ACS Pharmacology and Translational Science最新文献

GPR68 is a pH-sensing G protein-coupled receptor widely expressed throughout the body. It has been implicated in various diseases, including neurodegeneration, chronic inflammation, and cancer, emphasizing its role in pathophysiology. The identities of the structural elements essential for pH sensing have been controversial, as experiments and sequence analyses have been interpreted to suggest that an extracellular histidine cluster or, by contrast, an internal carboxylic triad, senses the extracellular pH. Recent molecular simulations and hydrogen-bond network analyses suggested instead a unifying mechanism whereby the extracellular histidine residues and the internal carboxylic triad couple to each other via a protonation-sensitive hydrogen-bond network that includes a fourth internal carboxylic group, E103^3.34. However, without experimental verification, there remains a gap in our understanding of the mechanism by which GPR68 is activated by protons. To this aim, here we have studied 16 GPR68 mutations, which we selected based on the hydrogen-bond network analyses and conservation of key residues. We implemented a cell-based homogeneous time-resolved fluorescence assay to monitor the Gα_q/11 and Gα_s coupled signaling pathways. We used this assay to study how each mutation alters the basal activity levels, half-maximal activation values, and reactivation at lower pH values. Our data identify E103^3.34Q as a gain-of-function mutant essential for the proton-sensing mechanism of GPR68. We further discovered that E149^4.53Q, a constitutively active mutant, prefers Gα_s coupling over Gα_q/11 and that wild-type GPR68 is more sensitive toward Gα_q/11 coupling over Gα_s.

To find potential biomarkers and related metabolic pathways based on metabonomics of rat joint tissue, the rats with stable collagen-induced arthritis (CIA) model were taken as the research object. A rat rheumatoid arthritis model was established by injecting type II collagen. Treatment groups received oral doses of puerarin (40 mg/kg), puerarin–gadolinium (40 mg/kg), gadolinium chloride (40 mg/kg), or methotrexate (0.5 mg/kg), while control groups received saline. After 28 days, joint tissue metabolomics was analyzed using UPLC-MS (Shimadzu LC-30A and SCIEX TripleTOF 6600+), revealing significant metabolite changes and altered metabolic pathways. For the animal experiment part, based on observed changes in morphological, histopathological, and biochemical indicators, puerarin–Gd demonstrated significant therapeutic efficacy, surpassing that of puerarin, gadolinium chloride, and the positive control group. For the metabolomics part, compared with the blank group, the number of significantly different metabolites in the model group was 238, and most of the expressions were upregulated. Compared with the model group, the number of significantly different metabolites in the puerarin–gadolinium treatment group was 165, but most of them were downregulated. The KEGG enrichment pathway showed that the differential metabolites enrichment pathways of the puerarin–gadolinium treatment group and model group were mainly: linoleic acid metabolism, α-linolenic acid metabolism, arachidonic acid metabolism, choline metabolism in cancer, retrograde endogenous cannabinoid signal transduction, and glycerol phosphate metabolism pathway. Puerarin–gadolinium has a good therapeutic effect on rheumatoid arthritis rats, and its mechanism may be related to the inhibition of ferroptosis and the regulation of lipid metabolism.

Cancer remains one of the most significant global health challenges, characterized by an increasing incidence and mortality rate worldwide. In vitro models play a significant role in the initial investigations of cancer biology, drug screening, and therapeutic development. However, although widely used, conventional 2D cell cultures fail to replicate the complex tumor microenvironment, leading to discrepancies in drug response and therapeutic efficacy. This perspective explores the transition from 2D to 3D culture models, highlighting their advantages, limitations, and impact on cancer research. Various 3D culture approaches, including scaffold-based systems, hydrogels, 3D-printed models, microfluidics, and organ-on-a-chip technologies, are discussed in terms of their relevance to cancer modeling and drug testing. Additionally, the review also highlights the integration of theranostic nano- and microparticles in cancer treatment, focusing on their application in drug delivery and interactions with 3D tumor spheroids and organoids. A comparative analysis of uptake mechanisms and interactions between particles and 3D models is presented along with advanced techniques for probing nanoparticle behavior and drug screening in these models. By bridging the gap between in vitro assays and clinical outcomes, 3D culture systems integrated with nanotechnology offer promising tools for improving cancer therapeutics.

