ACS Pharmacology and Translational Science最新文献

Recent models of GPCR signaling extend beyond the canonical paradigm of ligand-induced activation primarily mediated by G-proteins and β-arrestins. Indeed, the various outcomes of GPCR activation are precisely regulated by several factors, including GPCR-interacting proteins, isoform diversity, and GPCR phosphorylation patterns catalyzed by kinases. One underexplored source contributing to the pleiotropic activities of GPCRs is the dynamic recruitment and dissociation of 14-3-3 proteins, a family of ubiquitously expressed adaptor proteins that modulate key cellular processes. Recent studies have shown that GPCR-14-3-3 protein interactions are isoform-specific and can be modulated in a spatiotemporal manner by ligands; however, an investigation of 14-3-3 dynamics across all seven human isoforms and throughout a large panel of GPCRs has yet to be conducted. Thus, this work provides the first characterization of proximal GPCR-14-3-3 protein interactions at a GPCRome scale, achieved by constructing stable reporter cell lines expressing all human 14-3-3 isoforms and performing cell-based high-throughput screening to probe 82 GPCR interactomes. Among the notable findings from this screen, the NK3 receptor emerged as a promising candidate for an in-depth examination of 14-3-3 protein modulation of GPCR activity, especially given the limited literature regarding NK3 signaling, including its relation to G-proteins or β-arrestins. Our findings suggest that different combinations of hetero- and homodimers of 14-3-3 isoforms result in varying functional outcomes at NK3, such as upregulation of NK3 surface expression by 14-3-3γ. Furthermore, 14-3-3γ was found to modulate canonical signaling pathways by attenuating G-protein dissociation and enhancing β-arrestin recruitment signals.

This narrative review provides an overview of benign FAPI-PET/CT or PET/MRI findings and studies investigating molecular imaging in nononcological diseases. Although the current focus of [68Ga]Ga-FAPI PET/CT is on oncologic indications, there is growing interest in the potential of FAPI PET/CT for nononcologic applications. Taking into account all-in-one, clinical, and preclinical studies, and the priorities of FAPI imaging over 2-[18F]FDG, the future direction of growing interest in the potential of FAPI tracer PET/CT as a promising technique in targeting fibroblast activation protein can be classified into some main fields for imaging and treatment monitoring. (1) Imaging of fibrotic disease, (2) cardiovascular imaging, (3) inflammatory and infectious diseases, (4) bone disease, (5) neuroimaging, and (6) organ transplantation imaging. The FAPI-radioligand shows promise as a targeted tracer for identifying and monitoring nononcological conditions, but current evidence is mainly based on small, heterogeneous retrospective analyses and case reports. Therefore, prospective studies are needed to reach reliable conclusions.

Permeation enhancers (PEs) are excipients used in oral biotherapeutic formulations to facilitate the transport of bioactive compounds across the intestinal barrier and prevent their degradation. Concerns associated with the chronic use of PEs demand comprehensive approaches to elucidate their potential toxicity mechanisms. A recent publication from our group reported nephrotoxicity in beagles after daily administration of enteric-coated (EC) tablets containing propyl gallate (PG) as a PE. To further characterize EC–PG-mediated nephrotoxicity mechanisms, we conducted a longitudinal mass spectrometry (MS)-based multiomics analysis of the dog plasma lipidome and proteome. Time-course analyses revealed elevation across multiple lipid classes and, in particular, species containing arachidonic acid, which may reflect EC–PG treatment-induced inflammation. At the protein level, alterations in biological processes associated with coagulation, complement activation, protein degradation and metabolism, and lipid transport and metabolism were observed. Integrative multiomics analyses provided additional insights into toxicity mechanisms at the interface between lipids and proteins. This holistic approach highlighted lipid transport and metabolism, oxidative stress, and inflammation as altered biological processes by EC–PG administration. Altogether, longitudinal multiomics profiling and integrative analysis provided additional mechanistic hypotheses for EC–PG induced renal toxicity, demonstrating the value of such an approach to investigate mechanisms relevant to drug safety.

dTAG-13 is a heterobifunctional molecule that induces proteasomal degradation of FKBP12^F36V-tagged proteins and is widely used in the dTAG system. To better understand its in vivo behavior, we investigated its metabolism in vitro and its metabolism, pharmacokinetics, and tissue distribution time-course in mice. dTAG-13 was rapidly absorbed within 40 min and distributed to most tissues (although not brain) with a half-life of 3.1 h. We identified 20 metabolites that resulted from demethylation, amide hydrolysis, O-dealkylation, ester cleavage, and hydroxylation products. No phase II metabolites were detected. Demethylation was predominant in the liver, while hydrolysis metabolites were abundant in plasma and widely distributed. Both NADPH-dependent and hydrolysis pathways contributed to its metabolism, with CYP3A playing a moderate role in dTAG-13 degradation. These findings support the suitability of dTAG-13 for short-term protein degradation studies, while its limited brain penetration and rapid clearance highlight the need for improved analogs. This study provides foundational insights into dTAG-13 disposition for rational in vivo use.

