ACS Pharmacology and Translational Science最新文献

Opioid agonist ligands bind opioid receptors and stimulate downstream signaling cascades for various biological processes including pain and reward. Historically, before cloning the receptors, muscle contraction assays using isolated organ tissues were used followed by radiolabel ligand binding assays on native tissues. Upon cloning of the opioid G protein-coupled receptors (GPCRs), cell assays using transfected opioid receptor DNA plasmids became the standard practice including ³⁵S-GTPγS functional and cAMP based assays. A number of research laboratories have studied key "tool" reference opioid receptor ligands for decades and used them as control reference compounds. Some, but not all, of these commonly used tool compounds have been characterized and compared side by side in parallel assays for selectivity profiles at the different human opioid receptors isoforms. Herein, we performed the standard FLIPR calcium mobilization assay using HEK293 cells engineered to stably express the Gα_Δ6qi4myr in parallel, at human MOR, KOR, DOR, and NOP opioid receptors. The following tool compounds: morphine, fentanyl, oxycodone, DAMGO, DPDPE, U69593, deltorphin II, and nociceptin, were examined herein. These included the substance use disorder (SUD) compounds morphine, fentanyl, and oxycodone. Additionally, the antagonist tool compounds naloxone, NTI, norBNI, and β-FNA were assayed in parallel at the human MOR, KOR, DOR, and NOP opioid receptors. Furthermore, the agonist tool compounds were tested in the same in vivo tail-flick antinociception assays via intrathecal injection for ED₅₀ potencies. These data provide both in vitro comparative pharmacology as a reference for cellular activities and in vivo antinociception profiles for these tool compounds.

Leishmaniasis is a chronic inflammatory zoonotic illness caused by protozoan flagellates belonging to the Leishmania genus. Current data suggest that over 1 billion people worldwide are susceptible to infection, primarily in tropical and subtropical countries, where up to 2 million new cases are reported annually. Therefore, the development of a vaccine is crucial to combating this disease. This study employed immunoinformatics approaches to design a multiepitope anti-Leishmania vaccine, GH₁₈-cpLeish, based on a cluster of six glycosyl hydrolases 18. We identified six helper T lymphocyte (HTL) epitopes and twenty-six cytotoxic T lymphocyte (CTL) epitopes with IC₅₀ values <50 nM, indicating high affinity. Additionally, we also identified 20 continuous and twenty-six discontinuous B-cell epitopes. Analysis for allergenicity and toxicity showed no potential to induce these phenomena. All data obtained from in silico tools suggest that physicochemical and biological studies indicate that the GH₁₈-cpLeish chimeric protein is a promising candidate for an anti-Leishmania vaccine. Docking analysis showed that the Pep₁-cpLeish::TLR₁, Pep₁-cpLeish::TLR₂, Pep₁-cpLeish::/TLR₃, and Pep₁-cpLeish::/TLR₄ complexes maintained a stable form. The best interaction cluster score was observed in the complex Pep₁-cpLeish::TLR₂ (center = -622.6 and lowest energy = -841.7 kcal.mol^-1) followed by the complexes Pep₁-cpLeish::TLR₄ (center = -590.3 and lowest energy = -590.3 kcal.mol^-1), Pep₁-cpLeish::TLR₃ (center = -589.1 and lowest energy = -657.0 kcal.mol^-1), and Pep₁-cpLeish::TLR₁ (center = -504.1 and lowest energy = -602.9 kcal.mol^-1), respectively. This study suggests that GH₁₈-cpLeish may be suitable for constructing second-generation anti-Leishmania and even third-generation vaccines, given that its gene sequence is optimized for this purpose.

Sigma 1 receptor (S1R) is a multifunctional, ligand-activated protein located in the membranes of the endoplasmic reticulum (ER). It mediates a variety of neurological disorders, including epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease. The wide neuroprotective effects of S1R agonists are achieved by a variety of pro-survival and antiapoptotic S1R-mediated signaling functions. Nonetheless, relatively little is known about the specific molecular mechanisms underlying S1R activity. Many studies on S1R protein have highlighted the importance of maintaining normal cellular homeostasis through its control of calcium and lipid exchange between the ER and mitochondria, ER-stress response, and many other mechanisms. In this review, we will discuss S1R different cellular localization and explain S1R-associated biological activity, such as its localization in the ER-plasma membrane and Mitochondrion-Associated ER Membrane interfaces. While outlining the cellular mechanisms and important binding partners involved in these processes, we also explained how the dysregulation of these pathways contributes to neurodegenerative disorders.

