attenuated conjunctival hyperemia effect of (S)-10e is related to the NO-induced protein S-nitrosation of phosphodiesterase 3A (PDE3A). Overall, (S)-10e is a promising candidate for glaucoma treatment.

Assessing Darkness of the Human Kinome from a Medicinal Chemistry Perspective.

In drug discovery, human protein kinases (PKs) represent one of the major target classes due to their central role in cellular signaling, implication in various diseases as a consequence of deregulated signaling, and notable druggability. Individual PKs and their disease biology have been explored to different degrees, giving rise to heterogeneous functional knowledge and disease associations across the human kinome. The U.S. National Institutes of Health previously designated 162 understudied ("dark") human PKs and lipid kinases due to the lack of functional annotations and high-quality molecular probes for functional investigations. Given the large volumes of available PK inhibitors (PKIs) and activity data, we have systematically analyzed the distribution of PKIs and associated data at different confidence levels across the human kinome and distinguished between chemically explored, underexplored, and unexplored PKs. The analysis provides a medicinal chemistry-centric view of PK exploration and further extends prior assessment of the dark kinome.

In Vivo Effect of Halicin on Methicillin-Resistant Staphylococcus aureus-Infected Caenorhabditis elegans and Its Clinical Potential.

Recently, the high proportion of methicillin-resistant Staphylococcus aureus infections worldwide has highlighted the urgent need for novel antibiotics to combat this crisis. The recent progress in computational techniques for use in health and medicine, especially artificial intelligence (AI), has created new and potential approaches to combat antibiotic-resistant bacteria, such as repurposing existing drugs, optimizing current agents, and designing novel compounds. Halicin was previously used as a diabetic medication, acting as a c-Jun N-terminal protein kinase (JNK) inhibitor, and has recently demonstrated unexpected antibacterial activity. Although previous efforts have highlighted halicin's potential as a promising antibiotic, evidence regarding its effectiveness against clinical strains remains limited, with insufficient proof of its clinical applicability. In this study, we sought to investigate the antibacterial activity of halicin against MRSA clinical strains to validate its clinical applicability, and a C. elegans model infected by MRSA was employed to evaluate the in vivo effect of halicin against MRSA. Our findings revealed the antibacterial activity of halicin against methicillin-resistant S. aureus clinical strains with MICs ranging from 2 to 4 µg/mL. Our study is also the first work to evaluate the in vivo effect of halicin against S. aureus using a C. elegans model, supporting its further development as an antibiotic.

Clarithromycin Modulates Neutrophilic Inflammation Induced by Prevotella intermedia in Human Airway Epithelial Cells.

Objectives: In the present study, we aimed to clarify the mechanisms by which periodontal pathogens, particularly Prevotella intermedia, induce severe neutrophilic inflammation. In addition, we aimed to test the efficacy of macrolides, which has not been resolved in the neutrophilic inflammation induced by P. intermedia. Methods: NCl-H292 human airway epithelial cells were pre-incubated with clarithromycin for 2 h before incubation with P. intermedia supernatants. Then, C-X-C motif chemokine ligand 8 (CXCL8) transcription and interleukin (IL)-8 production were measured. To elucidate the signaling pathway, mitogen-activated protein kinase inhibitors were added to the cell culture, and the cells were subjected to Western blotting. Results:P. intermedia supernatants promoted CXCL8 transcription and IL-8 production, and the reactions were significantly suppressed by clarithromycin pretreatment. Only trametinib, the selective mitogen-activated extracellular signal-regulated kinase inhibitor, downregulated CXCL8 transcription and IL-8 production. Furthermore, Western blotting revealed that stimulation with P. intermedia supernatants specifically induces extracellular signal-regulated kinases (ERK) 1/2 phosphorylation, which is suppressed by clarithromycin pretreatment. Notably, the interference analysis revealed that ERK3 might be dispensable for IL-8 production under the stimulation of P. intermedia supernatants. Conclusions: Our results provide new insight into the mechanism underlying P. intermedia-induced production of IL-8 from human airway epithelial cells. Furthermore, macrolides might have therapeutic potential in regulating periodontal pathogen-induced neutrophilic inflammation in the lungs.