Targeting Protein-protein Interactions Research Articles

The immunotherapy mechanism of Hedyotis Diffusae Herba in treating liver cancer: a study based on network pharmacology, bioinformatics, and experimental validation.

Liver cancer is a malignant tumor that develops on or inside the liver. Hedyotis diffusa Willd (HDW) plays a significant role in anti-tumor activities; however, its mechanism against liver cancer remains unclear. This study aims to evaluate the immunotherapeutic mechanism of HDW in treating liver cancer through network pharmacology, bioinformatics analysis, and experimental validation. Network pharmacology was utilized to identify the active components and potential targets of HDW from the TCMSP database. A potential target protein-protein interaction (PPI) network was constructed using the STRING database, followed by function and pathway enrichment analysis of the targets using GO and KEGG methods. In addition, the key targets for HDW against liver cancer were identified using five different algorithms in Cytoscape. The TCGA and HPA databases were used to assess the mRNA and protein expression of core target genes in normal liver and liver cancer tissues and their relationship with overall survival in liver cancer, as well as their role in immune infiltration. Molecular docking between the core components of HDW and the core targets was performed using PyMOL software. The effects of HDW on the proliferation and apoptosis of liver cancer cells were examined using MTT and flow cytometry. The regulatory effects of the core component quercetin on core targets were validated using RT-qPCR and Western blot. A total of 163 potential targets were identified by searching for intersections among 7 types of active components and all potential and liver cancer targets. PPI network analysis revealed the core targets IL6 and TNF. GO enrichment analysis involved 2089 biological processes, 76 cellular components, and 196 molecular functions. KEGG enrichment analysis suggested that the anti-cancer effects of HDW might be mediated by the AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, and NF-κB signaling pathway. Database validation of key targets showed that mRNA and protein expression results for the IL6 gene were contradictory, while those for the TNF gene were consistent, both being underexpressed in liver cancer. Importantly, the expression of IL6 and TNF was related to the infiltration of 24 types of immune cells, with the highest correlation with macrophages. Molecular docking showed that IL6 and TNF had high binding stability with quercetin, with binding energies of - 7.4 and - 6.0kJ∙mol-1, respectively. Experimental validation showed that quercetin inhibited liver cancer cell proliferation and promoted apoptosis in a dose-dependent manner, with protein results indicating that quercetin downregulated the mRNA and protein expression of IL6 and TNF, and upregulated key proteins in the AGE-RAGE signaling pathway, AGEs, and RAGE. This study comprehensively elucidates the activity, potential targets, and molecular mechanisms of HDW against liver cancer, providing a promising strategy for the scientific basis and treatment mechanism of traditional Chinese medicine in treating liver cancer.

Impact of the near-physiological temperature on the in vitro maturation of bovine oocytes: A comparative proteomic approach

In vivo, the temperature inside preovulatory follicles of cows is approximately 1 °C lower than rectal temperature. However, standard bovine oocyte in vitro maturation (IVM) protocols use 38.5 °C based on rectal temperature. This study evaluated the effect of reducing IVM temperature to 37.5 °C on the proteomic profile of oocytes compared to the routine 38.5 °C. Nuclear maturation rate and cumulus cell (CC) expansion (30 COCs per group, 21 replicates) were assessed by observing the first polar body and using a subjective scoring method (0–4). Total nitrite concentrations in the culture medium were measured using the Griess method. Differential proteomics was performed using LC-MS/MS on pooled oocyte samples (500 matured oocytes per group, three replicates), followed by gene ontology enrichment, protein-protein interaction, and putative miRNA target analyses. No significant differences were observed between the groups in nuclear maturation, CC expansion, or nitrite concentration (P > 0.05). A total of 806 proteins were identified, with 7 up-regulated and 12 down-regulated in the treatment group compared to the control. Additionally, 12 proteins were unique to the control group, and 8 were unique to the treatment group. IVM at 37.5 °C resulted in the upregulation of proteins involved in protein folding and GTP binding, and the downregulation of enzymes with oxidoreductase activity and proteins involved in cytoskeletal fiber formation. Furthermore, 43 bovine miRNAs potentially regulating these genes (DES, HMOX2, KRT75, FARSA, IDH2, CARHSP1) were identified. We conclude that IVM of bovine oocytes at 37.5 °C induces significant proteomic changes without impacting nuclear maturation, cumulus cell expansion, or nitrite concentration in the IVM medium.

Combining Network Pharmacology and Experimental Verification to Ascertain the Mechanism of Action of Asparagus officinalis Against the Brain Damage Caused by Fluorosis.

