bioRxiv - Pharmacology and Toxicology最新文献

{"title":"From Virtual Screens to Cellular Target Engagement: New Small Molecule Ligands for the Immune Checkpoint LAG-3","authors":"Natalie Fuchs, Laura Calvo-Barreiro, Valerij Talagayev, Szymon Pach, Gerhard Wolber, Moustafa T. Gabr","doi":"10.1101/2024.08.04.604031","DOIUrl":"https://doi.org/10.1101/2024.08.04.604031","url":null,"abstract":"Herein, we performed a virtual screening study to discover new scaffolds for small molecule-based ligands of the immune checkpoint lymphocyte-activation gene 3 (LAG-3). Molecular dynamics (MD) simulations using the LAG-3 structure revealed two putative binding sites for small molecules: the antibody interface and a lipophilic canyon. A 3D pharmacophore screening resulted in the identification of potential ligands for these binding sites and afforded a library of 25 compounds. We then evaluated the screening hits for LAG-3 binding via microscale thermophoresis (MST) and surface plasmon resonance (SPR). Our biophysical screening identified two binders with <em>K</em>_D values in the low micromolar range, compounds 3 (antibody interface) and <strong>25</strong> (lipophilic canyon). Furthermore, we investigated the ability of LAG-3 hits to en-gage LAG-3 on a cellular level using a cellular thermal shift assay (CETSA), where compound <strong>3</strong> emerged as a promising candidate for future development.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933154","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of the first-efficacious A2AR negative allosteric modulators for high adenosine cancer immunotherapies","authors":"Margot Boujut, Margaux Héritier, Aurélie Gouiller, Camille Süess, Alessandro Scapozza, Thibaut De Smedt, Maxime Guibert, Sébastien Tardy, Hesham Hamed, David Pejoski, Leonardo Scapozza","doi":"10.1101/2024.08.05.606339","DOIUrl":"https://doi.org/10.1101/2024.08.05.606339","url":null,"abstract":"Inhibition of adenosine 2A receptor (A2AR) is recognized as a promising immunotherapeutic strategy but is challenged by the ubiquity of A2AR function in the immune system. To develop a safe yet efficacious immunotherapy, the discovery of a novel negative allosteric modulator (NAM) was preferred. Leveraging an in-house, sensitive, high-throughput screening cellular assay, novel A2AR NAM scaffolds were identified followed by an extensive structure-activity relationship (SAR) study, leading to the discovery of potent 2-amino-3,5-dicyanopyridine derivatives. Allosteric mode of action of active compounds was confirmed by shift assay, non-linearity of the Schild plot analysis, biophysical measurements, and retained satisfactory potencies in high-adenosine concentrations. Further correlation of A2AR engagement and downstream signaling was done in a human blood translational assay, clearly showcasing the potential of A2AR allosteric modulation as a novel approach for efficient and safer cancer immunotherapies.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"193 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of ICOS-targeted small molecules using affinity selection mass spectrometry screening","authors":"Longfei Zhang, Laura Calvo-Barreiro, Victor de Sousa Batista, Katarzyna Świderek, Moustafa T. Gabr","doi":"10.1101/2024.08.04.606538","DOIUrl":"https://doi.org/10.1101/2024.08.04.606538","url":null,"abstract":"Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound <strong>9</strong> with a promising ICOS/ICOS-L inhibitory profile (IC₅₀ = 29.38 ± 3.41 µM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound <strong>9</strong> in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Peucedanol ameliorates LPS-induced inflammation in RAW264.7 cells and CLP-induced sepsis in mice by inhibiting TLR4/myD88/NF-κB pathway","authors":"Qi Yao, Bo-tao Chang","doi":"10.1101/2024.08.02.606445","DOIUrl":"https://doi.org/10.1101/2024.08.02.606445","url":null,"abstract":"<strong>Background</strong> Previously, it has reported that Peucedanol (PEU) possesses anti-bacterial activity. However, its effect and mechanism against inflammation remains unclear.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"178 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design and Biophysical Characterization of Second-Generation Cyclic Peptide LAG-3 Inhibitors for Cancer Immunotherapy","authors":"Laura Calvo-Barreiro, Longfei Zhang, Yaser Ali, Ashfaq Ur Rehman, Moustafa Gabr","doi":"10.1101/2024.08.04.606540","DOIUrl":"https://doi.org/10.1101/2024.08.04.606540","url":null,"abstract":"Lymphocyte activation gene 3 (LAG-3) is an inhibitory immune checkpoint crucial for suppressing the immune response against cancer. Blocking LAG-3 interactions enables T cells to recover their cytotoxic capabilities and diminishes the immunosuppressive effects of regulatory T cells. A cyclic peptide (Cys-Val-Pro-Met-Thr-Tyr-Arg-Ala-Cys, disulfide bridge: 1-9) was recently reported as a LAG-3 inhibitor. Based on this peptide, we designed 19 derivatives by substituting tyrosine residue to maximize LAG-3 inhibition. Screening via TR-FRET assay identified 8 outperforming derivatives, with cyclic peptides 12 [Tyr6(L-3-CN-Phe)], 13 [Tyr6(L-4-NH2-Phe)], and 17 [Tyr6(L-3,5-DiF-Phe)] as top candidates. Cyclic peptide 12 exhibited the highest inhibition (IC50 = 4.45 ± 1.36 μM). MST analysis showed cyclic peptides 12 and 13 bound LAG-3 with KD values of 2.66 ± 2.06 μM and 1.81 ± 1.42 μM, respectively, surpassing the original peptide (9.94 ± 4.13 μM). Docking simulations indicated enhanced binding for cyclic peptide 12, with a docking score of -7.236 kcal/mol compared to -5.236 kcal/mol for the original peptide.