ACS Pharmacology and Translational Science最新文献

Inhibitors of the Bruton’s tyrosine kinase (BTK) are of broad utility in the treatment of multiple diseases including several B-cell malignancies via effective blockade of oncogenic B-cell receptor (BCR) signaling. BTK is a cytoplasmic tyrosine kinase which harbors a targetable cysteine residue (Cys481) and the majority of BTK inhibitors are covalent modifiers directed at this position. Despite possessing a common mechanism of action, BTK inhibitors differ in key attributes including off-target kinome profiles, tolerability, pharmacokinetics and the underlying BTK inhibition kinetics. These characteristics play a significant role in the ultimate utility of these drugs. Herein, we compare several clinically active BTK inhibitors in biochemical and in vitro assays to gain a broader appreciation of the similarities and differences that govern the success of this important drug class. The combined datasets highlight that each agent has excellent on-target potency and good BTK selectivity. The data further suggests an association between optimized BTK inhibition kinetics and in vitro cytotoxicity profiles.

Gastric ulcers (GUs), a frequent gastrointestinal condition, cause mucosal injury and inflammation. We aimed to investigate the protective effects and underlying mechanisms of ezetimibe, statins, and their combination in indomethacin-induced GUs. Rats were assigned into six groups (n = 8, each): (I) normal (control), (II) gastric ulcer induced with a single oral dose of indomethacin (30 mg/kg body weight), (III) rats received oral simvastatin (40 mg/kg/day) for 14 days, (IV) rats received oral ezetimibe (10 mg/kg/day) for 14 days, (V) the combination group received both oral simvastatin and ezetimibe, and (VI) standard group received oral famotidine (20 mg/kg). On day 14, gastric ulcers were induced by a single oral dose of indomethacin (30 mg/kg), and the animals were sacrificed 6 h later for sample collection and tissue analysis. Tissue levels of malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), Kelch-like ECH-associated protein 1 (Keap1), superoxide dismutase (SOD), nuclear erythroid factor 2 (Nrf-2) and hem-oxygenase 1 (HO-1) were measured. Ezetimibe, simvastatin, and their combination prevented GU. The combination therapy significantly reduced MDA, TNF-α, Keap1, IL-1, and serum C-reactive protein (CRP). However, Nrf-2, HO-1, and SOD were significantly increased when compared to the GU group and monotherapy. Histological investigations demonstrated that the combination therapy reduced GU severity and preserved stomach tissue. Simvastatin plus ezetimibe exerted synergistic gastroprotective effects in rats, associated with Nrf2/HO-1 activation and suppression of Keap1, oxidative stress, and pro-inflammatory cytokines. This combination may represent a novel therapeutic approach for preventing NSAID-induced GUs, meriting further mechanistic and translational studies.

Histone deacetylases (HDACs) regulate gene expression and are promising targets in oncology. Especially the class I isoforms HDAC1 and HDAC2 are overexpressed in cancer. However, while ortho-aminoanilides with a suitable (het)aryl substitution are well-characterized HDAC1/HDAC2 inhibitors, the corresponding phenol analogs have not been sufficiently investigated so far. To this end, we compared the ortho-hydroxyanilide derivative ST13 with the pan-HDAC inhibitor vorinostat and Cpd-60, an ortho-aminoanilide with high HDAC1/HDAC2 selectivity. ST13 was further developed into a light-activatable prodrug (ST17) by masking its zinc-binding group with a photoremovable 4,5-dimethoxy-2-nitrobenzyl protecting group. Overall, we verified that ST13 is a selective, slow- and tight-binding HDAC1/HDAC2 inhibitor with antiproliferative activity. Furthermore, we demonstrated that the light-activatable prodrug ST17 readily releases ST13 upon irradiation, thereby allowing to precisely control its antiproliferative properties. These findings validate ortho-hydroxyanilides as effective HDAC1/HDAC2-selective inhibitors and highlight photocaging as a promising strategy to achieve spatiotemporal control of epigenetic therapies in cancer.

Sulforaphane (SFN) is a phytoderived compound abundant in cruciferous plants that possesses a broad spectrum of anticancer properties. We showed that SFN-induced caspase-mediated apoptosis in grade IV bone metastasis-derived androgen-insensitive PC-3 (IC₅₀ = 4.2 μM), and lymph node metastasis-derived androgen-sensitive LNCaP (IC₅₀ = 2.8 μM) prostate adenocarcinoma cells. SFN-mediated cardiotoxic side effects were tested in a preclinical in vitro model that enables the study simultaneously of the impact of drugs on cancer cell death and contractile properties of engineered heart tissues generated from human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM EHT). Thereby, SFN exposure induced PC-3 cell death without affecting the contractile force of hiPSC-CM EHT. Interestingly, the irregular beating pattern of hiPSC-CM EHT observed in the presence of PC-3 coculture was normalized compared to vehicle treatment. Overall, this in vitro coculture model of hiPSC-CM EHT and cancer cells could facilitate the study of cardiotoxic cancer drug side-effects.

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.