Asparagus officinalis (ASP) has antioxidation, anti-inflammatory, antiaging, and immune system-enhancing effects. We explored the preventive and therapeutic consequences of ASP on the brain damage elicited by fluorosis through network pharmacology and in vivo experimental validation. We ascertained the pharmaceutically active ingredients and drug targets of ASP from the Traditional Chinese Medicine Systems Pharmacology database, predicted the disease targets of fluorosis-induced brain injury using GeneCards and Online Mendelian Inheritance in Man databases, obtained target protein-protein interaction networks in the Search Tool for the Retrieval of Interacting Genes/Proteins database, used Cytoscape to obtain key targets and active ingredients, and conducted enrichment analyses of key targets in the Database for Annotation, Visualization and Integrated Discovery. Enrichment analyses showed that "mitogen-activated protein kinase" (MAPK), "phosphoinositide 3-kinase/protein kinase B" (PI3K-Akt), "nuclear factor-kappa B" (NF-κB), and the "neurotrophin signaling pathway" were the most enriched biological processes and signaling pathways. ASP could alleviate fluorosis-based injury, improve brain-tissue damage, increase urinary fluoride content, and improve oxidation levels and inflammatory-factor levels in the body. ASP could also reduce dental fluorosis, bone damage, fluoride concentrations in blood and bone, and accumulation of lipid peroxide. Upon ASP treatment, expression of silent information regulator (SIRT)1, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), MAPK, NF-κB, PI3K, Akt, and B-cell lymphoma-2 in rat brain tissue increased gradually, whereas that of Bax, caspase-3, and p53 decreased gradually. We demonstrated that ASP could regulate the brain damage caused by fluorosis through the SIRT1/BDNF/TrkB signaling pathway, and reported the possible part played by ASP in preventing and treating fluorosis.

Targeting protein-protein interactions for therapeutic intervention in Parkinson's disease to delay progression.

Background: Parkinson's disease (PD) is a neurodegenerative condition with few treatments to slow or stop development. Protein-protein interactions (PPIs) are intriguing therapeutic targets. This research examined the safety and effectiveness of a new Parkinson's disease medication targeting PPIs. Methods: After enrollment, 60 individuals were randomly assigned to the treatment and control groups. MDS-UPDRS Part III score change from baseline to week 12 was the main outcome measure. Secondary outcome measures were the Hoehn and Yahr scale, NMSS, and Montreal Cognitive Assessment. Study-wide adverse events were tracked. Results: Compared to the control group, the treatment group exhibited a substantial improvement in MDS-UPDRS Part III score (p < 0.001). Additionally, the therapy group showed substantial improvements in Hoehn and Yahr stage, NMSS score, and MoCA score compared to the control group (p < 0.001). No significant adverse effects were documented with the experimental medication. Conclusion: This research suggests that targeting PPIs may treat Parkinson's disease. No harmful side effects were detected with the experimental medication, which improved motor and non-motor symptoms in PD patients. These results require more study to determine the long-term safety and effectiveness of targeting PPIs in Parkinson's disease.

Mechanism of ergosterol peroxide on MCF-7 breast cancer cells based on network pharmacology and in vitro experiments

This study investigated the effects of ergosterol peroxide(EP) on the proliferation and apoptosis of MCF-7 breast cancer cells, explored its possible mechanisms of action, and verified the effects and mechanisms by in vitro experiments. Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI) networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects. The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation, and the colony formation assay was used to assess the cell cloning ability. Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis, mitochondrial membrane potential and reactive oxygen species(ROS) levels. Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cytochrome C(Cyt C), caspase-7, cleaved caspase-7, phosphatidylinositol 3-kinase(PI3K), and se-rine/threonine kinase B(AKT) in MCF-7 cells treated with EP. The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer; the results of Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway, PI3K-AKT signaling pathway, cellular senescence signaling pathway, and viral carcinogenesis pathway; and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group, exhibiting a time-and concentration-dependent trend, and EP can inhibit colony formation of MCF-7 breast cancer cells. Treatment with 10, 20, and 40 μmol·L~(-1) EP for 24 h resulted in a significant increase in the total apoptosis rate of MCF-7 cells, a significant decrease in mitochondrial membrane potential, and a significant increase in ROS levels. In addition, treatment with EP led to an upregulation of Cyt C, Bax, and cleaved caspase-7 protein expression, and a downregulation of p-PI3K, p-AKT, and Bcl-2 protein expression in MCF-7 cells. Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway mediating the mitochondrial apoptotic pathway.

