ACS Pharmacology and Translational Science最新文献

The P2X purinergic receptor 7 (P2X7) has an essential role in inflammation, innate immunity, tumor progression, neurodegenerative diseases, and several other diseases, leading subsequently to the development of P2X7 modulators. AZ11645373 is a frequently studied P2X7 antagonist tool compound but always used as a racemic mixture. Racemic AZ11645373 can be separated into its respective enantiomers by chiral chromatography, albeit in small batches, and these were stereochemically intact over two years later, by chiral high-performance liquid chromatography (HPLC) analysis. On a higher scale, significant decomposition is observed during purification. One of the enantiomers was crystallized as a palladium complex, and its (R)-configuration was determined by single-crystal X-ray diffraction, further confirmed, in solution, by vibrational circular dichroism. Biological studies demonstrated that both (S)- and (R)-forms were able to fully inhibit human P2X7, but (R)-AZ11645373 was more potent, with an IC₅₀ of 32.9 nM. Contrary to its effect on human P2X7, (S)-AZ11645373 was ineffective on mouse P2X7, while the (R)-AZ11645373 enantiomer was a full antagonist. These results demonstrated that the antagonistic effects of racemic AZ11645373 are mainly due to its (R)-enantiomer. Site-directed mutagenesis and molecular dynamics simulations indicated that the (R)-enantiomer may form specific interactions with Phe95 and the antagonists bound to other P2X7 monomers. Phe95 is situated in the allosteric binding site at the edge of the upper vestibule and appears to be the pivotal molecular gateway between AZ11645373 allosteric binding and locking of the closed state of the P2X7 channel. All together, these structure–function relationships should be helpful for drug design of P2X7 modulators.

The P2X purinergic receptor 7 (P2X7) has an essential role in inflammation, innate immunity, tumor progression, neurodegenerative diseases, and several other diseases, leading subsequently to the development of P2X7 modulators. AZ11645373 is a frequently studied P2X7 antagonist tool compound but always used as a racemic mixture. Racemic AZ11645373 can be separated into its respective enantiomers by chiral chromatography, albeit in small batches, and these were stereochemically intact over two years later, by chiral high-performance liquid chromatography (HPLC) analysis. On a higher scale, significant decomposition is observed during purification. One of the enantiomers was crystallized as a palladium complex, and its (R)-configuration was determined by single-crystal X-ray diffraction, further confirmed, in solution, by vibrational circular dichroism. Biological studies demonstrated that both (S)- and (R)-forms were able to fully inhibit human P2X7, but (R)-AZ11645373 was more potent, with an IC₅₀ of 32.9 nM. Contrary to its effect on human P2X7, (S)-AZ11645373 was ineffective on mouse P2X7, while the (R)-AZ11645373 enantiomer was a full antagonist. These results demonstrated that the antagonistic effects of racemic AZ11645373 are mainly due to its (R)-enantiomer. Site-directed mutagenesis and molecular dynamics simulations indicated that the (R)-enantiomer may form specific interactions with Phe95 and the antagonists bound to other P2X7 monomers. Phe95 is situated in the allosteric binding site at the edge of the upper vestibule and appears to be the pivotal molecular gateway between AZ11645373 allosteric binding and locking of the closed state of the P2X7 channel. All together, these structure-function relationships should be helpful for drug design of P2X7 modulators.

The rapid and organized healing of the cornea, while maintaining optical clarity, is essential for patient health and quality of life following corneal injuries. Oxygen plays a critical role in regulating cell migration and proliferation during wound repair, and the application of stem cell-derived exosomes offers potential therapeutic benefits due to their antioxidant and antiscarring properties. In this study, we developed oxygenated exosome-coated hemoglobin nanoparticles (OExo NPs) designed for effective oxygen delivery to enhance corneal re-epithelialization, reduce inflammation, and mitigate scarring. These OExo NPs exhibit a uniform average diameter of 130 nm and demonstrate consistent oxygen release capabilities. In vitro assays using human corneal epithelial cells-transformed (HCE-T) revealed that OExo NPs significantly promote cell proliferation and accelerate migration in scratch wound assays. Fluorescence imaging confirmed the successful internalization of OExo NPs into HCE-T cells and increased intracellular oxygen levels under hypoxic conditions. Gene expression analyses indicated a downregulation of critical wound healing markers, including HIF-1α, VEGF, IL-8, and FAK, suggesting effective alleviation of hypoxia, inhibition of angiogenesis, suppression of inflammation, and reduction of scar formation. These results highlight the potential of OExo NPs as a promising therapeutic approach for topical treatment of corneal wounds.

