Targeting Protein-protein Interactions Research Articles

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-paired 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.

Targeting protein-protein interaction interfaces with antiviral N protein inhibitor in SARS-CoV-2

Coronaviruses not only pose significant global public health threats but also cause extensive damage to livestock-based industries. Previous studies have shown that 5-benzyloxygramine (P3) targets the Middle East respiratory syndrome coronavirus (MERS-CoV) nucleocapsid (N) protein N-terminal domain (N-NTD), inducing non-native protein-protein interactions (PPIs) that impair N protein function. Moreover, P3 exhibits broad-spectrum antiviral activity against CoVs. The sequence similarity of N proteins is relatively low among CoVs, further exhibiting notable variations in the hydrophobic residue responsible for non-native PPIs in the N-NTD. Therefore, to ascertain the mechanism by which P3 demonstrates broad-spectrum anti-CoV activity, we determined the crystal structure of the SARS-CoV-2 N-NTD:P3 complex. We found that P3 was positioned in the dimeric N-NTD via hydrophobic contacts. Compared with the interfaces in MERS-CoV N-NTD, P3 had a reversed orientation in SARS-CoV-2 N-NTD. The Phe residue in the MERS-CoV N-NTD:P3 complex stabilized both P3 moieties. However, in the SARS-CoV-2 N-NTD:P3 complex, the Ile residue formed only one interaction with the P3 benzene ring. Moreover, the pocket in the SARS-CoV-2 N-NTD:P3 complex was more hydrophobic, favoring the insertion of the P3 benzene ring into the complex. Nevertheless, hydrophobic interactions remained the primary stabilizing force in both complexes. These findings suggested that despite the differences in the sequence, P3 can accommodate a hydrophobic pocket in N-NTD to mediate a non-native PPI, enabling its effectiveness against various CoVs.

Immune checkpoint inhibitors and cancer immunotherapy by aptamers: an overview.

Efforts in cancer immunotherapy aim to counteract evasion mechanisms and stimulate the immune system to recognise and attack cancer cells effectively. Combination therapies that target multiple aspects of immune evasion are being investigated to enhance the overall efficacy of cancer immunotherapy. PD-1 (Programmed Cell Death Protein 1), CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), LAG-3 (Lymphocyte-Activation Gene 3), and TIM-3 (T Cell Immunoglobulin and Mucin Domain-Containing Protein3) are all immune checkpoint receptors that play crucial roles in regulating the immune response and maintaining self-tolerance often exploited by cancer cells to evade immune surveillance. Antibodies targeted against immune checkpoint inhibitors such as anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab), anti-CTLA-4 antibodies (e.g., Ipilimumab), and experimental drugs targeting LAG-3 and TIM-3, aim to block these interactions and unleash the immune system's ability to recognise and destroy cancer cells. The US FDA has approved different categories of immune checkpoint inhibitors that have been utilised successfully in some patients with metastatic melanoma, renal cell carcinoma, head and neck cancers, and non-small lung cancer. Although several immune checkpoint inhibitor antibodies have been developed, they exhibited immune-related adverse effects, resulting in hypophysitis, diabetes, and neurological issues. These adverse effects of antibodies can be reduced by developing aptamer against the target. Aptamers offer several advantages over traditional antibodies, such as improved specificity, reduced immunogenicity, and flexible design for reduced adverse effects that specifically target and block protein-protein or receptor-ligand interactions involved in immune checkpoint pathways. The current study aims to review the function of particular immune checkpoint inhibitors along with developed aptamer-mediated antitumor cytotoxicity in cancer treatment.

A library-derived peptide inhibitor of the BZLF1 transcription factor.

Transcription factor dysregulation is associated with many diseases, including cancer. Peptide-based molecules are increasingly recognised as important modulators of difficult intracellular protein-protein interaction targets, with peptide library screening consequently proven to be a viable strategy in developing inhibitors against a wide range of transcription factors (TFs). However, current strategies simply select the highest affinity of binding to a target TF rather than the ability to inhibit TF function. Here, we utilise our Transcription Block Survival (TBS) screening platform to enable high-throughput identification of peptides that inhibit TFs from binding to cognate DNA sites, hence inhibiting functionality. In this study, we explore whether the TBS can be expanded to derive a potent and functional peptide inhibitor of the BZLF1 transcription factor. The library-derived peptide, AcidicW, is shown to form a more stable dimer with BZLF1 than the BZLF1 homodimer, with a thermal denaturation temperature exceeding 80°C. AcidicW can also functionally inhibit the BZLF1:TRE DNA interaction with high potency and an IC50 of 612 nM.

A Multimodal Deep Learning Framework for Predicting PPI-Modulator Interactions.