Research on the mechanism of Ursolic acid for treating Parkinson's disease by network pharmacology and experimental verification

The objective of this study was to investigate the potential targets and mechanisms of UA in the treatment of PD. The efficacy of UA in PD was assessed through network pharmacology, molecular docking, and experimental methods. Common target protein-protein interaction (PPI) networks were constructed and visualized using Cytoscape. As a result, 9 key genes, namely CASP3, IL6, IL1B, PTGS2, CREB1, TNF, MAPK3, JUN, and CASP8, were selected. Molecular docking simulations were performed using Discovery Studio 2019 to validate the correlation between UA and the core targets. The results demonstrated a favorable binding affinity between UA and CASP8, IL1B, CASP3, TNF, MAPK3 and IL6. In vivo studies showed UA ameliorated motor dysfunction, and UA can significantly increase the protein expression of tyrosine hydroxylase (TH) in PD mice model. In addition, in vitro experiments confirmed that UA effectively reduced the protein expression of CASP8, CASP3 and MAPK3 in PD cell models and suppressed the gene expression of TNF-α, IL-6, and IL-1β. These findings indicate that the therapeutic effects of UA on PD could be due to its influence on various targets within both the apoptosis and neuroinflammatory signaling pathways. Consequently, this study provides a methodological and theoretical foundation for further elucidating the pharmacological mechanism of UA.

Abstract PO-009: AC676 a BTK Chimeric Degrader: Phase 1 study in patients with B-cell Malignancies

Abstract Study Background: Bruton tyrosine kinase (BTK) is a key signaling molecule that plays a central role in B-cell receptor transduction. The development of agents that inhibit BTK has transformed the management of patients with B-cell malignancies. However, therapy using covalent BTK inhibitors such as ibrutinib, as well as non-covalent inhibitors such as pirtobrutinib, can still result in resistance, primarily due to the development of mutations in BTK including residues C481 and L528. AC676 is designed as a chimeric degrader to target and degrade BTK using Accutar's proprietary Protein-Protein Interaction Targeting Chimeras (PPI-TAC) platform. By effectively linking a BTK ligand to the cereblon E3-ligase recruiting ligand, AC676 brings BTK in proximity to cereblon, thereby inducing subsequent ubiquitination and degradation of BTK. AC676 degrades BTK proteins irrespective of mutations; including C481, kinase dead L528 and others, and thus may be effective for the treatment of patients who have progressed on both covalent and non-covalent BTK inhibitors. Notably, it is also effective in cell lines expressing gain of function PLCG2 mutants, suggesting that it removes BTK’s scaffolding function. AC676 does not degrade cereblon neo-substrates, so neutropenia is not expected to be an on-target effect. This abstract describes an ongoing first in human Phase 1 trial of AC676. Study Description: AC676-001 is a Phase 1 dose-escalation study of AC676 administered orally once daily as monotherapy in patients with relapsed and refractory B-cell malignancies. Approximately 60 patients may be enrolled. Eligible patients must be ≥ 18 years, and have one of the following histologically confirmed relapsed or refractory disease types: chronic lymphocytic leukemia and small lymphocytic lymphoma, diffuse large B cell lymphoma – non-GCB subtype, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma including Waldenstrom macroglobulinemia. Patients must have received at least two prior systemic therapies or have no other standard of care therapies to provide significant clinical benefit. Patients must have measurable disease per disease-specific response criteria and Eastern Cooperative Oncology Group performance status ≤ 1. Dose-escalation will begin with an accelerated titration phase, followed by a standard 3+3 phase. AC676 is administered orally once daily on a 28 day per cycle schedule at doses ranging from 50mg to 600mg. The primary objective of the study is to evaluate the safety and tolerability of AC676. Secondary objectives include the evaluation of anti-tumor activity and the pharmacokinetic profile following single and multiple doses. Study enrollment began April in 2023 with five sites currently open in the United States (NCT05780034). Citation Format: Manish R Patel, Michael Tees, Farrukh T Awan, Nadia Khan, Nancy Mota, Hui Zhang, Su Young Kim, Jennifer Woyach. AC676 a BTK Chimeric Degrader: Phase 1 study in patients with B-cell Malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-009.

Abstract PO3-19-07: AC699-001, a first in human Phase 1 trial utilizing a novel estrogen receptor chimeric degrader in patients with advanced or metastatic breast cancer