The accumulation of ceramides and related metabolites has emerged as a pivotal mechanism contributing to the onset of age-related diseases. However, small molecule inhibitors targeting the ceramide de novo synthesis pathway for clinical use are currently unavailable. We synthesized a safe and orally bioavailable inhibitor, termed ALT-007, targeting the rate-limiting enzyme of ceramide de novo synthesis, serine palmitoyltransferase (SPT). In a mouse model of age-related sarcopenia, ALT-007, administered through the diet, effectively restored muscle mass and function compromised by aging. Mechanistic studies revealed that ALT-007 enhances protein homeostasis in Caenorhabditis elegans and mouse models of aging and age-related diseases, such as sarcopenia and inclusion body myositis (IBM); this effect is mediated by a specific reduction in very-long chain 1-deoxy-sphingolipid species, which accumulate in both muscle and brain tissues of aged mice and in muscle cells from IBM patients. These findings unveil a promising therapeutic avenue for developing safe ceramide inhibitors to address age-related neuromuscular diseases.

Current in vitro cell-based methods, relying on single cell types, have structural and functional limitations in determining lung drug permeability, which is a contributing factor affecting both local and systemic drug levels. To address this issue, we investigated a 3D human lung airway model generated using a cell culture insert, wherein primary human lung epithelial and endothelial cells were cocultured at an air-liquid interface (ALI). To ensure that the cell culture mimics the physiological and functional characteristics of airway tissue, the model was characterized by evaluating several parameters such as cellular confluency, ciliation, tight junctions, mucus-layer formation, transepithelial electrical resistance, and barrier function through assaying fluorescein isothiocyanate-dextran permeability. To understand how the characterized ALI quality attributes influenced the absorption of inhaled drugs through the epithelial-endothelial barrier, we measured the permeability and epithelial intracellular concentrations of albuterol sulfate (AL), formoterol fumarate (FO), and fluticasone furoate (FL). The presented characterization results overall demonstrate that this culture platform mimicked the airway-specific structure and barrier function. An apparent permeability (P _app) of 5.7 × 10^-6 cm/s and an intracellular concentration below 1% were quantified for AL over 3 h. The P _app of FO was 8.5 × 10^-6 cm/s, with an intracellular concentration of 3.8%. Due to its high lipophilicity, FL showed a higher intracellular concentration (17.4%) compared to AL and FO, but also a 73.1% loss of the compound over 3 h due to nonspecific binding, with a P _app as low as 1.3 × 10^-7 cm/s. While the model exhibited physiologically relevant properties, its utility in estimating the permeability of inhaled drugs may be drug-specific, warranting further optimization and study.

Idiopathic pulmonary fibrosis (IPF) is a debilitating, incurable, and life-threatening disease that lacks effective therapy. The overexpression of phosphodiesterase 10A (PDE10A) plays a vital role in pulmonary fibrosis (PF). However, the impact of selective PDE10A inhibitors on the tumor growth factor-β (TGF-β)/small mother against decapentaplegic (Smad) signaling pathway remains unclear. Herein, we have exploited a novel carbonyl sulfide (COS)/hydrogen sulfide (H₂S)-donor hybrid PDE10A inhibitor called COS-2080 with a well-defined mechanism of H₂S-releasing action. It exhibited highly potent inhibitory activity against PDE10A and excellent PDE subfamily selectivity. Moreover, COS-2080 demonstrated significant antifibrotic effects by inhibiting cell proliferation and mitigating fibroblast-to-myofibroblast transition (FMT). A dry powder inhalation formulation called COS-2080-DPI has been developed using the ultrasonic spray freeze drying (USFD) technique, demonstrating significant antifibrotic efficacy in mice with bleomycin-induced PF at a dosage approximately 600 times lower than pirfenidone. This remarkable antifibrotic efficacy of COS-2080 on TGF-β1-induced FMT could be primarily attributed to its inhibition of the Smad2/Smad3 phosphorylation. Moreover, COS-2080 effectively attenuated fibrosis in MRC-5 cells by activating the cAMP/protein kinase A (PKA)/CREB pathway and potentially increasing levels of p53 protein. Our findings suggest that effective inhibition of PDE10A potentially confers a protective effect on FMT in PF by impeding TGF-β signaling and activating the cAMP/PKA/CREB/p53 axis.