Protein-protein interactions (PPIs) are essential for various biological processes and diseases. However, most existing computational methods for identifying PPI modulators require either target structure or reference modulators, which restricts their applicability to novel PPI targets. To address this challenge, we propose MultiPPIMI, a sequence-based deep learning framework that predicts the interaction between any given PPI target and modulator. MultiPPIMI integrates multimodal representations of PPI targets and modulators and uses a bilinear attention network to capture intermolecular interactions. Experimental results on our curated benchmark data set show that MultiPPIMI achieves an average AUROC of 0.837 in three cold-start scenarios and an AUROC of 0.994 in the random-split scenario. Furthermore, the case study shows that MultiPPIMI can assist molecular docking simulations in screening inhibitors of Keap1/Nrf2 PPI interactions. We believe that the proposed method provides a promising way to screen PPI-targeted modulators.

Differentiation of Acute Myeloid Leukemia Cells upon Pharmacological Inhibition of LSD1 Requires Its N-Terminal Intrinsically Disordered Region

Lysine-specific histone demethylase 1 (LSD1/KDM1A) governs hematopoiesis by regulating hematopoietic stem cell renewal and proper differentiation. Additionally, LSD1 is required for the maintenance of acute myeloid leukemia (AML) cells and has emerged as a key therapeutic target. Prior work from our group and others have revealed that the LSD1 demethylase activity is not required for AML proliferation. Instead, the interaction between LSD1 and GFI1/GFI1B is necessary to sustain AML cell survival. Thus, beyond its canonical demethylase activity, LSD1 has nonenzymatic, transcription factor (TF)-specific scaffolding functions critical for its function. Most LSD1 inhibitors function by blocking protein-protein interactions between LSD1 and TFs, even though they were originally designed as enzyme inhibitors. While studies indicate that LSD1 has important scaffolding functions, how LSD1 can coordinate with TFs to control gene expression programs is not well understood. Here, we used CRISPR-suppressor scanning to identify regions of LSD1 outside the active site that when mutated can lead to resistance to the covalent LSD1 inhibitor, GSK-LSD1. The understudied N-terminal intrinsically disordered region (IDR) of LSD1 was among the most enriched regions. To explore this further, we generated clonal AML cell lines harboring in-frame deletions within the IDR. These IDR-mutant cell lines were drug resistant and failed to differentiate upon GSK-LSD1 treatment. Moreover, knockdown of endogenous LSD1 followed by overexpression of LSD1 IDR-mutants rescued growth in the presence of GSK-LSD1, confirming that a fully functional IDR is necessary for differentiation. We performed LSD1 co-immunoprecipitation followed by mass spectrometry in wild-type and IDR-mutant cells to measure changes in LSD1 interactors upon GSK-LSD1 treatment. GSK-LSD1 treatment disrupted the LSD1-GFI1B interaction in both wild-type and mutant cells at comparable levels. Since GSK-LSD1 still induces dissociation of the mutant complex but has minimal effect on proliferation, our results suggest that the IDR-mutant cells no longer require GFI1B. In support, knockout of GFI1B inhibited growth of wild-type but not IDR-mutant cells. Strikingly, GSK-LSD1 treatment promoted LSD1's interaction with several master regulator TFs in hematopoiesis, including C/EBPα, PU.1, and RUNX1. Notably, C/EBPα was the most up-regulated interactor in both cell lines, with modest enrichment in IDR-mutant over wild-type. Using live-cell imaging assays, we found that deletions within the LSD1 IDR enhance association with these TFs. These results suggest that GSK-LSD1 reprograms LSD1-TF interactions, disrupting LSD1-GFI1B to induce LSD1-C/EBPα association, and that the IDR mutations alters this LSD1 redistribution to other TF partners. LSD1 is known to silence gene enhancers during differentiation in a process termed “enhancer decommissioning”. The mechanisms by which LSD1 occupies and controls enhancers remain elusive. To test if the LSD1 IDR regulates enhancer elements, we profiled LSD1 and H3K27ac using ChIP-seq in wild-type and IDR-mutant cells upon GSK-LSD1 treatment. Upon GSK-LSD1 treatment, disruption of LSD1-GFI1B binding was accompanied by increased LSD1 levels at other TF and H3K27ac sites, supporting the notion that loss of LSD1-GFI1B interaction induces LSD1 redistribution to other TF partner sites. A concomitant increase in H3K27ac levels was observed across LSD1 sites co-bound by C/EBPα, PU.1, or RUNX1 in wild-type cells. By contrast, in IDR-mutant cells, increases in H3K27ac levels were blocked at these sites upon GSK-LSD1 treatment, suggesting that full enhancer activation is impaired by mutations in the IDR, potentially due to altered association with master regulator TFs. This points to a function of LSD1 IDR in controlling enhancer activation for differentiation. Collectively, our study reveals a role for IDR in regulating LSD1-TF interactions, controlling enhancer activation that is necessary for AML cell differentiation. These data provide deep functional and mechanistic insights into the role of LSD1 IDR and offer important considerations for pharmacological targeting of protein-protein interactions.

