Small Molecule Screening Research Articles

Abstract PO-077: High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC

Abstract Cancer metastasis is a complex cascade that involves activation of cancer cell invasion and migration. This activation is greatly attributed by multiple factors, including the tumor-microenvironment (TME). Cancer-associated fibroblasts (CAF), a prominent cell type within the TME, have been shown to promote cancer cell invasion and migration, causing metastasis in various cancers including head and neck squamous cell carcinoma (HNSCC). The molecular mechanisms of how CAFs promote HNSCC invasion remain elusive. Our research goal is to understand how CAFs influence HNSCC cells to become invasive and potentially metastatic. Using a top-down research approach, our aim is to identify novel therapeutic chemical probe/drug regimens that potentially target CAF-dependent HNSCC cancer cell-invasion. In-order to perform high-throughput small molecule screens, we have established a 384-well format, three-dimensional (3D) spheroid invasion assay, as a powerful tool to study CAF-dependent HNSCC cancer cell invasion. This platform is currently being used to screen small molecule libraries and identify putative molecular targets, providing insights into underlying mechanisms of CAF-induced cancer cell invasion and candidate therapeutic strategies. Citation Format: Kunal Karve, Stephanie Poon, Panagiotis Prinos, Laurie Ailles. High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-077.

Bio-activity prediction of drug candidate compounds targeting SARS-Cov-2 using machine learning approaches.

The SARS-CoV-2 3CLpro protein is one of the key therapeutic targets of interest for COVID-19 due to its critical role in viral replication, various high-quality protein crystal structures, and as a basis for computationally screening for compounds with improved inhibitory activity, bioavailability, and ADMETox properties. The ChEMBL and PubChem database contains experimental data from screening small molecules against SARS-CoV-2 3CLpro, which expands the opportunity to learn the pattern and design a computational model that can predict the potency of any drug compound against coronavirus before in-vitro and in-vivo testing. In this study, Utilizing several descriptors, we evaluated 27 machine learning classifiers. We also developed a neural network model that can correctly identify bioactive and inactive chemicals with 91% accuracy, on CheMBL data and 93% accuracy on combined data on both CheMBL and Pubchem. The F1-score for inactive and active compounds was 93% and 94%, respectively. SHAP (SHapley Additive exPlanations) on XGB classifier to find important fingerprints from the PaDEL descriptors for this task. The results indicated that the PaDEL descriptors were effective in predicting bioactivity, the proposed neural network design was efficient, and the Explanatory factor through SHAP correctly identified the important fingertips. In addition, we validated the effectiveness of our proposed model using a large dataset encompassing over 100,000 molecules. This research employed various molecular descriptors to discover the optimal one for this task. To evaluate the effectiveness of these possible medications against SARS-CoV-2, more in-vitro and in-vivo research is required.

A novel small molecule inhibitor of p38⍺ MAP kinase augments cardiomyocyte cell cycle entry in response to direct cell cycle stimulation.

Myocardial infarction (MI) is the leading cause of mortality globally due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we demonstrated that overexpression of four-cell cycle factors, CDK1, CDK4, cyclin B1 and cyclin D1 (4F), induced cell division in ~20% of the post-mitotic CMs overexpressed 4F. The current study aims to identify a small molecule that augments 4F-induced CM cycle induction. Screening of small molecules with a potential to augment 4F-induced cell-cycle induction in 60-day-old mature human induced pluripotent cardiomyocytes (hiPS-CMs) revealed N-(4,6-Dimethylpyridin-2-yl)-4-(pyridine-4-yl)piperazine-1-carbothioamide (NDPPC), which activates cell cycle progression in 4F-transduced hiPS-CMs. Autodock tool and Autodock vina computational methods showed that NDPPC has a potential interaction with the binding site at the human p38⍺ mitogen-activated protein kinase (p38⍺ MAP kinase), a critical negative regulator of the mammalian cell cycle. A p38 MAP kinase activity assay showed that NDPPC inhibits p38⍺ with 5-10 times lower IC50 compared to the other P38 isoforms in a dose-dependent manner. Overexpression of p38⍺ MAP kinase in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect. NDPPC is a novel inhibitor for p38 MAP kinase and is a promising drug to augment CM cell cycle response to the 4F. NDPPC could become an adjunct treatment with other cell cycle activators for heart failure treatment.

Identification and verification of diagnostic biomarkers in recurrent pregnancy loss via machine learning algorithm and WGCNA.