Abstract Background Estrogen receptors (ER) are hormone-regulated transcription factors that play a critical role in breast cancer initiation and proliferation. Modulation of estrogen activity with therapeutics such as tamoxifen and aromatase inhibitors have been the mainstay therapeutic strategy for ER-positive breast cancer. Several ER-directed therapies have been developed to antagonize the oncogenic ER function, including Selective ER Degraders (SERDs) such as fulvestrant which is approved to treat patients with advanced or metastatic breast cancer. However, it requires intramuscular injection and has poor solubility, thereby limiting its administered dose and therefore its efficacy. In addition to SERDs, a newly emerging technology, E3 ligase-engaged chimeric degraders, has been used to induce potent and deeper ER degradation. Empowered by Accutar's proprietary Protein-Protein Interaction Targeting Chimeras (PPI-TAC) platform, AC699 was meticulously designed as a chimeric degrader to target and degrade the protein of interest, ERα. By effectively linking an ER ligand to an E3-ligase recruiting ligand, AC699 brings ERα in proximity to an E3 ligase, thereby inducing subsequent ubiquitination and degradation of ERα. Notably, chimeric ER degraders possess the unique advantage of degrading the ER protein without the inherent risk of activating an ER signal. Moreover, these molecules are not degraded alongside the target protein allowing for their efficient recycling within the cell. This direct mechanism enables chimeric ER degraders to achieve potent ER degradation with increased specificity, thereby potentially providing a higher therapeutic index compared to SERDs. This abstract describes a first-in-human Phase 1 trial of AC699, a novel ER degrader. Study Description The AC699-001 Phase 1 dose escalation study will enroll up to 60 patients with locally advanced or metastatic ER-positive, human epidermal growth factor receptor 2-negative, breast cancer. Patients must have progressed on standard treatment including at least two prior endocrine regimens or at least one prior line of therapy if combined with a CDK4/6 inhibitor. Prior chemotherapy is not required but must not exceed three prior cytotoxic regimens. Patients must have at least one measurable lesion or at least one predominantly lytic bone lesion. AC699 is given orally, once daily, with doses ranging from 100 mg to 600 mg in a standard 3+3 dose escalation design. The primary objective is to evaluate the safety and tolerability of AC699 using CTCAE 5.0. Secondary objectives include assessing preliminary anti-tumor activity according to RECIST 1.1 and characterizing the pharmacokinetic profile of single and multiple doses of AC699. Enrollment began in December 2022 with five sites in the United States planned. NCT05654532. Citation Format: Manish R Patel, Rachel Layman, Joy Nolte Fong, Hui Zhang, Su Kim, Erika Hamilton. AC699-001, a first in human Phase 1 trial utilizing a novel estrogen receptor chimeric degrader in patients with advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-19-07.

Network Pharmacology Study on Herb Pair Bletilla striata-Galla chinensis in the Treatment of Chronic Skin Ulcers.

Herb pair Bletilla striata-Galla chinensis (BS-GC) is a classic combination of topical traditional Chinese medicine formulae in the treatment of chronic skin ulcers (CSUs). The aim of this study is to explore the effective active ingredients of BS-GC, as well as the core targets and signal transduction pathways of its action on CSUs. The ingredients of BS-GC were obtained from TCMSP and HERB databases. The targets of all active ingredients were retrieved from the SwissTargetPrediction database. The targets of CSUs were obtained from OMIM, GeneCards, Drugbank, and DisGeNET databases. A drug-disease target protein-protein interaction (PPI) network was constructed to select the most core targets, and an herb-ingredient-target network was built by utilizing Cytoscape 3.7.2. Furthermore, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes database (KEGG) analysis and verified the results of network pharmacology through molecular docking. A total of 40 active ingredients from the herb pair BS-GC were initially screened, and a total of 528 targets were retrieved. Meanwhile, the total number of CSU targets was 1032. Then, the number of common targets between BS-GC and CSUs was 107. The 13 core targets of herb pair BS-GC with CSUs were filtered out according to the PPI network, including AKT1, TNF, EGFR, BCL2, HIF1A, MMP-9, etc. The 5 main core active ingredients were 1-(4-Hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene-4,7-diol, 1-(4- Hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol, physcion, dihydromyricetin, and myricetin. The main biological processes were inflammation, oxidative stress, and immune response, involving the AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, NF-κB signaling pathway, and calcium signaling pathway. Molecular docking results showed good binding activity between the 5 main core active ingredients and 13 core targets. This study predicted the core targets and signal transduction pathways in the treatment of CSUs to provide a reference for further molecular mechanism research.

Evaluation of Solubility and Membrane Permeability of Middle-Molecule Compounds Using Artificial Membranes and Living Cells

In contrast to small molecules, middle molecules present a promising therapeutic modality owing to their elevated specificity, minimal adverse effects, capacity to target protein-protein interactions, and, unlike antibody-based drugs, their suitability for oral administration and intracellular target engagement. Post-oral administration, the paramount considerations encompass solubility and membrane permeability during the initial phase until the drug attains systemic circulation. Furthermore, penetration of the cell membrane is essential to accessing intracellular targets. We evaluated the solubility and membrane permeability of 965 compounds sourced from middle molecule libraries affiliated with Hokkaido University, Kitasato University, and the University of Tokyo. To gauge membrane permeability, we employed both the parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell monolayers. Notably, while membrane permeability in Caco-2 cells exhibited an approximate threefold increase in comparison to PAMPA measurements, certain compounds demonstrated permeability levels less than one-third of those observed in Caco-2 cells. Recognizing the potential involvement of efflux transporters expressed in Caco-2 cells in these variations, we conducted additional assessments involving directional transport in the presence of a transporter inhibitor. Our findings suggest that nearly 80% of these compounds serve as substrates for efflux transporters. Considering the relevance of intracellular targets, we shifted our focus from membrane permeation to intracellular uptake, conducting simulations tailored to assess cellular uptake.