Lipopolysaccharide (LPS)-neutralizing peptides are emerging as new potential therapeutic modalities to treat sepsis and skin infections. Purinergic ligand-gated ion channels (P2X receptors) play a critical role in various biological processes, including inflammation. Recent drug development efforts have significantly focused on the modulation of P2X receptors. Here, we investigated the effects of the synthetic LPS-neutralizing peptide Pep19-2.5 on human P2X receptors in cells of the innate immune system. Pep19-2.5 concentration-dependently triggered Ca²⁺ influx, interleukin (IL)-1β, and lactate dehydrogenase (LDH) release in Toll-like receptor-stimulated human macrophages and monocytes. Ca²⁺ influx was mediated at least partially by P2X7 receptors, and IL-1β and LDH release by P2X7 receptors, respectively. Confocal microscopy confirmed the colocalization of Pep19-2.5 with P2X7 receptors. Pep19-2.5-induced IL-1β release in primed cells was dependent on K⁺ efflux, caspase-1, and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 inflammasome. In the presence of the P2X7 receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate, Pep19-2.5 reduced IL-1β and LDH release. In 1321N1, astrocytoma cells stably transfected with human P2X receptors, Pep19-2.5 potently modulated P2X7 and P2X4 receptors (IC₅₀ values of 0.346 and 0.146 μM, respectively) but showed less (P2X1, P2X3) or no activity (P2X2) at other P2X receptor subtypes. Our findings underline the potential of LPS-neutralizing peptides as modulators of P2X receptors, thus expanding their applicability beyond the treatment of sepsis to the treatment of inflammatory diseases.

Acetyl coenzyme A (acetyl-CoA), a pivotal regulatory metabolite, is a product of numerous catabolic reactions and a substrate for various anabolic responses. Its role extends to crucial physiological processes, such as glucose homeostasis and free fatty acid utilization. Moreover, acetyl-CoA plays a significant part in reshaping the metabolic microenvironment and influencing the progression of several diseases and conditions, including cancer, insulin resistance, diabetes, heart failure, fear, and neuropathic pain. This Review delves into the role of acetyl-CoA in both physiological and pathological conditions, shedding light on the key players in its formation within the cytosol. We specifically focus on the physiological impact of malonyl-CoA decarboxylase (MCD), acetyl-CoA synthetase2 (ACSS2), and ATP-citrate lyase (ACLY) on metabolism, glucose homeostasis, free fatty acid utilization, and post-translational modification cellular processes. Additionally, we present the pathological implications of MCD, ACSS2, and ACLY in various clinical manifestations. This Review also explores the potential and limitations of targeting MCD, ACSS2, and ACLY using small molecules in different clinical settings.

[This corrects the article DOI: 10.1021/acsptsci.1c00230.].

Antibody-drug conjugates (ADCs) are a promising drug modality substantially expanding in both the discovery space and clinical development. Assessing the biotransformation of ADCs in vitro and in vivo is important in understanding their stability and pharmacokinetic properties. We previously reported biotransformation pathways for the anti-B7H4 topoisomerase I inhibitor ADC, AZD8205, puxitatug samrotecan, that underpin its structural stability in vivo using an intact protein liquid chromatography-high resolution mass spectrometry (LC-HRMS) approach. Herein, we employed a LC-high resolution multiple reaction monitoring (LC-MRM^HR) approach using both collision-induced dissociation (CID) and electron-activated dissociation (EAD) methods, confirming our earlier findings. Furthermore, we were able to obtain additional detailed structural information on the biotransformation products expanding on earlier intact analyses. We also highlight the high sensitivity of LC-MRM^HR for successfully identifying minor biotransformation products at low concentrations that were not detectable using the intact protein LC-HRMS workflow. Especially, EAD aided in the confirmation of biotransformation species that contain newly formed disulfide bonds due to the preferential dissociation of disulfide bonds using this method. We observed biotransformation reactions that vary between linker-payload (PL) conjugation sites on the antibody. For example, the trend toward constitutional isomerism in thio-succinimide linker hydrolysis, and the resulting positional isomers from thiol adduct formation following linker-PL deconjugation. The reported orthogonal analytical approaches highly complement and fortify the intact protein LC-HRMS data. This study sheds further light on detailed structural characterization of various ADC species and validates the proposed biotransformation pathways explaining the stability of AZD8205 in vivo.