l-Glutamine (Gln) suppresses inflammation via upregulation of mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1; however, high dosages required may cause serious side effects. Here, we developed a glutaminase-resistant less-hydrolyzable Gln derivative, α, δ-N-acetyl-glutamine (α, δ-NAG). Oral administration of α, δ-NAG and Gln to ovalbumin-induced asthma model mice suppressed asthmatic parameters at 0.2 and 2 g/kg body weight, respectively. α, δ-NAG upregulated MKP-1 in an extracellular signal-regulated kinase (ERK) MAPK-dependent manner. MKP-1 siRNA abrogated the beneficial effects of α, δ-NAG. α, δ-NAG transiently increased intracellular calcium ([Ca²⁺]i), resulting in increased Ras activity. Inhibition of Gαq, a G protein subfamily, abrogated the effects of α, δ-NAG on [Ca²⁺]i and Ras activity. Inhibition of Gαq, Ca²⁺, and Ras abrogated the α, δ-NAG effects, such as ERK phosphorylation, MKP-1 upregulation, and neutrophilia/Th1 responses, in asthmatic mice. Overall, α, δ-NAG exhibited ∼10,000-fold stronger anti-inflammatory activity than Gln, likely attributable to its upregulation of MKP-1 by activating the G protein-coupled receptor (GPCR)/Gαq/Ca²⁺/Ras/ERK cascade.

The COVID-19 pandemic incited a global health crisis that accelerated the development of antiviral therapeutics. One successful avenue for inhibiting SARS-CoV-2 has been through targeting the main protease (M^pro; 3CL^pro), a key enzyme for the viral lifecycle that cleaves at 11 sites in the viral polyprotein pp1a and pp1ab. Although potent inhibitors of M^pro have been discovered, including the FDA-approved drug Paxlovid, the potential emergence of resistant variants requires continued antiviral development efforts. The current methods to characterize binders of M^pro, such as SPR or ITC, are costly and time-consuming. To improve the speed and feasibility of M^pro inhibitor development, we developed a competitive miniaturized fluorescence polarization (FP) binding assay. We repurposed small molecules from a DNA-encoded library screen into FP probes by appending a fluorophore with various linkers. After identifying a probe that exhibited potent M^pro binding (K_D ∼43 nM), we optimized buffer conditions, pH, and additives. Assay validation revealed that our competitive fluorescence polarization assay was robust, with a Z′-score of 0.79 and a signal window of 23. This assay can be used as a single-point assay for high-throughput screening or to triage small molecules by generating K_i values for binding. Efforts from this work resulted in an M^pro binding assay that requires minimal protein consumption, low sample volumes, short incubation times (30 min), and operates at room temperature. In conclusion, we developed a robust FP assay that can be used to rapidly characterize M^pro-binding small molecules to support the development of new SARS-CoV-2 antivirals.

The antiviral activity of compounds can be enhanced synergistically when used in combination; however, the underlying mechanisms remain poorly understood. This study aimed to identify the key properties contributing to these synergistic effects. Residual titers of influenza A virus were measured after treatment with a combination of one to three compounds. In parallel, the effects of each compound on the physical properties of lipid bilayers, specifically membrane fluidity, permeability, and solubilization, were assessed. Partial least-squares regression models were constructed to predict the log reduction in residual viral titers based on the summary statistics of the membrane property changes induced by the individual compounds in each combination. These models demonstrated high predictive accuracy. Analysis of the regression coefficients revealed that combinations producing diverse membrane effects, such as (1) increased permeability, (2) decreased fluidity, (3) apparent reduction in permeability (likely due to interactions with the fluorescent probe), and (4) both increased and decreased fluidity over time (depending on whether the compounds initially induced a significant change in fluidity and whether the effect was mitigated over time), were associated with enhanced antiviral activity. Because a single compound is unlikely to produce all these effects simultaneously, combining multiple compounds may be necessary to achieve synergistic antiviral action. Furthermore, canonical correlation analyses revealed strong associations between the changes in membrane properties and the molecular structures of the compounds.

The orexin-2 receptor (OX₂R), a G protein-coupled receptor activated by the neuropeptides, orexin A and B, plays an integral role in orchestrating motivation, feeding behavior, and the sleep-wake cycle. Pharmacological modulation of OX₂R has shown therapeutic potential for a variety of central nervous system (CNS) diseases, most notably narcolepsy and insomnia. Noninvasive imaging of OX₂R could enable the visualization of its regional distribution, facilitate assessments of target engagement, and support the development of OX₂R-directed therapies. Nonetheless, there are currently no suitable radioligands available for imaging OX₂R with positron emission tomography (PET). Herein, we report the design and evaluation of two novel PET ligand candidates, [¹⁸F]1 ([¹⁸F]OX₂-2303) and [¹⁸F]2 ([¹⁸F]OX₂-2304), as potential imaging probes for OX₂R. Both candidates exhibit excellent OX₂R binding affinity (K_i = 0.1 and 1 nM, respectively) and remarkable selectivity over OX₁R (>600-fold). In vitro autoradiography confirmed robust and selective binding to OX₂R in rat brain sections. In vivo PET imaging revealed low brain uptake at baseline, attributed to active efflux by P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP). Furthermore, pharmacological inhibition of these efflux transporters markedly enhanced brain penetration and OX₂R antagonists demonstrated notable blocking effects to OX₂R tracers during these conditions. Collectively, [¹⁸F]1 ([¹⁸F]OX₂-2303) and [¹⁸F]2 ([¹⁸F]OX₂-2304) constitute promising chemical starting points for the development of OX₂R PET radioligands, although further medicinal chemistry optimization will be required to overcome transporter-mediated efflux from the brain.

