ACS Pharmacology and Translational Science最新文献

The opioid crisis presents a significant public health issue and underscores the urgency of developing effective treatments for opioid use disorder (OUD). Mitragynine (MTG), the major active alkaloid found in kratom (Mitragyna speciosa), presents as a potential OUD therapy. A physiologically based pharmacokinetic (PBPK) model has been established to support first-in-human (FIH) dose selection and assess potential drug–drug interactions (DDIs). Extensive physicochemical and in vitro studies were performed to define MTG’s pharmacokinetic properties for the model. The model was validated through in vivo pharmacokinetic studies (intravenous and oral) in both male and female Sprague–Dawley rats, revealing sex-related pharmacokinetic differences. Further validation in nonrodent models included pharmacokinetic studies in female beagle dogs. Utilizing this model, single and multiple-dose simulations of MTG (either as the pure compound or as the major alkaloid present in kratom) administration in humans were conducted, predicting the plasma concentration–time profiles of MTG and its active metabolite, 7-hydroxymitragynine (7-HMG) to facilitate dose selection. The model also evaluates MTG’s potential as both a victim and perpetrator in drug interactions, considering its effects with CYP3A4 and CYP2D6 inhibitors and substrates. Simulation results indicate that potent CYP3A4 and CYP2D6 inhibitors have minimal impact on MTG exposure. However, coadministration with CYP3A4 inhibitors leads to a reduction in 7-HMG formation. As a perpetrator, MTG has negligible effects on CYP2D6 substrates but increases midazolam exposure by 2.2 to 2.7-fold. This comprehensive model supports the therapeutic development of MTG.

Peroxynitrite causes oxidative DNA damage that can lead to cancer, cardiovascular, and neurodegenerative diseases. Polyphenol compounds have been extensively studied for their ability to prevent oxidative stress and DNA damage, but the cellular mechanisms for this behavior remain uncertain. We examined the ability of polyphenol compounds to inhibit peroxynitrite-mediated DNA damage in vitro and in Escherichia coli (AB1157) using gel electrophoresis, UV–vis spectroscopy, cellular DNA damage assays, and cell viability assays. Plasmid DNA gel electrophoresis studies show that polyphenols inhibit peroxynitrite-mediated DNA damage (IC₅₀ values of 13.4–1122 μM), activity that directly correlates with polyphenol redox potential. UV–vis kinetic studies also show peroxynitrite decomposition rates 2 to 27 times faster upon polyphenol addition. Polyphenols with gallol groups (IC₅₀ values of 13.4–158 μM) prevent significantly more DNA damage than their catechol analogs (IC₅₀ values of 68–1122 μM). Polyphenols with carboxylic acid groups decompose peroxynitrite five times faster than their methyl esters, indicating that carboxylate groups accelerate peroxynitrite decomposition. Polyphenol antioxidants also prevent cell death and cellular DNA damage upon peroxynitrite challenge in E. coli, confirming that polyphenols promote cell survival primarily by preventing DNA damage. This work represents the first combined in vitro and cellular study to determine the relationships between polyphenol structure and antioxidant prevention of peroxynitrite-mediated oxidative stress, information crucial for guiding studies of polyphenol antioxidant disease prevention.

Dopamine D4 receptor (D₄R) signaling affects decision-making, memory formation, cognition, and attention. Previously developed D₄R-selective ligands were metabolically unstable in vivo due to amide bond linker hydrolysis. In this study, analog compounds were synthesized using click chemistry, bioisosterically replacing amides with a 1,2,3-triazole linker. Herein, we report 1,2,3-triazole analogs maintained high D₄R affinity and subtype selectivity but had slightly reduced functional efficacy in cAMP and β-arrestin recruitment assays. Using rat and human liver microsomes to evaluate phase I metabolism, we determined that amide ligands were more metabolically unstable in rat microsomes, and the triazole substitutions enhanced compound stability. Four compounds were evaluated in rat pharmacokinetics studies. In particular, 17 (antagonist) and 18 (low-efficacy partial agonist) had desirable results in plasma half-life and brain exposure measures. These new analogs are suitable for behavioral studies in rats and represent improved molecular tools to further explore D₄R signaling in rodent models.

Somatostatin receptor 2 (SSTR2) is a valuable target for the treatment of patients with neuroendocrine tumors (NETs). [¹⁷⁷Lu]Lu-DOTA-TATE, an SSTR2-targeted ligand, is clinically used for therapy in patients with NETs; however, a low-absorbed dose in tumors remains a limitation. Albumin binders (ALBs) are commonly used to enhance the tumor accumulation of radioligands, but the effect of modulating ALB affinity has not been thoroughly investigated for DOTA-TATE-based SSTR2-targeted radioligands. In this study, we newly developed three DOTA-TATE-based radioligands with 4-(p-iodophenyl)butyric acid ([¹¹¹In]In-TATE-DA-I), 4-(p-bromophenyl)butyric acid ([¹¹¹In]In-TATE-DA-Br), and 4-(p-tolyl)butyric acid ([¹¹¹In]In-TATE-DA-CH₃) as ALB moieties. In the albumin-binding assay, the order of albumin-binding potency of each radioligand was consistent with that of the corresponding ALB moieties alone. In the biodistribution study, the radioligands with stronger albumin-binding potencies showed higher area under the curve (AUC) values in the blood. All ALB-containing radioligands exhibited higher tumor AUC values than [¹¹¹In]In-DOTA-TATE, and the order of tumor AUC values corresponded with those of blood AUC values and albumin-binding potencies. Among them, [¹¹¹In]In-TATE-DA-I showed the highest tumor AUC value and clearly visualized the SSTR2-positive tumor in SPECT/CT imaging. These findings highlight the importance of structural modification in the ALB moiety to enhance tumor accumulation of DOTA-TATE-based radioligands.