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"29 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Machine learning enables high-throughput, low-replicate screening for novel anti-seizure targets and compounds using combined movement and calcium fluorescence in larval zebrafish","authors":"Christopher Michael McGraw, Annapurna Poduri","doi":"10.1101/2024.08.01.606228","DOIUrl":"https://doi.org/10.1101/2024.08.01.606228","url":null,"abstract":"Identifying new, more efficacious anti-seizure medications (ASMs) is challenging, partly due to limitations in animal-based assays. Zebrafish (Danio rerio) can serve as a model of chemical and genetic seizures, but methods for detecting seizure-like activity in zebrafish, though powerful, have been hampered by low sensitivity (locomotor/behavioral assays) or low-throughput (tectal electrophysiology or calcium fluorescence microscopy). To address these issues, we developed a novel approach to assay seizure-like activity using combined locomotor and calcium fluorescence features, measured simultaneously from unrestrained larval zebrafish using a 96-well fluorescent plate reader. Using custom software to track fish movement and changes in fluorescence (deltaF/F0) from high-speed time-series (12.6Hz), we trained logistic classifiers using elastic net regression to distinguish seizure-like activity from non-seizure related changes based on event-specific and subject-specific features in response to the GABAAR antagonist, pentylenetetrazole (PTZ). We demonstrate that a classifier trained on combined movement and fluorescence data achieves high accuracy (\"PTZ M+F\"; area-under-curve receiver-operator characteristic (AUC-ROC): 0.98; F1 score: 0.912) and out-performs classifiers trained on movement (\"PTZ M\"; AUC-ROC: 0.9, F1: 0.9) or fluorescence features alone (\"PTZ F\"; AUC-ROC 0.96; F1: 0.87). The rate of classified seizure-like events increases as a dose-response to PTZ (serial dose escalation, 0, 2.5mM, 15mM) and is strongly suppressed by ASM treatment (valproic acid, VPA; tiagabine, TGB). At high-dose PTZ, we show that VPA reduces seizure-like activity defined by either \"PTZ M+F\" or \"PTZ M\" classifiers. Meanwhile, TGB selectively reduces events defined by the \"PTZ M+F\" classifier, paralleling previous reports that TGB reduces electrographic but not locomotor seizures and highlighting the potential for our approach to combine features of previously orthogonal assays. Using ASM benchmark data, we employ bootstrap simulation to calculate the expected statistical power of our method as a function of sample size. We demonstrate that anti-seizure responses (robust strictly standardized mean difference, RSSMD, versus control) with magnitudes similar to those associated with VPA or TGB can be reliably detected (true positive rate (TPR) &gt; 90%) with as few as N=4 biological replicates per group, while maintaining a 5% false positive rate. In a prospective test screen with 3-6 replicates per group and on-plate controls, the anti-seizure effect of 4 out of 5 tested ASMs (CBZ, LEV, LZP, TGB) was detected. In summary, we demonstrate a simple high-throughput approach to whole organism anti-seizure phenotyping combining two previously reported metrics to facilitate screens for novel anti-seizure interventions in zebrafish.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933160","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Drug Metabolism and Transport Capacity of Endothelial Cells, Pericytes, and Astrocytes: Implications for CNS Drug Disposition","authors":"Hannah N. Wilkins, Stephen A. Knerler, Ahmed Warshanna, Rodnie Colón Ortiz, Kate Haas, Benjamin C. Orsburn, Dionna W. Williams","doi":"10.1101/2024.08.01.606165","DOIUrl":"https://doi.org/10.1101/2024.08.01.606165","url":null,"abstract":"Therapeutically targeting the brain requires interactions with endothelial cells, pericytes, and astrocytes at the blood brain barrier (BBB). We evaluated regional and cell-type specific drug metabolism and transport mechanisms using rhesus macaques and <em>in vitro</em> treatment of primary human cells. Here, we report heterogenous distribution of representative drugs, tenofovir (TFV), emtricitabine (FTC), and their active metabolites, which cerebrospinal fluid measures could not reflect. We found that all BBB cell types possessed functional drug metabolizing enzymes and transporters that promoted TFV and FTC uptake and pharmacologic activation. Pericytes and astrocytes emerged as pharmacologically dynamic cells that rivaled hepatocytes and were uniquely susceptible to modulation by disease and treatment. Together, our findings demonstrate the importance of considering the BBB as a unique pharmacologic entity, rather than viewing it as an extension of the liver, as each cell type possesses distinct drug metabolism and transport capacities that contribute to differential brain drug disposition.