The rapid and organized healing of the cornea, while maintaining optical clarity, is essential for patient health and quality of life following corneal injuries. Oxygen plays a critical role in regulating cell migration and proliferation during wound repair, and the application of stem cell-derived exosomes offers potential therapeutic benefits due to their antioxidant and antiscarring properties. In this study, we developed oxygenated exosome-coated hemoglobin nanoparticles (OExo NPs) designed for effective oxygen delivery to enhance corneal re-epithelialization, reduce inflammation, and mitigate scarring. These OExo NPs exhibit a uniform average diameter of 130 nm and demonstrate consistent oxygen release capabilities. In vitro assays using human corneal epithelial cells-transformed (HCE-T) revealed that OExo NPs significantly promote cell proliferation and accelerate migration in scratch wound assays. Fluorescence imaging confirmed the successful internalization of OExo NPs into HCE-T cells and increased intracellular oxygen levels under hypoxic conditions. Gene expression analyses indicated a downregulation of critical wound healing markers, including HIF-1α, VEGF, IL-8, and FAK, suggesting effective alleviation of hypoxia, inhibition of angiogenesis, suppression of inflammation, and reduction of scar formation. These results highlight the potential of OExo NPs as a promising therapeutic approach for topical treatment of corneal wounds.

Kinase inhibitors are small-molecule drugs designed to target oncogenic mutations in cancer treatment. Although less toxic than conventional chemotherapy drugs, they can cause severe adverse effects in some patients, resulting in dose reduction and cessation. To evaluate if therapeutic drug monitoring of kinase inhibitors and their metabolites can improve toxicity assessment in patients, we developed and evaluated the analytical performance of two parallel methods utilizing liquid chromatography (LC) and paper spray (PS) ionization coupled with a triple quadrupole mass spectrometer (MS) for the measurement of dabrafenib, its major metabolite OH-dabrafenib, and trametinib in patient plasma samples. The PS-MS method yielded a faster sample analysis time (2 min) compared to the LC separation (9 min). The two methods shared the same analytical measurement range (AMR) for dabrafenib and OH-dabrafenib (10-3500 and 10-1250 ng/mL), but the AMR differed for trametinib (LC-MS: 0.5-50 ng/mL; PS-MS: 5.0-50 ng/mL). The imprecision across their respective AMR was 1.3-6.5% (dabrafenib), 3.0-9.7% (OH-dabrafenib), and 1.3-5.1% (trametinib) for the LC-MS method and 3.8-6.7% (dabrafenib), 4.0-8.9% (OH-dabrafenib), and 3.2-9.9% (trametinib) for the PS-MS method. Using authentic patient samples, the quantification results were comparable between the two methods: dabrafenib (correlation coefficient r = 0.9977), OH-dabrafenib (r = 0.885), and trametinib (r = 0.9807). Nonetheless, the PS-MS method displayed significantly higher variations compared with the LC-MS method. Based on the LC-MS method, we were able to profile the concentrations and metabolism patterns of dabrafenib and trametinib in patients who were receiving the drugs for BRAF V600 mutation-driven malignancies.

Kinase inhibitors are small-molecule drugs designed to target oncogenic mutations in cancer treatment. Although less toxic than conventional chemotherapy drugs, they can cause severe adverse effects in some patients, resulting in dose reduction and cessation. To evaluate if therapeutic drug monitoring of kinase inhibitors and their metabolites can improve toxicity assessment in patients, we developed and evaluated the analytical performance of two parallel methods utilizing liquid chromatography (LC) and paper spray (PS) ionization coupled with a triple quadrupole mass spectrometer (MS) for the measurement of dabrafenib, its major metabolite OH-dabrafenib, and trametinib in patient plasma samples. The PS–MS method yielded a faster sample analysis time (2 min) compared to the LC separation (9 min). The two methods shared the same analytical measurement range (AMR) for dabrafenib and OH-dabrafenib (10–3500 and 10–1250 ng/mL), but the AMR differed for trametinib (LC–MS: 0.5–50 ng/mL; PS–MS: 5.0–50 ng/mL). The imprecision across their respective AMR was 1.3–6.5% (dabrafenib), 3.0–9.7% (OH-dabrafenib), and 1.3–5.1% (trametinib) for the LC–MS method and 3.8–6.7% (dabrafenib), 4.0–8.9% (OH-dabrafenib), and 3.2–9.9% (trametinib) for the PS–MS method. Using authentic patient samples, the quantification results were comparable between the two methods: dabrafenib (correlation coefficient r = 0.9977), OH-dabrafenib (r = 0.885), and trametinib (r = 0.9807). Nonetheless, the PS–MS method displayed significantly higher variations compared with the LC–MS method. Based on the LC–MS method, we were able to profile the concentrations and metabolism patterns of dabrafenib and trametinib in patients who were receiving the drugs for BRAF V600 mutation-driven malignancies.

