ACS Pharmacology and Translational Science最新文献

This study evaluates the potential of a ¹⁷⁷Lu-labeled GRPR-targeting antagonist as a radiotherapeutic agent for tumors expressing the gastrin-releasing peptide receptor (GRPR). The therapeutic effect of the radioligand was investigated both as a monotherapy and in combination with the mTOR inhibitor everolimus. The GRPR antagonist, LF1 (AAZTA⁵-Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂), was synthesized using the chelator AAZTA⁵ linked via a 4-amino-1-carboxymethylpiperidine (Pip) spacer and radiolabeled with lutetium-177. The preclinical evaluation included assessments of binding kinetics, blood and organ clearance, plasma protein binding, and metabolic stability. SPECT/CT imaging and biodistribution studies were performed in mice bearing PC3 xenograft tumors. To assess its therapeutic efficacy, PC-3-mice were treated with [¹⁷⁷Lu]Lu-LF1 either alone or following everolimus pretreatment. [¹⁷⁷Lu]Lu-LF1 showed high binding affinity (K_d = 0.12 ± 0.01 nM) and favorable pharmacokinetics, including rapid blood clearance and low plasma protein binding (2–3% at 5 and 15 min p.i.). Although subject to enzymatic degradation, the radioligand demonstrated high, sustained, and specific tumor uptake (42 ± 5.0% IA/g at 1 h and 3.9 ± 1.1% IA/g at 144 h p.i.). Pancreatic uptake cleared quickly, allowing for high-contrast SPECT/CT imaging. Therapeutically, tumors treated with 93 MBq of [¹⁷⁷Lu]Lu-LF1 grew more slowly than those treated with 41 MBq. The combination of everolimus and [¹⁷⁷Lu]Lu-LF1 resulted in significant tumor growth inhibition, compared to the relevant monotherapies with either [¹⁷⁷Lu]Lu-LF1 or everolimus. [¹⁷⁷Lu]Lu-LF1 shows promise as a therapeutic radioligand for GRPR-positive prostate cancer, offering high tumor uptake and rapid clearance from nontarget tissues. Mice bearing PC3 xenograft tumors were well tolerated and demonstrated enhanced therapeutic efficacy when combined with everolimus.

Tumor-associated macrophages (TAMs) critically shape the multiple myeloma (MM) microenvironment, yet the molecular programs linking immune signaling to MM dissemination remain unclear. Here, we identify a TAM-derived IL6-STAT3-PIM2-cMyc-FN1 axis that governs cell adhesion and epithelial-mesenchymal transition (EMT) in MM. Proviral Integration Site for Moloney murine leukemia virus 2 (PIM2) acts as a central effector by transcriptionally suppressing fibronectin 1 (FN1) via stabilization of c-Myc, thereby reducing MM-stromal adhesion and promoting migratory capacity. IL6-family cytokines secreted by M2-like TAMs activate STAT3 to induce PIM2 expression, forming a feed-forward loop that reinforces the EMT-like phenotype. Functional assays confirm that PIM2 knockdown restores FN1, increases adhesion, and impairs cell migration, while the dual silencing of FN1 reverses these effects. Analysis of patient biopsies and xenograft models revealed a reciprocal pattern of PIM2 and FN1 expression. These findings delineate a TAM-controlled signaling circuit that integrates inflammatory cues with adhesion loss and invasive behavior, highlighting the IL6-STAT3-PIM2-cMyc-FN1 axis as a potential target in MM therapy.

Gangliosides are sialic acid-enriched glycosphingolipids that play a vital role in regulating multiple signaling pathways during cancer progression. The diversity in their cell- and tissue-specific expression and dysregulations in cancer cells contributes to the unique pathophysiology of triple-negative breast cancer (TNBC). In this study, we follow up on our previously established hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (DTX-CPT-Gel therapy) that ensured effective tumor regression in multiple murine syngeneic and xenograft tumor models. Here, we demonstrate that DTX-CPT-Gel therapy downregulates GM3/GD3/GM1 gangliosides by targeting different ganglioside metabolic genes at the transcriptional and translational levels. DTX-CPT-Gel therapy-mediated alterations in ganglioside metabolism affect the activity of key growth factor receptor-mediated signaling pathways, including the epidermal growth factor receptor (EGFR) and cMET/hepatic growth factor receptor (HGFR), which positively impact tumor mitigation. Our work on DTX-CPT-Gel therapy, in continuum, highlights the potential of this therapy for TNBC treatment by intercepting multiple lipid-mediated signaling pathways and reinforces GD3 synthase/ST8SIA1 as a promising target for TNBC therapy.

The noncompetitive lysine-specific demethylase 1 (LSD1) inhibitors SP-2509 and SP-2577 are N′-(1-phenylethylidene)benzohydrazides that display potent activity in Ewing sarcoma. They block transcriptional regulation of the causative oncogenic fusion protein EWSR1::FLI1 and cause cell death. However, SP-2509 and SP-2577 are the only LSD1 inhibitors active in Ewing sarcoma; other LSD1 inhibitors have little effect. Studies from our group and others suggest that SP-2509 activity may result from off-target activity affecting the mitochondria. Here, we identified potential off-target mechanisms of N′-(1-phenylethylidene)benzohydrazides using an unbiased approach, cellular thermal shift assay coupled to mass spectrometry. Interestingly, this revealed significant destabilization of the electron transport chain complex III protein ubiquinol-cytochrome c reductase (UQCRFS1). We find that UQCRFS1 destabilization is likely linked to impaired iron–sulfur (Fe–S) cofactor binding and that SP-2509 broadly destabilizes cellular Fe–S proteins. Using both chemical and genetic tools, we show that SP-2509 mediated cell death is LSD1 independent and instead requires a N′-(2-hydroxybenzylidene)benzohydrazide. Our studies suggest that this core moiety alters iron metabolism in the cell. Importantly, we also find that the reversal of EWSR1::FLI1 transcriptional regulation by SP-2509 is independent of LSD1 inhibition. This unique activity is instead associated with the N′-(2-hydroxybenzylidene)benzohydrazide core and destabilization of Fe–S proteins. These findings reveal a novel mechanism of action for this class of compounds and raise additional questions regarding how EWSR1::FLI1 transcriptional regulation is linked to Fe–S biogenesis, the precise mechanisms of cell death, the biological features of susceptible cancer cells, and strategies for clinical translation.

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.