Recurrent pregnancy loss defined as the occurrence of two or more pregnancy losses before 20-24 weeks of gestation, is a prevalent and significant pathological condition that impacts human reproductive health. However, the underlying mechanism of RPL remains unclear. This study aimed to investigate the biomarkers and molecular mechanisms associated with RPL and explore novel treatment strategies for clinical applications. The GEO database was utilized to retrieve the RPL gene expression profile GSE165004. This profile underwent differential expression analysis, WGCNA, functional enrichment, and subsequent analysis of RPL gene expression using LASSO regression, SVM-RFE, and RandomForest algorithms for hub gene screening. ANN model were constructed to assess the performance of hub genes in the dataset. The expression of hub genes in both the RPL and control group samples was validated using RT-qPCR. The immune cell infiltration level of RPL was assessed using CIBERSORT. Additionally, pan-cancer analysis was conducted using Sangerbox, and small-molecule drug screening was performed using CMap. A total of 352 DEGs were identified, including 198 up-regulated genes and 154 down-regulated genes. Enrichment analysis indicated that the DEGs were primarily associated with Fc gamma R-mediated phagocytosis, the Fc epsilon RI signaling pathway, and various metabolism-related pathways. The turquoise module, which showed the highest relevance to clinical symptoms based on WGCNA results, contained 104 DEGs. Three hub genes, WBP11, ACTR2, and NCSTN, were identified using machine learning algorithms. ROC curves demonstrated a strong diagnostic value when the three hub genes were combined. RT-qPCR confirmed the low expression of WBP11 and ACTR2 in RPL, whereas NCSTN exhibited high expression. The immune cell infiltration analysis results indicated an imbalance of macrophages in RPL. Meanwhile, these three hub genes exhibited aberrant expression in multiple malignancies and were associated with a poor prognosis. Furthermore, we identified several small-molecule drugs. This study identifies and validates hub genes in RPL, which may lead to significant advancements in understanding the molecular mechanisms and treatment strategies for this condition.

Identification of the natural chalcone glycoside hydroxysafflor yellow A as a suppressor of P53 overactivation-associated hematopoietic defects.

Enhanced P53 signaling may lead to hematopoietic disorders, yet an effective therapeutic strategy is still lacking. Our study, along with previous research, suggests that P53 overactivation and hematopoietic defects are major consequences of zinc deficiency. However, the relationship between these two pathological processes remains unclear. In this study, we observed a severe reduction in the number of hematopoietic stem cells (HSCs) and multi-lineage progenitor cells in zebrafish treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and showed the indispensable role of P53 signaling in the process. Next, we took advantage of HSCs-labeled transgenic zebrafish and conducted a highly efficient phenotypic screening for small molecules against P53-dependent hematopoietic disorders. Hydroxysafflor yellow A (HSYA), a natural chalcone glycoside, exhibited potent protection against hematopoietic failure in zinc-deficient zebrafish and strongly inhibited the P53 pathway. We confirmed the protective effect of HSYA in zinc-deficient mice bone marrow nucleated cells, which showed a significant suppression of P53 signaling and oxidative stress. Furthermore, the hematopoietic-protective activity of HSYA was validated using a mice model of myelotoxicity induced by 5-FU. In summary, our work provides an effective phenotypic screening strategy for identifying hematopoietic-protective agents and reveals the novel role of HSYA as a promising lead compound in rescuing hematopoietic disorders associated with P53 overactivation.

Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

Low circulating phosphate (Pi) leads to rickets, characterized by expansion of the hypertrophic chondrocytes (HCs) in the growth plate due to impaired HC apoptosis. Studies in HCs demonstrate that Pi activates the Raf/MEK/ERK1/2 and mitochondrial apoptotic pathways. To determine how Pi activates these pathways, a small-molecule screen was undertaken to identify inhibitors of Pi-induced ERK1/2 phosphorylation in HCs. Vascular endothelial growth factor receptor 2 (VEGFR2) was identified as a target. Invitro studies in HCs demonstrate that VEGFR2 inhibitors block Pi-induced pERK1/2 and caspase-9 cleavage. Like Pi, rhVEGF activates ERK1/2 and caspase-9 in HCs and induces phosphorylation of VEGFR2, confirming that Pi activates this signaling pathway in HCs. Chondrocyte-specific depletion of VEGFR2 leads to an increase in HCs, impaired vascular invasion, and a decrease in HC apoptosis. Thus, these studies define a role for VEGFR2 in transducing Pi signals and mediating its effects on growth plate maturation.