Mast cells are tissue-resident immune cells that are critical for the pathogenesis of allergic and inflammatory disorders. Their physiological functions include host defense against parasites and, more recently, food quality control through antigen avoidance. The purine nucleoside adenosine (ADO), like other mast cell activators, such as antigens or Mrgprb2 agonists, increases intracellular Ca²⁺ concentration; however, it fails to induce degranulation of preformed mediators when applied to mast cells alone, and there is limited knowledge about whether ADO evokes the de novo synthesis and release of inflammatory mediators in tissue mast cells. An unbiased genome-wide analysis of gene expression triggered by various mast cell activators should enable the identification of the gene program specifically activated by ADO in mast cells and thereby reveal new components of the associated inflammatory responses. Here, we performed bulk RNA sequencing on primary murine peritoneal mast cells (PMCs) representing connective tissue mast cells. By comparing responses evoked by ADO stimulation with those of the Mrgprb2 agonist compound 48/80 and antigens activating FcεRI receptors, we identified 393 genes uniquely regulated by ADO, including genes encoding the de novo synthesized mediators transforming growth factor α and interleukin 7. Transcription factor activity inference, protein classification, functional enrichment analysis, protein interaction network analysis, and topology analysis revealed a distinct ADO-specific transcriptional gene program involved in phosphoinositide signaling, vesicle trafficking, glycolysis, mitochondrial activity, and cell cycle arrest. The functional relevance of the identified de novo synthesized mediators for ADO-evoked inflammatory reactions can be evaluated in future studies.

ACKR3 is a class A G protein-coupled receptor that is considered as an atypical chemokine receptor. It does not activate G proteins but efficiently recruits β-arrestin and mediates ligand internalization and was thus proposed as a scavenger receptor. Besides chemokines, ACKR3 internalizes a variety of endogenous opioid peptides, including adrenorphin and dynorphin A. By reducing their availability to the classical opioid receptors, ACKR3 is proposed to participate in the endogenous pain management system, suggesting it as a new potential target for a new class of analgesics. Available structural data for ACKR3 are focused on the binding of chemokines (e.g., CXCL12), but how opioid peptides bind at ACKR3 remains enigmatic. Here, we structurally modeled opioid peptide binding at ACKR3 with a focus on adrenorphin, its ACKR3 selective variant LIH383, and dynorphin A. By combining molecular dynamics simulations with pharmacophore analysis, we analyze the opioid peptides’ binding modes and compare them with binding to classical opioid receptors (MOR, KOR, and DOR). We apply our model to rationally explain previously reported structure–activity relationships for adrenorphin derivatives, which also supports the model’s validation. Moreover, we include in vitro ACKR3 mutational experiments on both the receptor and LIH383 to further strengthen our structural model. Taken together, we systematically combine in silico observations and in vitro readouts to contribute to the understanding of ACKR3's ligand binding profile and set the basis for further ACKR3 ligand development.

Although animal models offer the physiology of the entire organism, various cell populations, and circuit-level behaviors, their predictive ability for polygenic neuropsychiatric disorders may be limited by species-specific neurodevelopment and genetics. Consequently, despite decades of neuropharmacological research, many CNS-targeted drug candidates still fail in late-stage clinical trials. This review summarizes how neuronal-engineering platforms, especially patient-derived induced pluripotent stem-cell (iPSC) organoids and neuron-glia cocultures, enable high-throughput screening (HTS) pipelines with greater clinical fidelity. This review focuses explicitly on neuropsychiatric disorders such as major depressive disorder, schizophrenia, bipolar disorder, and anxiety, and emphasizes human cell-derived organoid and neuron-glia coculture models tailored to their circuit-level pathophysiology. Organoid-enabled HTS couples human genetics with automated phenotyping, accelerating identification of circuit-level drug effects while reducing animal use. The remaining issues are integrating multiomics data, vascularization, and batch variability. These gaps will be filled, and precision psychiatry will become attainable with the continued advancements in biomaterials, single-cell analytics, and machine learning, by highlighting how human iPSC-derived organoids and advanced neuronal engineering recapitulate pathology and enable scalable drug screening. This review addresses a critical bottleneck in psychiatric drug development and outlines how these innovations can help close the bench-to-bedside gap in neuropsychiatric drug discovery.

High-throughput analysis has become a critical component in chemical biology and analytical chemistry due to the large libraries of compounds that are screened every day for drug development. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein modifications, both chemically deposited and biological post-translational modifications (PTMs). With the advent of antibody drug conjugates (ADCs) and other novel protein-based conjugates, the demand for such an analysis has skyrocketed. Here, we present a new protocol that achieves quantitative data for modified peptides in approximately 30 s of MS acquisition time. This platform includes a direct injection MS approach coupled with new software named iFishMass to extract targeted signals from hundreds of runs. iFishMass automatically generates plots and statistics. This platform will enable a faster analysis of synthetic modifications installed on monoclonal antibodies to create ADCs, and it is potentially scalable to biological PTMs. Sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates, paving the way to an inexpensive but effective platform for high-throughput screening of conjugation sites on proteins.

The Hamm laboratory recently published a cohort of PAR4 antagonists that were effective against the tethered ligand activation of PAR4. These compounds were generated from an ultralarge virtual screen using a homology model of PAR4. Upon further investigation, it appears the protease-activated receptor antagonists highlighted in this work have some thrombin liability. The Hamm laboratory further characterized the activity of these compounds using various methods, including a fluorescent thrombin activity assay, a chromogenic thrombin activity assay, and flow cytometry assays. We conclude that they do indeed antagonize PAR4, but thrombin is an additional target.