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sulfoglycodendron Antivirals with Scalable Architectures and Activities","authors":"PETR KRAL, Francesco Coppola, Roya Jafari, Katherine D. McReynolds","doi":"10.1101/2024.08.01.606251","DOIUrl":"https://doi.org/10.1101/2024.08.01.606251","url":null,"abstract":"Many viruses initiate their cell-entry by binding their multi-protein receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglyco-dendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multi-protein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed detail nature of binding between these antivirals and viral protein receptors, and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141881674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Salidroside Mitigates Malignant Arrhythmias by Restoring Sodium Channel Function During Ultra-Acute Myocardial Infarction","authors":"Gongxin Wang, Yilin Zhao, Chenchen Zhang, Xiuming Dong, Siyu Sun, Xiulong Wang, Dongxu Li, Xuefang Li, Huan Li, Chieh-Ju Lu, Yimei Du, Zhigang Chen, Fei Lin, Guoliang Hao","doi":"10.1101/2024.07.31.606101","DOIUrl":"https://doi.org/10.1101/2024.07.31.606101","url":null,"abstract":"Background: The ultra-acute phase (Phase 1a) of acute myocardial infarction (AMI) is marked by a high incidence of malignant arrhythmias, often occurring during the prehospital period. Currently, there are no effective treatment options available for managing these arrhythmias at this early stage. Methods and Results: Using dual-channel optical mapping, we simultaneously recorded membrane potentials and calcium transients during acute myocardial infarction. Calcium transient duration maps accurately localized the infarcted region, and action potential activation time maps revealed conduction heterogeneity in the infarcted zone. Patch-clamp recordings showed that Salidroside (Sal) (1 ug/mL) significantly increased sodium current density from -59.27 ± 2.15 pA/pF to -83.46 ± 3.19 pA/pF (P&lt;0.01) and shifted the Nav1.5 activation curve leftward (V1/2 from -37.27 ± 0.5 mV to -44.55 ± 0.7 mV, P&lt;0.01). In rat and rabbit AMI models, Sal pre-treatment reduced conduction heterogeneity and arrhythmia incidence compared to controls. Optical mapping showed improved conduction velocity and uniformity in the Sal group. Conclusions: Sal restores electrophysiological function in damaged myocardium by modulating sodium currents, reducing conduction heterogeneity, and decreasing malignant arrhythmia incidence during the ultra-acute phase of AMI. These findings suggest a novel therapeutic strategy for AMI, addressing a critical unmet need in antiarrhythmic therapy.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141881798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and application of species ID and insecticide resistance assays, for monitoring sand fly Leishmania vectors in the Mediterranean basin and in the Middle East","authors":"Sofia Balaska, Jahangir Khajehali, Konstantinos Mavridis, Mustafa Akiner, Kyriaki Maria Papapostolou, Latifa Remadi, Ilias Kioulos, Michail Miaoulis, Emmanouil Alexandros Fotakis, Alexandra Chaskopoulou, John Vontas","doi":"10.1101/2024.07.30.605769","DOIUrl":"https://doi.org/10.1101/2024.07.30.605769","url":null,"abstract":"Background\u0000Development of insecticide resistance (IR) in sand fly populations is an upcoming issue of public health concern, threatening leishmaniasis mitigation efforts by insecticide-based vector control. There is a major knowledge gap in the IR status of wild populations worldwide, possibly attributed to the unavailability of specialized tools, such as bioassay protocols, species baseline susceptibility to insecticides and molecular markers, to monitor such phenomena in sand flies.\u0000Methodology/ Principal findings\u0000Several sand fly populations from (semi-)rural regions of Greece, Turkey and Iran were sampled and identified to species, showing populations structure in accordance with previously reported data. Genotyping of known pyrethroid resistance-associated loci revealed the occurrence of voltage-gated sodium channel (vgsc) mutations in all surveyed countries. Knock-down resistance (kdr) mutation L1014F was prevalent in Turkish regions and L1014F and L1014S were recorded for the first time in Iran and in Turkey and Greece, respectively, yet in low frequencies. Moreover, CDC bottle bioassays against pyrethroids in mixed species populations from Greece indicated full susceptibility, using though the mosquito discriminating doses. In parallel, we established a novel individual bioassay protocol and applied it comparatively among distinct Phlebotomus species populations, to detect any possible divergent species-specific response to insecticides. Indeed, a significantly different knock-down rate between P. simici and P. perfiliewi was observed upon exposure to deltamethrin.\u0000Conclusions/Significance\u0000IR in sand flies is increasingly reported in leishmaniasis endemic regions, highlighting the necessity to generate additional monitoring tools, that could be implemented in relevant eco-epidemiological settings, in the context of IR management. Our molecular and phenotypic data add to the IR map in a macroarea with otherwise limited data coverage.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"216 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141881675","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}