Abstract GS.04: Myocardial Tricarboxylic Acid Cycle Suppression Disrupts The Epigenetic Landscape In Human Heart Failure With Preserved Ejection Fraction

Heart Failure with Preserved Ejection Fraction (HFpEF) represents the greatest unmet therapeutic need in cardiology and is intimately related to obesity and metabolic syndrome. Using a bedside-to-bench approach, we investigated the hypothesis that suppressed tricarboxylic acid cycle (TCA) enzymes and metabolic intermediates in HFpEF influence epigenetic histone post-translational modifications (PTMs) that regulate genes involved in mitochondrial OxPhos and fatty acid metabolism. Endomyocardial biopsies were obtained from human HFpEF patients (n=32) and donor controls (n=12) and integrated proteomics, metabolomics, ChIP-seq, and RNAseq analyses were performed. Small molecule histone PTM modulators were screened in vitro in neonatal rat ventricular cardiomyocytes (NRVMs) to identify potential therapeutic targets. Proteomics demonstrates reduced TCA cycle enzyme abundance consistent with our recently published metabolomics data showing reduced succinate, fumarate, and malate. Histone PTM profiling found H3K27ac and H3K36me3 upregulated (p<0.001) and inversely correlated with TCA cycle intermediates succinate and fumerate. ChIP-seq promoter gene ontology analysis revealed enrichment of mitochondrial metabolism and fatty acid uptake ontologies (CD36), as well as major cardiac transcription factors such as GATA4. Small-molecule inhibitor proteomics screens of histone writers in neonatal rat ventricular myocytes showed that H3K27ac inhibition upregulates all TCA cycle enzymes 1.5-4 fold and may rescue TCA cycle function. Our multi-omics approach suggests an association between H3K27ac, H3K36me3, and TCA cycle intermediates. Inhibition of myocardial H3K27ac deposition represents a novel therapeutic target for HFpEF.

Targeting an allosteric site in dynamin-related protein 1 to inhibit Fis1-mediated mitochondrial dysfunction

The large cytosolic GTPase, dynamin-related protein 1 (Drp1), mediates both physiological and pathological mitochondrial fission. Cell stress triggers Drp1 binding to mitochondrial Fis1 and subsequently, mitochondrial fragmentation, ROS production, metabolic collapse, and cell death. Because Drp1 also mediates physiological fission by binding to mitochondrial Mff, therapeutics that inhibit pathological fission should spare physiological mitochondrial fission. P110, a peptide inhibitor of Drp1-Fis1 interaction, reduces pathology in numerous models of neurodegeneration, ischemia, and sepsis without blocking the physiological functions of Drp1. Since peptides have pharmacokinetic limitations, we set out to identify small molecules that mimic P110’s benefit. We map the P110-binding site to a switch I-adjacent grove (SWAG) on Drp1. Screening for SWAG-binding small molecules identifies SC9, which mimics P110’s benefits in cells and a mouse model of endotoxemia. We suggest that the SWAG-binding small molecules discovered in this study may reduce the burden of Drp1-mediated pathologies and potentially pathologies associated with other members of the GTPase family.

A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

In Silico Structural-based Virtual Screening and Molecular Docking Analysis of Plant-derived Small Molecules for Treatment of COVID-19

Background and Objectives Despite many studies on the new Coronavirus disease 2019 (COVID-19), there is still a rate of disease mortality, which has made researchers to focus more on finding successful antiviral drugs. In this regard, small molecule inhibitors have been suggested for their higher safety, lower toxicity, and cost-effectiveness. In this study, we applied virtual screening and docking analysis to identify the prospective inhibitors of 3CL protease and ACE-2 receptors Subjects and Methods In this study, 1,600 natural compounds were assessed by virtual screening. The ligands with a high affinity to bind to active site residues of target proteins were identified using the glide docking method and then were included in the induced-fit docking analysis in Schrödinger-Maestro software. Results The found compounds such as Theaflavin and Delphinidin had a high affinity to bind to the receptors. They had higher binding energy and a potent inhibitory effect compared to common drugs such as chloroquine. Conclusion The introduced natural compounds can be used to suppress COVID-19. The results may help develop new drugs or formulations to combat COVID-19; however, clinical trials are needed to examine the potential of these small molecules alone or in combination with other medical procedures.

Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening

The MUS81-EME1/2 structure-specific endonucleases play a crucial role in the processing of stalled replication forks and recombination intermediates, and have been recognized as an attractive drug target to potentiate the anti-cancer efficacy of DNA-damaging agents. Currently, no bioactive small-molecule inhibitors of MUS81 are available. Here, we performed a high-throughput small-molecule inhibitors screening, using the FRET-based DNA cleavage assay. From 7920 compounds, we identified dyngo-4a as a potent inhibitor of MUS81 complexes. Dyngo-4a effectively inhibits the endonuclease activities of both MUS81-EME1 and MUS81-EME2 complexes, with IC50 values of 0.57 μM and 2.90 μM, respectively. Surface plasmon resonance (SPR) and electrophoretic mobility shift assay (EMSA) assays reveal that dyngo-4a directly binds to MUS81 complexes (KD ∼ 0.61 μM) and prevents them from binding to DNA substrates. In HeLa cells, dyngo-4a significantly suppresses bleomycin-triggered H2AX serine 139 phosphorylation (γH2AX). Together, our results demonstrate that dyngo-4a is a potent MUS81 inhibitor, which could be further developed as a potentially valuable chemical tool to explore more physiological roles of MUS81 in the cells.

Huangqi-Danshen decoction reshapes renal glucose metabolism profiles that delays chronic kidney disease progression

Huangqi-Danshen decoction (HDD), a Chinese herbal preparation, is effective in clinical treatment of chronic kidney disease (CKD). However, the underlying mechanism remains to be clarified. In this study, we aimed to investigate the role of HDD in the regulation of renal glucose metabolism in a CKD mouse model. The 0.2% adenine-induced CKD mouse model was administered HDD extract at a dose of 6.8 g/kg/day for 4 weeks. Detection of renal glucose metabolites was performed by ultra-performance liquid chromatography-tandem mass spectrometry. The expression of renal fibrosis and glucose metabolism-related proteins was tested by Western blotting, immunohistochemistry, and immunofluorescence. The results showed that HDD treatment could significantly reduce serum creatinine (0.36 ± 0.10 mg/dL vs. 0.51 ± 0.07 mg/dL, P < 0.05) and blood urea nitrogen (40.02 ± 3.73 mg/dL vs. 62.91 ± 10 mg/dL, P < 0.001) levels, and improve renal pathological injury and fibrosis. Aberrant glucose metabolism was found in the kidneys of CKD mice, manifested by enhanced glycolysis and pentose phosphate pathway, and tricarboxylic acid cycle inhibition, which could be partially restored by HDD treatment. Furthermore, HDD regulated the expression of hexokinase 2, phosphofructokinase, pyruvate kinase M2, pyruvate dehydrogenase E1, oxoglutarate dehydrogenase, and glucose-6-phosphate dehydrogenase in CKD mice. In conclusion, HDD protected against adenine-induced CKD, reshaped glucose metabolism profiles, and restored the expression of key enzymes of glucose metabolism in the kidneys of CKD mice. This study sheds light on targeting glucose metabolism for the treatment of CKD and screening small molecule compounds from herbal medicine to slow CKD progression.

Expression of Cell-Adhesion Molecules in E. coli: A High Throughput Screening to Identify Paracellular Modulators.

Cell-adhesion molecules (CAMs) are responsible for cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Claudins (CLDNs), occludin (OCLN), and junctional adhesion molecules (JAMs) are CAMs' components of the tight junction (TJ), the single protein structure tasked with safeguarding the paracellular space. The TJ is responsible for controlling paracellular permeability according to size and charge. Currently, there are no therapeutic solutions to modulate the TJ. Here, we describe the expression of CLDN proteins in the outer membrane of E. coli and report its consequences. When the expression is induced, the unicellular behavior of E. coli is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). Our method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows high-throughput screening (HTS) of small-molecules for interactions with CAMs. Here, we focused on using iCLASP to identify paracellular modulators for CLDN2. Furthermore, we validated those compounds in the mammalian cell line A549 as a proof-of-concept for the iCLASP method.

Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy

High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.

Small-molecule High-throughput Screening Identifies an MEK Inhibitor PD198306 that Enhances Sorafenib Efficacy via MCL-1 and BIM in Hepatocellular Carcinoma Cells.

Sorafenib is the most widely used systematic therapy drug for treating unresectable Hepatocellular Carcinoma (HCC) but showed dissatisfactory efficacy in clinical applications. We conducted a combinational quantitative small-molecule high-throughput screening (qHTS) to identify potential candidates to enhance the treatment effectiveness of sorafenib. First, using a Hep3B human HCC cell line, 7051 approved drugs and bioactive compounds were screened, then the primary hits were tested with/without 0.5 μM sorafenib respectively, the compound has the half maximal Inhibitory Concentration (IC50) shift value greater than 1.5 was thought to have the synergistic effect with sorafenib. Furthermore, the MEK inhibitor PD198306 was selected for the further mechanistic study. 12 effective compounds were identified, including kinase inhibitors targeting MEK, AURKB, CAMK, ROCK2, BRAF, PI3K, AKT and EGFR, and a μ-opioid receptor agonist and a Ltype calcium channel blocker. The mechanistic research of the combination of sorafenib plus PD198306 showed that the two compounds synergistically inhibited MEK-ERK and mTORC1- 4EBP1 and induced apoptosis in HCC cells, which can be attributed to the transcriptional and posttranslational regulation of MCL-1 and BIM. Small-molecule qHTS identifies MEK inhibitor PD1938306 as a potent sorafenib enhancer, together with several novel combination strategies that are valuable for further studies.