Potential acridinedione derivatives for the development of a heterobifunctional PROTAC for targeted degradation of PCSK9 protein

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) interacts with the low-density lipoprotein (LDL) receptor by protein-protein interaction (PPI). The inhibition of this PPI lowers the LDL cholesterol levels, thereby reducing the risks of cardiovascular diseases. We identified in-house synthesized acridinedione scaffolds as binders of PCSK9 through microsecond timescale conventional and biased molecular dynamics (MD) simulations. The free energy of binding was calculated by the Molecular Mechanics Poisson-Boltzmann Surface Area and umbrella sampling methods for acridinedione molecules and compared with reference molecules (co-crystallized inhibitors). Three acridinedione molecules (DSPD-2, DSPD-4, and DSPD-6) were identified as potential binders of the PCSK9 allosteric binding site from preliminary docking and steered MD simulations. The stability of molecules inside the allosteric pocket was established by the low values of root mean square deviations of backbone C-α atoms. The acridinedione derivatives also showed a shorter distance of hydrogen bonds as compared to the standard molecules. The molecule DSPD-6 showed the lowest binding free energy as compared to other selected and standard molecules. The binding of the selected molecules at the allosteric binding pocket had no effect on structural parameters of LDL receptor-binding site of PCSK9 protein. Finally, a proteolysis targeting chimera (PROTAC) was developed by attaching a proteasome recruiting tag to DSPD-6 (lowest binding affinity) to achieve ligand-induced protein degradation. These results qualify the development of the DSPD-6 molecule as a heterobifunctional PROTAC for targeted degradation of PCSK9 protein. Moreover, our computational strategy provides a framework for the identification of molecules against conventionally undruggable PPI targets.

Network pharmacology and molecular docking analysis on the mechanism of Tripterygium wilfordii Hook in the treatment of Sjögren syndrome.

Tripterygium wilfordii Hook. F (TWH) has significant anti-inflammatory and immunosuppressive effects, and is widely used in the inflammatory response mediated by autoimmune diseases. However, the multi-target mechanism of TWH action in Sjögren syndrome (SS) remains unclear. Therefore, the aim of this study was to explore the molecular mechanism of TWH in the treatment of SS using network pharmacology and molecular docking methods. TWH active components and target proteins were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. SS-related targets were obtained from the GeneCards database. After overlap, the therapeutic targets of TWH in the treatment of SS were screened. Protein-protein interaction and core target analysis were performed by STRING network platform and Cytoscape software. In addition, the affinity between TWH and the disease target was confirmed by molecular docking. Finally, the DAVID (visualization and integrated) database was used for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of overlapping targets. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database shows that TWH contains 30 active components for the treatment of SS. Protein-protein interaction and core target analysis suggested that TNF, MMP9, TGFB1, AKT1, and BCL2 were the key targets of TWH in the treatment of SS. In addition, the molecular docking method confirmed that the bioactive molecules of TWH had a high affinity with the target of SS. Enrichment analysis showed that TWH active components were involved in multiple signaling pathways. Pathways in cancer, Lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications is the main pathway. It is associated with a variety of biological processes such as inflammation, apoptosis, immune injury, and cancer. Based on data mining network pharmacology, and molecular docking method validation, TWH is likely to be a promising candidate for the treatment of SS drug, but still need to be further verified experiment.

Abstract CT156: A Phase I study evaluating AC676, an innovative BTK chimeric degrader, in patients with relapsed and refractory B-cell malignancies

