Small-molecule Compound Library Research Articles

Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast.

The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.

Genetically encoded fluorescent sensors for visualizing polyamine levels, uptake, and distribution.

Polyamines are abundant and physiologically essential biomolecules that play a role in numerous processes, but are disrupted in diseases such as cancer, and cardiovascular and neurological disorders. Despite their importance, measuring free polyamine concentrations and monitoring their metabolism and uptake in cells in real-time remains impossible due to the lack of appropriate biosensors. Here we engineered, characterized, and validated the first genetically encoded biosensors for polyamines, named iPASnFRs. We demonstrate the utility of iPASnFR for detecting polyamine import into mammalian cells, to the cytoplasm, mitochondria, and the nucleus. We demonstrate that these sensors are useful to probe the activity of polyamine transporters and to uncover biochemical pathways underlying the distribution of polyamines amongst organelles. The sensors powered a high-throughput small molecule compound library screen, revealing multiple compounds in different chemical classes that strongly modulate cellular polyamine levels. These sensors will be powerful tools to investigate the complex interplay between polyamine uptake and metabolic pathways, address open questions about their role in health and disease, and enable screening for therapeutic polyamine modulators.

Effect of ac4C acetyltransferase NAT10 on tumor progression via ATF4/ASNS mediated asparagine biosynthesis in osteosarcoma.

234 Background: Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, characterized by high malignancy, high mortality, and high disability rates. Investigating the mechanism of osteosarcoma progression, identifying new targets, and developing effective therapy are of great significance for improving patient clinical outcomes. NAT10-mediated ac4C modification is widely present in mRNA and plays important roles in multiple biological processes of mRNA. NAT10 and ac4C modification also play a crucial role in the occurrence and development of malignant tumors such as bladder cancer, esophageal cancer, and breast cancer. Methods: Through RNA-seq and functional screening, NAT10 was identified as the potential therapeutic target. The biological functions of NAT10 and ac4C modification in osteosarcoma were explored in vivo and in vitro. Downstream target of NAT10 was identified through acRIP-seq combined with RNA-seq. The ac4C modification of downstream target gene were detected using RIP-qPCR and its regulatory effect on mRNA stability was explored using RNA half-life analysis. Drug screening was performed using FDA-approved drugs and a small molecule compound library to identify NAT10 inhibitors. The efficacy of inhibitors in osteosarcoma treatment is validated through in vivo, in vitro experiments and patient-derived tumor xenograft (PDX) models. Results: Functional screening targeting RNA modification regulatory factors demonstrated that inhibiting NAT10 significantly reduced the proliferation, migration and invasion of osteosarcoma cells. NAT10 knockout significantly inhibited the proliferation and metastasis of osteosarcoma cells, as well as the malignant progression in vivo. The acRIP-seq and RNA-seq revealed that ATF4 is a downstream target gene regulated by NAT10 in osteosarcoma. NAT10 promoted ac4C modification of ATF4 mRNA, enhanced the stability of ATF4 mRNA, and ATF4 promoted the transcription and expression of ASNS. ASNS catalyzes the synthesis of asparagine (Asn), thereby promoting the malignant progression of osteosarcoma. Drug screening identified Paliperidone and AG-401 as potential inhibitors of NAT10. Through cell functional experiments, orthotopic osteosarcoma models, organoids model, and PDX models, it was demonstrated that Paliperidone and AG-401 can inhibit the malignant progression of osteosarcoma. Conclusions: NAT10 was highly expressed in osteosarcoma and associated with patient prognosis. Through ac4C modification, NAT10 regulated the synthesis of asparagine mediated by ATF4/ASNS, providing materials for protein and nucleotide synthesis in tumor cells, thus promoting the malignant progression of osteosarcoma. Paliperidone and AG-401 were identified as potential NAT10 inhibitors and targeting NAT10 could be a promosing strategy in osteosarcoma treatment.

Marine fungal diversity unlocks potent antivirals against monkeypox through methyltransferase inhibition revealed by molecular dynamics and free energy landscape

The escalating threat posed by the Monkeypox virus (MPXV) to global health necessitates the urgent discovery of effective antiviral agents, as there are currently no specific drugs available for its treatment, and existing inhibitors are hindered by toxicity and poor pharmacokinetic profiles. This study aimed to identify potent MPXV inhibitors by screening a diverse library of small molecule compounds derived from marine fungi, focusing on the viral protein VP39, a key methyltransferase involved in viral replication. An extensive virtual screening process identified four promising compounds—CMNPD15724, CMNPD28811, CMNPD30883, and CMNPD18569—alongside a control molecule. Rigorous evaluations, including re-docking, molecular dynamics (MD) simulations, and hydrogen bond analysis, were conducted to assess their inhibitory potential against MPXV VP39. CMNPD15724 and CMNPD30883, in particular, demonstrated a superior binding affinity and stable interactions within the target protein's active site throughout the MD simulations, suggesting a capacity to overcome the limitations associated with sinefungin. The stability of these VP39-compound complexes, corroborated by MD simulations, provided crucial insights into the dynamic behavior of these interactions. Furthermore, Principal Component Analysis (PCA) based free energy landscape assessments offered a detailed understanding of the dynamic conformational changes and energetic profiles underlying these compounds' functional disruption of VP39. These findings establish CMNPD15724, CMNPD28811, CMNPD30883, and CMNPD18569 as promising MPXV inhibitors and highlight marine fungi as a valuable source of novel antiviral agents. These compounds represent potential candidates for further experimental validation, advancing the development of safer and more effective therapeutic options to combat this emerging viral infection.