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Accumulation of NCOA1 dependent on HERC3 deficiency transactivates matrix metallopeptidases and promotes extracellular matrix degradation in intervertebral disc degeneration.

Matrix metallopeptidases (MMPs) are critical matrix-degrading molecules and they are frequently overexpressed in degenerative discs. This study aimed to investigate the mechanism for MMP upregulation. Immunoblot and RT-qPCR were used for detecting protein and gene expression levels. 4-month-old and 24-month-old C57BL/6 mice were used for evaluating intervertebral disc degeneration (IDD). An ubiquitination assay was used to determine protein modification. Immunoprecipitation and mass spectrometry were used for identifying protein complex members. We identified the elevation of 14 MMPs among 23 members in aged mice with IDD. Eleven of these 14 MMP gene promoters contained a Runx2 (runt-related transcription factor 2) binding site. Biochemical analyses revealed that Runx2 recruited a histone acetyltransferase p300 and a coactivator NCOA1 (nuclear receptor coactivator 1) to assemble a complex, transactivating MMP expression. The deficiency of an E3 ligase called HERC3 (HECT and RLD domain containing E3 ubiquitin-protein ligase 3) resulted in the accumulation of NCOA1 in the inflammatory microenvironment. High throughput screening of small molecules that specifically target the NCOA1-p300 interaction identified a compound SMTNP-191, which showed an inhibitory effect on suppressing MMP expression and attenuating the IDD process in aged mice. Our data support a model in which deficiency of HERC3 fails to ubiquitinate NCOA1, leading to the assembly of NCOA1-p300-Runx2 and causing the transactivation of MMPs. These findings offer new insight into inflammation-mediated MMP accumulation and also provide a new therapeutic strategy to retard the IDD process.

THE APPLICATION OF ARTIFICIAL INTELLIGENCE IN ANTIBIOTIC DISCOVERY: AN OVERVIEW OF CURRENT AND FUTURE PERSPECTIVE

Innovative techniques for antibiotic discovery are required to hasten the development of effective therapies as the worldwide burden of antibiotic resistance rises. Artificial intelligence, a technological revolution in full swing, offers a hopeful future by removing bottlenecks in the pipeline for discovering new antibiotics. This review highlights how improvements in artificial intelligence are reviving the use of earlier antibiotic discovery approaches, including small molecule screening and natural product exploration. The use of modern machine learning techniques in new fields of antibiotic discovery, such as antibacterial systems biology, drug combination creation, antimicrobial peptide identification, and mechanism of action prediction, is then examined. Finally, we make a call to action for multidisciplinary collaboration and free access to high-quality screening datasets to hasten the development of novel antibiotic medications and the training of machine learning models.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin-or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20μM with a Vmax of 0.079nmol/(μg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

Streamlined DNA-encoded small molecule library screening and validation for the discovery of novel chemotypes targeting BET proteins

Targeting aberrant epigenetic programs that drive tumorigenesis is a promising approach to cancer therapy. DNA-encoded library (DEL) screening is a core platform technology increasingly used to identify drugs that bind to protein targets. Here, we use DEL screening against bromodomain and extra-terminal motif (BET) proteins to identify inhibitors with new chemotypes, and successfully identified BBC1115 as a selective BET inhibitor. While BBC1115 does not structurally resemble OTX-015, a clinically active pan-BET inhibitor, our intensive biological characterization revealed that BBC1115 binds to BET proteins, including BRD4, and suppresses aberrant cell fate programs. Phenotypically, BBC1115-mediated BET inhibition impaired proliferation in acute myeloid leukemia, pancreatic, colorectal, and ovarian cancer cells invitro. Moreover, intravenous administration of BBC1115 inhibited subcutaneous tumor xenograft growth with minimal toxicity and favorable pharmacokinetic properties invivo. Since epigenetic regulations are ubiquitously distributed across normal and malignant cells, it will be critical to evaluate if BBC1115 affects normal cell function. Nonetheless, our study shows integrating DEL-based small-molecule compound screening and multi-step biological validation represents a reliable strategy to discover new chemotypes with selectivity, efficacy, and safety profiles for targeting proteins involved in epigenetic regulation in human malignancies.