Abstract Study Background Bruton tyrosine kinase (BTK) is a key signaling molecule that plays a central role in B-cell receptor transduction. The development of drugs that inhibit BTK has transformed the management of patients with B-cell malignancies. However, therapy using covalent BTK inhibitors such as ibrutinib, and non-covalent inhibitors such as pirtobrutinib, can still result in resistance, primarily due to the development of mutations in BTK including residues C481 and L528. AC676 was designed as a chimeric degrader to target and degrade BTK using Accutar's proprietary Protein-Protein Interaction Targeting Chimeras (PPI-TAC) platform. By effectively linking a BTK ligand to the cereblon E3-ligase recruiting ligand, AC676 brings BTK in proximity to cereblon, thereby inducing subsequent ubiquitination and degradation of BTK. AC676 degrades BTK proteins irrespective of mutations; including C481, kinase dead L528 and others, and thus may be effective for the treatment of patients who progress on both covalent and non-covalent BTK inhibitors. Notably, it is also effective in cell lines expressing gain of function PLCG2 mutants, suggesting that it removes BTK’s scaffolding function. AC676 does not degrade cereblon neo-substrates, so neutropenia is not expected to be an on-target effect. This abstract describes an ongoing first in human Phase 1 trial of AC676. Study Description AC676-001 is a Phase 1 dose-escalation study of AC676 administered orally once daily as monotherapy in patients with relapsed and refractory B-cell malignancies. Approximately 60 patients may be enrolled. Eligible patients must be ≥ 18 years, and have one of the following histologically confirmed relapsed or refractory disease types: Chronic lymphocytic leukemia and small lymphocytic lymphoma, Diffuse large B cell lymphoma - non-GCB subtype, Follicular lymphoma, Mantle cell lymphoma, Marginal zone lymphoma, Lymphoplasmacytic lymphoma including Waldenstrom macroglobulinemia. Patients must have received at least two prior systemic therapies or have no other standard of care therapies to provide significant clinical benefit. Patients must have measurable disease per disease-specific response criteria and Eastern Cooperative Oncology Group performance status≤ 1. Dose-escalation will begin with an accelerated titration phase, followed by a standard 3+3 phase. AC676 is administered orally once daily on a 28 day per cycle schedule at doses ranging from 50mg to 600mg. The primary objective of the study is to evaluate the safety and tolerability of AC676. Secondary objectives include the evaluation of anti-tumor activity and the pharmacokinetic profile following single and multiple doses. Study enrollment began in April 2023 with three sites currently open in the United States (NCT05780034). Citation Format: Jennifer Woyach, Michael Tees, Nancy Mota, Gladys Brown, Su Young Kim, Manish R. Patel. A Phase I study evaluating AC676, an innovative BTK chimeric degrader, in patients with relapsed and refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT156.

Abstract CT075: A Phase 1 trial evaluating AC699, an orally bioavailable chimeric estrogen receptor degrader, in patients with advanced or metastatic breast cancer

Abstract Background The role of estrogen receptor (ER) signaling in driving breast carcinogenesis is well established, and endocrine therapy has been a mainstay in the treatment of patients with ER-positive advanced or metastatic breast cancer for decades. Aromatase inhibitors, selective estrogen receptor modulators, and selective ER degraders (SERDs) such as fulvestrant, and more recently elacestrant, are integral components of the therapeutic strategy for patients with ER-positive breast cancer. However, unmet medical need persists in this patient population, due to suboptimal clinical outcomes and the development of resistance to therapy. E3 ligase-engaged chimeric degraders represent a technological advance with the potential to induce potent and deeper ER degradation. Utilizing Accutar's proprietary Protein-Protein Interaction Targeting Chimeras (PPI-TAC) platform, AC699 was designed as a novel chimeric degrader targeting ERα. By effectively linking an ER ligand to an E3-ligase recruiting ligand, AC699 brings ERα in proximity to an E3 ligase, resulting in ubiquitination and subsequent degradation of ERα. Chimeric ER degraders possess the unique advantage of degrading the ER protein without the inherent risk of activating an ER signal. Moreover, these molecules are not degraded alongside the target protein, allowing for their efficient recycling within the cell. This direct mechanism enables chimeric ER degraders to achieve potent ER degradation with increased specificity, thereby potentially providing a higher therapeutic index compared to SERDs. Study Description The AC699-001 trial is a first in human Phase 1 dose escalation study (NCT05654532) which will enroll up to 60 patients with locally advanced or metastatic ER-positive, human epidermal growth factor receptor 2-negative, breast cancer. Patients must have received at least two prior lines of endocrine treatment, or at least one prior line, if combined with a CDK4/6 inhibitor. Prior chemotherapy is not required but must not exceed three prior lines of cytotoxic treatment. Patients must have at least one measurable lesion or at least one predominantly lytic bone lesion. AC699 is administered orally, once daily, with doses ranging from 100 mg to 600 mg in a standard 3+3 dose escalation design. The primary objective is evaluation of the safety and tolerability of AC699. Secondary objectives include assessment of preliminary anti-tumor activity and characterization of the pharmacokinetic profile of single and multiple doses of AC699. Enrollment began in December 2022 and a total of 5 sites have been activated in the United States. Citation Format: Rachel M. Layman, Manish R. Patel, David Cosgrove, Michael Danso, Nancy Mota, Marjorie E. Zettler, Katherine C. Pehlivan, Erika Hamilton. A Phase 1 trial evaluating AC699, an orally bioavailable chimeric estrogen receptor degrader, in patients with advanced or metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT075.