Adenosine triphosphate (ATP) and its breakdown products, including adenosine, play key roles in regulating immune responses. Altered ATP and adenosine levels in blood may reflect the presence or development of various pathologies; however, their rapid metabolism and clearance makes accurate measurement of their concentrations difficult. Not surprisingly, studies simultaneously monitoring ATP and its breakdown products are sparse and show conflicting results, and the workflows used are difficult to implement in clinical routine. Here, we present the simultaneous measurement of ATP and its metabolites in blood samples from healthy donors by combining a liquid chromatography–mass spectrometry-based quantification method with various procedures of blood sampling. We find that ATP and adenosine are best preserved in an ethylenediaminetetraacetic acid (EDTA) blood collection tube containing ectonucleotidase and nucleoside transporter inhibitors. In contrast, inosine and its downstream metabolites are detected in a serum collection tube without inhibitors. Therefore, we propose the use of these two sampling tubes to obtain a faithful determination of ATP and its degradation products. Overall, our approach provides a valuable and reliable tool to monitor changes in the concentration of ATP metabolites that can be easily implemented for biobanking purposes in the context of clinical trials.

Biotechnology and biomedical advances have driven the development of novel biopharmaceuticals to meet growing clinical demands. Among approved biologics, native Escherichia coli asparaginase has been under continuous optimization to improve thermostability, half-life, resistance to human proteases, and reduce adverse effects, particularly allergenicity. Here, we engineered an antileukemic biobetter by combining the substitutions P40S/S206C─previously identified by our group as less immunogenic and with extended bloodstream activity in mice─with N24S, reported to enhance in vitro stability. The purified triple mutant enzyme was biochemically characterized, and its cytotoxicity against leukemic cell lines and antigenic properties in Balb/c SPF mice were evaluated. TM displayed robust asparaginase activity, a >3-fold reduction in K_M for asparagine, superior thermostability, enhanced proteolytic resistance, and a lower in silico immunogenicity score compared to wild-type. In vivo, compared to wild-type, TM showed no apparent toxicity, a lower decrease in platelet counts, reduced induction of antiasparaginase IgE antibodies, and a preserved pharmacokinetic profile. In conclusion, combined mutations conferred substantial biochemical and immunological improvements, supporting the strategy of targeted amino acid substitutions to advance next-generation asparaginase biopharmaceuticals.

Pseudomonas aeruginosa is considered one of the most threatening pathogens worldwide, due to its high adaptability, which leads to resistance to classical antimicrobials. This fact has driven the development of new therapeutic strategies to reduce multiresistant strains and to minimize infection progression. In this context, the protective effect of a monoclonal antibody (mAb122) specific to pyocyanin (PYO), a key virulence factor of P. aeruginosa, was studied in vitro. Quenching PYO may reduce P. aeruginosa pathogenesis and partially lessen host immune dysregulation by impairing cytokine production. With this aim, murine macrophages were challenged with different PYO concentrations to assess their cytotoxicity by evaluating different cell viability hallmarks. Subsequently, the protective effect of mAb122 was studied on the PYO-treated cells. The addition of mAb122 significantly increased the percentage of viable cells compared to those treated just with the virulence factor (4.34- to 11.07-fold increase in MH-S and RAW 264.7 cells, respectively). Moreover, the PYO immunomodulatory effect and the outcome of mAb122 addition on the host response were also studied by measuring relevant cytokines in cell media. Results showed that mAb122 treatment, rather than reversing PYO impairment in cytokine production, either maintained the levels or triggered an increase, depending on the specific cytokine examined. Thus, the significant rise in cell viability and the nontoxic effect of mAb122 itself in vitro place PYO mAb as a promising candidate for in vivo testing as a potential therapeutic agent. However, its effects on the host immune system should be carefully studied and minimized.

Fragile X-associated tremor/ataxia syndrome (FXTAS), a nucleotide repeat expansion disorder, arises from CGG repeat expansions in the 5′ untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene, leading to RNA foci formation and toxic protein aggregation via repeat-associated non-AUG (RAN) translation. These fundamental mechanisms often lead to a series of consequences, including splicing defects, neuroinflammation, mitochondrial dysfunction, impaired autophagy, and cell death. Targeting toxic RNA repeats offers a promising therapeutic strategy. In this study, we identified Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, as a potential treatment for FXTAS. At first, we utilized various biophysical assays and molecular docking to confirm Celecoxib’s strong binding affinity toward the r(CGG)_exp RNA. Further studies in the cellular model demonstrated the potency of Celecoxib in reducing toxic protein aggregates and improving splicing defects. Notably, it significantly reduces FMR1PolyG aggregates in the Drosophila FXTAS model, leading to improved locomotor impairments and the mitigation of associated downstream pathological consequences. Moreover, Celecoxib treatment significantly extends the lifespan of the flies. Thus, these results collectively support the therapeutic potential of repurposing Celecoxib for the treatment of FXTAS.

Thrombotic diseases, classified as arterial or venous, remain one of the most important global health concerns. Myocardial infarction, ischemic stroke, and venous thromboembolism (VTE), which include deep vein thrombosis and pulmonary embolism, are prominent causes of illness and death. Antithrombotic agents, classified by their sites of action, are essential for preventing and treating thrombus formation. Transdermal drug delivery systems have emerged as promising alternatives for antithrombotic therapy by improving drug bioavailability, patient adherence, and therapeutic efficacy while reducing side effects. This systematic review, conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, identified 25 relevant articles through structured database searches. An additional search in clinical trial registries revealed no ongoing or completed clinical studies involving transdermal antithrombotic therapy. The literature focuses on transdermal formulations of heparins and acetylsalicylic acid, with fewer reports on direct oral anticoagulants and other agents. The literature search revealed that the most investigated delivery systems were microneedles (13), micro/nanoemulsions (2), ethosomes (1), hydrogels (5), polymeric patches (3), and liposomes (1). The ongoing interest in antithrombotic transdermal formulations highlights both their therapeutic importance and the difficulties still associated with traditional administration methods. While innovative transdermal formulations show promise, further research is necessary to develop scalable, effective, and cost-efficient technologies for clinical applications.