Monensin (MON) is a polyether ionophore antibiotic of natural origin and is an FDA-approved drug for veterinary use. Recent studies have highlighted its potential anticancer activity in various in vitro and in vivo models. In this study, we evaluated the anti-breast cancer activity of MON and 37 synthetic analog compounds using cell monolayer and organoid models. Through a mini-ring cell viability assay, several compounds were identified that were more potent and selective against breast cancer cells compared to non-cancerous cells, surpassing the activity of parent MON. MON and these compounds induced significant DNA fragmentation, reduced cell migration, and downregulated SOX2 expression. Furthermore, MON and the most potent analog, compound 12, reduced the percentage of CD44⁺/CD24^–/low stem-like cells and diminished colony formation properties. Proteomics analyses revealed that several pathways, including extracellular matrix organization, were significantly dysregulated by MON and compound 12 in breast cancer cells. Among these, TIMP2, a protein associated with the suppression of tumor growth and metastasis, was identified as one of the most prominently upregulated proteins by MON and compound 12 in MDA-MB-231 cells. This finding was also validated in other breast cancer and melanoma cell lines. To simulate breast cancer metastasis to the brain, a human hybrid organoid system: tumor in brain organoid (HOSTBO) model was developed. MON and compound 12 significantly reduced Ki-67 expression within the HOSTBOs, and compound 12 significantly downregulated SOX2 expression. Collectively, MON and compound 12 significantly reduced the proliferation of breast cancer stem-like cells in the organoid models, inhibited their migration, and dysregulated markers associated with stemness, demonstrating their potential as anti-metastatic agents and warranting further clinical development.

Parkinson’s disease (PD), the second most prevalent neurodegenerative disorder afflicting human health, is primarily characterized by the degeneration of dopaminergic neurons in the midbrain, leading to movement disorders as the main clinical manifestation. Extensive research has demonstrated that the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and its accompanying neuroinflammation play a pivotal role in the progression of PD. ST-4, namely 15-oxosteviol, an analogue of the diterpene oridonin, exhibits potent and specific inhibition of NLRP3 in in vitro experiments. The anti-inflammatory effects of ST-4 were evaluated in mouse models of chronic and progressive disorders, in which it showed significant efficacy in ameliorating obesity, type 2 diabetes, and peritonitis. In this study, the potential interest of ST-4 for the treatment of neuroinflammatory diseases was further investigated in a PD mouse model. ST-4 effectively suppressed the activation of the NLRP3 inflammasome induced by lipopolysaccharide in neuronal cells. Additionally, treatment with ST-4 significantly improved various aspects of PD pathology, including behavioral impairments, loss of dopaminergic neurons, alterations in cerebral neurophysiology, and dysregulated gene expression associated with metabolic dysfunction, highlighting its therapeutic potential for the treatment of Parkinson’s disease.

Therapy-induced senescence (TIS) is a reversible growth arrest induced by anticancer treatments, which may contribute to the development of long-term therapy resistance in tumor cells. Senotherapeutics, agents targeting senescent cells, are being tested in clinical trials to improve patient outcomes. Due to the transient nature of TIS, we hypothesized that senolytics would be most effective when administered at the appropriate time. We created a reliable TIS cell line model in triple-negative breast cancer (TNBC) using experimental drug YM155. We observed that a single dose of YM155 triggers a brief senescence, leading to a persistent drug-tolerant state that cannot be reversed by redosing. This reversibility is not limited to cancer cells. It extends to noncancerous human cells and live zebrafish larvae, suggesting a rapid adaptation mechanism against xenobiotics. We identified transforming growth factor-β (TGF-β), a cytokine linked to TNBC chemoresistance, as being expressed alongside the emergence of drug tolerance. We inhibited TGF-β signaling to eliminate the tolerant phenotype and promote the clearance of cancer cells by immune cells. However, this was most effective within a specific time window after TIS induction. We suggest that the timely use of senotherapeutics could improve the effectiveness of anticancer drugs in clinical settings.