Differential expression of serum miR-486 and miR-25 in ulcerative colitis and Crohn’s disease: Correlations with disease activity, extent, and location

Novel reliable biomarkers of inflammatory bowel disease (IBD) are clinically imperative due to potential limitations of endoscopic techniques. MicroRNAs (miRNAs) have emerged as non-invasive biomarkers of IBD; however, the full disease-specific miRNAs signature for IBD subtypes remains elusive. We evaluated the diagnostic role of circulating miR-486 and miR-25 in IBD patients and their potential ability to discriminate IBD subtypes; ulcerative colitis (UC) and Crohn’s disease (CD). Sixty UC patients, 60 CD patients, and 60 healthy controls were recruited. Serum miRNA expression was determined using RT-qPCR. Bioinformatics was employed for target gene and protein-protein interaction (PPI) network analyses. Serum miR-486 was upregulated in CD patients, but didn’t change in UC patients compared to controls. Conversely, serum miR-25 was decreased in both CD and UC patients compared to controls. Only miR-486 was differentially expressed between UC and CD patients. Receiver-operating characteristic analysis revealed that serum miR-486 was superior in CD diagnosis (AUC=0.945) and significantly distinguished CD and UC patients, whereas miR-25 showed discriminative potential for both UC and CD from controls. In the multivariate logistic analysis only miR-486 was associated with the risk of CD diagnosis. Serum miR-486 was correlated with CD activity index and location of disease in CD patients, whereas miR-25 was correlated with the type/extent of UC. PPI network analysis revealed common target genes and signaling pathways for both miRNAs. Conclusively, serum miR-486 and miR-25 might serve as new biomarkers of IBD, with serum miR-486 could be employed in risk stratification of IBD subtypes and has the ground for clinical utility in CD diagnosis, whereas miR-25 has potential for UC and CD diagnosis.

A network pharmacology approach and experimental validation to investigate the anticancer mechanism of Qi-Qin-Hu-Chang formula against colitis-associated colorectal cancer through induction of apoptosis via JNK/p38 MAPK signaling pathway

Ethnopharmacological relevanceThe Qi-Qin-Hu-Chang Formula (QQHCF) is a traditional Chinese medicine prescription that is clinically used at the Affiliated Hospital of Nanjing University of Chinese Medicine for the treatment of colitis-associated colorectal cancer (CAC). Aim of the studyTo evaluate the potential therapeutic effects of QQHCF on a CAC mouse model and investigate its underlying mechanisms using network pharmacology and experimental validation. Materials and methodsThe active components and potential targets of QQHCF were obtained from Traditional Chinese Medicine Systems Pharmacology (TCMSP) and herb-ingredient-targets gene network were constructed by Cytoscape 3.9.2. Target genes of CAC were obtained from GeneCards, Online Mendelian Inheritance in Man, and DrugBank database. The drug disease target protein-protein interaction (PPI) network was constructed and the core targets were visualized and identified using Cytoscape. The Metascape database was used for GO and KEGG enrichment analysis. UHPLC-MS/MS was used to further identify the active compounds in QQHCF. Subsequently, the therapeutic effects and potential mechanism of QQHCF against CAC were investigated in AOM/DSS-induced CAC mouse in vivo, and HT-29 and HCT116 cells in vitro. Finally, interactions between JNK, p38, and active ingredients were assessed by molecular docking. ResultsA total of 176 active compounds, 273 potential therapeutic targets, and 2460 CAC-related target genes were obtained. The number of common targets between QQHCF and CAC were 165. KEGG pathway analysis indicated that the MAPK signaling pathway was closely associated with CAC, which may be the potential mechanism of QQHCF against CAC. Network pharmacology and UHPLC-MS/MS analyses showed that the active compounds of QQHCF included quercetin, kaempferol, luteolin, wogonin, oxymatrine, lupanine, and baicalin. Animal experiments demonstrated that QQHCF reduced tumor load, number, and size in AOM/DSS-treated mice, and induced apoptosis in colon tissue. In vitro experiments further showed that QQHCF induced apoptosis and inhibited cell viability, migration, and invasion in HCT116 and HT-29 cells. Notably, QQHCF activated the JNK/p38 MAPK signaling pathway both in vivo and in vitro. Molecular docking analysis revealed an ability for the main components of QQHCF and JNK/p38 to bind. ConclusionThe present study demonstrated that QQHCF could ameliorate AOM/DSS-induced CAC in mice by activating the JNK/p38 MAPK signaling pathway. These results have important implications for the development of effective treatment strategies for CAC.