Residence time (RT) refers to the duration that a drug remains bound to its target, affecting its efficacy and pharmacokinetic properties. RTs are key factors in drug design, yet the structure-based design of ligands with desired RTs is still in its infancy. Here, we propose that a combination of cutting-edge molecular dynamics-based methods with classical computer-aided ligand design can help identify ligands that bind not only with high affinity to their target receptors but also with the required residence time to fully exert their beneficial action without causing undesired side effects.

Residence time (RT) refers to the duration that a drug remains bound to its target, affecting its efficacy and pharmacokinetic properties. RTs are key factors in drug design, yet the structure-based design of ligands with desired RTs is still in its infancy. Here, we propose that a combination of cutting-edge molecular dynamics-based methods with classical computer-aided ligand design can help identify ligands that bind not only with high affinity to their target receptors but also with the required residence time to fully exert their beneficial action without causing undesired side effects.

Drug discovery for central nervous system (CNS) targets is a high stakes process with estimated success rates below ten percent. Dose scaling, penetration through the blood–brain-barrier (BBB), and potency are among the various challenges involved in developing drugs for CNS targets. The standard approach to evaluate some of these parameters is dosing lead therapeutic compounds via intravenous delivery and assessing their brain levels via tissue homogenization and ex vivo quantification. Although a cost and time effective approach, brain homogenization lacks pharmacokinetic spatial resolution and normalizes drug levels to the entire brain volume. The brain, however, is known to have regional differences in cellular composition, transporters, BBB permeability, and drug-metabolizing enzymes, factors that could significantly affect pharmacological assessments during drug discovery. In this study we employ electrochemical aptamer-based sensors, a technology that allows in situ, real-time molecular monitoring in live animals, to reveal significant differences in the pharmacokinetics of drug uptake and accumulation in the brain of mice. Using vancomycin in the context of penetrating brain injury (PBI), our results highlight that potency may be significantly affected by PBI location. Additionally, more accurate dose scaling and delivery for deep brain wounds could be achieved by adjusting route of administration based on real-time-measured pharmacokinetic profiles, for example by changing delivery from intravenous to intracerebroventricular dosing. We emphasize the issue of establishing accurate pharmacological parameters during preclinical drug discovery efforts and underline the value of aptamer-based sensors for precise estimations of drug pharmacokinetics, transport across the BBB, and effective dose delivery during preclinical trials.

Drug discovery for central nervous system (CNS) targets is a high stakes process with estimated success rates below ten percent. Dose scaling, penetration through the blood-brain-barrier (BBB), and potency are among the various challenges involved in developing drugs for CNS targets. The standard approach to evaluate some of these parameters is dosing lead therapeutic compounds via intravenous delivery and assessing their brain levels via tissue homogenization and ex vivo quantification. Although a cost and time effective approach, brain homogenization lacks pharmacokinetic spatial resolution and normalizes drug levels to the entire brain volume. The brain, however, is known to have regional differences in cellular composition, transporters, BBB permeability, and drug-metabolizing enzymes, factors that could significantly affect pharmacological assessments during drug discovery. In this study we employ electrochemical aptamer-based sensors, a technology that allows in situ, real-time molecular monitoring in live animals, to reveal significant differences in the pharmacokinetics of drug uptake and accumulation in the brain of mice. Using vancomycin in the context of penetrating brain injury (PBI), our results highlight that potency may be significantly affected by PBI location. Additionally, more accurate dose scaling and delivery for deep brain wounds could be achieved by adjusting route of administration based on real-time-measured pharmacokinetic profiles, for example by changing delivery from intravenous to intracerebroventricular dosing. We emphasize the issue of establishing accurate pharmacological parameters during preclinical drug discovery efforts and underline the value of aptamer-based sensors for precise estimations of drug pharmacokinetics, transport across the BBB, and effective dose delivery during preclinical trials.