An economical, high-throughput protein-protein interaction modulator drug screening technique based on surface-enhanced Raman scattering

Increasing identification of protein-protein interaction (PPI) targets creates opportunities for drug discovery. However, the current drug screening strategies are costly and reagent-consuming, which hinders the progress in PPI modulator drug discovery. The primary obstacle lies in the inability to achieve molecular fishing with existing technology. Herein, we present a novel high-throughput, homogeneous-phase screening assay for PPI modulator discovery called “SERScreen”, based on a magnetic field-amplified surface-enhanced Raman spectroscopy (SERS). Two high-affinity proteins are fixed respectively on the magnetic beads (MBs) and the SERS tags, and the cross-linking of two nanoprobes induced by PPI enables strong SERS signals. Candidate modulators interfere with the PPI according to higher affinity toward one protein, resulting in a significant reduction in SERS intensity above the MBs. We established a potential drug screening platform for PD-1/PD-L1, and demonstrated its feasiblility not only with known inhibitors (Durvalumab and BMS-202), but also with a small-molecule combinatorial library, successfully identifying two new candidate inhibitors via the molecular fishing of SERScreen. Furthermore, the anticancer mechanism of two candidates was discussed. As an ultrasensitive, low reagent consumption (2 µL sample solution), high-throughput screening technique in the PPI modulator discovery, SERScreen offers an effective solution for molecular fishing from complex samples and displays high compatibility with automatic measurement equipment.

PPII-AEAT: Prediction of protein-protein interaction inhibitors based on autoencoders with adversarial training

Protein-protein interactions (PPIs) have shown increasing potential as novel drug targets. The design and development of small molecule inhibitors targeting specific PPIs are crucial for the prevention and treatment of related diseases. Accordingly, effective computational methods are highly desired to meet the emerging need for the large-scale accurate prediction of PPI inhibitors. However, existing machine learning models rely heavily on the manual screening of features and lack generalizability. Here, we propose a new PPI inhibitor prediction method based on autoencoders with adversarial training (named PPII-AEAT) that can adaptively learn molecule representation to cope with different PPI targets. First, Extended-connectivity fingerprints and Mordred descriptors are employed to extract the primary features of small molecular compounds. Then, an autoencoder architecture is trained in three phases to learn high-level representations and predict inhibitory scores. We evaluate PPII-AEAT on nine PPI targets and two different tasks, including the PPI inhibitor identification task and inhibitory potency prediction task. The experimental results show that our proposed PPII-AEAT outperforms state-of-the-art methods.

Effects of Viscum coloratum (Kom.) Nakai on collagen-induced rheumatoid arthritis

Ethnopharmacological relevanceViscum coloratum (Kom.) Nakai has been traditionally used in China for nearly a thousand years to treat rheumatic diseases. However, its efficacy and mechanisms in treating rheumatoid arthritis (RA) have not been demonstrated. Aim of the studyTo investigate the anti-arthritic effects and molecular mechanisms of Viscum coloratum (Kom.) Nakai on collagen-induced arthritic mice through network pharmacology technology and experimental validation. Materials and methodsFirst, the main ingredients of the extract of Viscum coloratum (Kom.) Nakai (EVC) were identified through chemical composition characterization using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS). Then, the collagen-induced arthritis (CIA) model was established in DBA/1 J mice and the ameliorative effects of EVC on the progression of CIA mice were evaluated by oral treatment with different doses of the EVC for 28 days. After that, cytokine antibody microarray assay was used to detect the levels of multiple inflammation-related cytokines and chemokines in each group, and performed Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis. Subsequently, the potential target for the effective chemical components of EVC in treating RA was identified using various databases. Additionally, a drug-disease target protein-protein interaction network (PPI) was conducted using Cytoscape for visualization and clustering, while GO and KEGG enrichment analyses were performed with the Metascape database. Finally, identified phenotypes and targets by network pharmacology analysis were experimentally validated in vivo. ResultsTreatment with EVC significantly suppressed the severity of CIA with a dramatic reduction of paw swelling, arthritis index, levels of IgGs (IgG, IgG1, IgG2a, and IgG2b), multi-inflammation-related cytokines and chemokines on the progression of CIA. Histopathological examinations showed EVC could markedly inhibit inflammatory cell infiltration, tartrate-resistant acid phosphatase (TRAP) activity of osteoclast, and bone destruction. Furthermore, GO and KEGG enrichment analyses revealed that EVC could ameliorate RA by inhibiting osteoclast differentiation and regulating multiple signaling pathways including Osteoclast differentiation, IL-17, and TNF. PPI network analysis demonstrated that AKT1, MMP9, MAPK3, and other genes were highly related to EVC in treating RA. Finally, we proved that EVC could inhibit the expression of NFTAc1, MMP9, Cathepsin K, and AKT which were closely related to osteoclast activity. ConclusionsEVC could treat RA through multiple components, multiple targets, and multiple pathways. The present study demonstrated the therapeutic efficacy of EVC and its molecular mechanisms in treating RA, indicating that it would be a potent candidate as a novel botanical drug for further investigation.

AI-guided pipeline for protein–protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

Protein–protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

Mechanisms of action of Shizhenqing granules for eczema treatment: Network pharmacology analysis and experimental validation

BackgroundJiuwan decoction has been used to treat chronic eczema since the Qing Dynasty. According to clinical experience, Shizhenqing granules (SZQG), derived from the Jiuwan decoction, exert beneficial clinical effects on acute eczema and reduce recurrence. Therefore, we elucidated the underlying mechanisms of SZQG through network pharmacology, molecular docking, and experimental validation. MethodsThe main chemical components of SZQG were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). And the targets of SZQG against eczema were screened out through online databases. Then, the regulatory network map of the “herbal compound–potential target” and the target protein-protein interaction (PPI) network was constructed. The Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using by R language. Additionally, the interaction between the active compounds and the targets was verified by molecular docking technology. Finally, an experiment in vivo was used to verify the effect and mechanism of SZQG on eczema. ResultsUsing UHPLC-MS/MS, 158 main chemical compounds of SZQG were identified, and 72 compounds were selected according to the criteria for further analysis. All 237 potential targets of SZQG in eczema were explored using multiple online databases. The network with 14 core targets was screened out, including STAT3, RELA, TNF, JUN, MAPK3, IL-6, PIK3CA, STAT1, MAPK14, MAPK1, IL-4, NFKBIA, IL1B, and MYC. KEGG analyses indicated that the therapeutic effects of SZQG on eczema were predominantly associated with cytokine-cytokine receptor interaction, TNF, MAPK, NF-κB, toll-like receptor, T cell receptor, and Th1 and Th2 cell differentiation signaling pathways. Furthermore, the good affinity between the core compounds and core targets was verified by molecular docking technology, particularly for RELA and MAPK. Animal experiments revealed that SZQG downregulated MAPK14, RELA, T-bet, and GATA3 mRNA expression, reduced immunoglobulin E (IgE) and interleukin-4 (IL-4) serum concentrations, and improved eczema-like lesions in model rats. ConclusionThis study identified potential targets and signaling pathways of SZQG in the treatment of eczema, whereby RELA and MAPK14 may constitute the main therapeutic targets of SZQG in cytokine regulation and reduction of inflammatory responses.

Deciphering Multi-target Pharmacological Mechanism of Cucurbita pepo Seeds against Kidney Stones: Network Pharmacology and Molecular Docking Approach.

Urolithiasis is a prevalent condition with significant morbidity and economic implications. The economic burden associated with urolithiasis primarily stems from medical expenses. Previous literature suggests that herbal plants, including Cucurbita pepo, have lithotriptic capabilities. C. pepo is an annual, herbaceous, widely grown, and monoecious vegetative plant known for its antioxidants, fibers, and fatty acids. Recent studies on C. pepo seeds have shown therapeutic potential in reducing bladder stones and urodynamic illnesses, like kidney stones. However, the precise molecular and pharmacological mechanisms are unclear. In this research, we employed network pharmacology and molecular docking to examine the active compounds and biological mechanisms of Cucurbita pepo against kidney stones. Active constituents were obtained from previous studies and the IMPPAT database, with their targets predicted using Swiss target prediction. Kidney stone-associated genes were collected from DisGeNET and GeneCards. The active constituent-target-pathway network was constructed using Cytoscape, and the target protein-protein interaction network was generated using the STRING database. Gene enrichment analysis of C. pepo core targets was conducted using DAVID. Molecular docking was performed to identify potential kidney stone-fighting agents. The findings revealed that Cucurbita pepo contains 18 active components and has 192 potential gene targets, including AR, EGFR, ESR1, AKT1, MAPK3, SRC, and MTOR. Network analysis demonstrated that C. pepo seeds may prevent kidney stones by influencing disease-related signaling pathways. Molecular docking indicated that key kidney stone targets (mTOR, EGFR, AR, and ESR1) effectively bind with active constituents of C. pepo. These findings provide insight into the anti-kidney stone effects of Cucurbita pepo at a molecular level. In conclusion, this study contributes to understanding the potential of Cucurbita pepo in combating kidney stones and lays the foundation for further research.