Topoisomerase Inhibitors and PIM1 Kinase Inhibitors Improve Gene Editing Efficiency Mediated by CRISPR-Cas9 and Homology-Directed Repair.

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.

A novel algorithm for the virtual screening of extensive small molecule libraries against ERCC1/XPF protein-protein interaction for the identification of resistance-bypassing potential anticancer molecules.

Cancer cell's innate chemotherapeutic resistance continues to be an obstacle in molecular oncology. This theory is firmly tied to the cancer cells' integral DNA repair mechanisms continuously neutralizing the effects of chemotherapy. Amidst these mechanisms, the nuclear excision repair pathway is crucial in renovating DNA lesions prompted by agents like Cisplatin. The ERCC1/XPF complex stands center-stage as a structure-specific endonuclease in this repair pathway. Targeting the ERCC1/XPF dimerization brings forth a strategy to augment chemotherapy by eschewing the resistance mechanism integral to cancer cells. This study tracks and identifies small anticancer molecules, with ERCC1/XPF inhibiting potential, within extensive small-molecule compound libraries. A novel hybrid virtual screening algorithm, conjoining ligand- and target-based approaches, was developed. All-atom molecular dynamics (MD) simulations were then run on the obtained hit molecules to reveal their structural and dynamic contributions within the binding site. MD simulations were followed by MM/GBSA calculations to qualify the change in binding free energies of the protein/ligand complexes throughout MD simulations. Conducted analyses highlight new potential inhibitors AN-487/40936989 from the SPECS SC library, K219-1359, and K786-1161 from the ChemDiv Representative Set library as showing better predicted activity than previously discovered ERCC1/XPF inhibitor, CHEMBL3617209. The algorithm implemented in this study expands our comprehension of chemotherapeutic resistance and how to overcome it through identifying ERCC1/XPF inhibitors with the aim of enhancing chemotherapeutic impact giving hope for ameliorated cancer treatment outcomes.

Screening, Deconvolution and Parallel Synthesis of Trisubstituted Piperazine and Trisubstituted 2,3-diketopierazine Libraries for the Rapid Identification of Antagonists of the Nuclear Retinoic Acid Receptor-related Orphan Receptor Gamma (RORγ)

Background: Genetic studies support a key role for RORγ and RORα in the differentiation of proinflammatory Th17 cells, and a growing body of evidence suggests a pathogenic role for Th17 in several autoimmune diseases, including MS, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis. RORγ antagonists have been shown to suppress Th17 differentiation and delay the onset of disease in an experimental autoimmune encephalomyelitis mouse model of MS. Objective: Given the high therapeutic interest of RORγ antagonists and the promising activity of currently known ligands, small molecules with higher potency and receptor selectivity (in particular within the ROR family) are highly desirable. We used our small molecule compound library to discover, characterize, and optimize novel RORγ antagonists for the treatment of autoimmune diseases from Mixture-based Combinatorial Chemical Libraries. Methods: We screened the FIU collection of small molecule libraries (>30 million compounds) composed of 75 molecular scaffolds systematically arranged in positional scanning and scaffold ranking formats. We identified scaffolds that selectively inhibit the binding of RORγ, RORγ, and RORβ but not RORα, and others that function as antagonists of all three receptors. Results: The deconvolution of selected PS-SCL mixtures led to the identification of novel chemical entities, trisubstituted piperazine and diketopiperazine that function as RORγ antagonists. Conclusion: The screening of a large complex library led to the rapid identification of novel trisubstituted piperazine and diketopiperazine antagonists of the nuclear retinoic acid receptor-related orphan receptor gamma (RORγ).

Abstract 3218: Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML

Abstract Background: Acute myeloid leukemia (AML) with KMT2A-rearrangements have a poor prognosis and so there is an urgent need for novel therapies. Menin inhibitors have shown promising anti-leukemic effects in KMT2A-rearranged and NPM1-mutated AML. However, the efficacy of menin inhibitors as single agents may be limited by the development of resistance mutations. Combining menin inhibitors with other epigenetic drugs may present a strategy to enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic drug library in KMT2A-rearranged (MV4;11) and NPM1-mutant (OCI-AML3) AML cell lines to identify synergistic therapies that can be used in conjunction with menin inhibitors. Methods: During assay optimization, MV4;11 and OCI-AML3 cells were treated with the menin inhibitor DSP-5336, and their viability was assessed. Quality control metrics were applied with Z’ values consistently greater than 0.5 for all assay plates and then the cells were subjected to a small molecule compound library screen containing a set of 932 epigenetic drugs. The screening process included a systematic evaluation of each drug's effect on cell viability in combination with the DSP-5336. Results: The combination drug screen demonstrated robust performance in both MV4;11 and OCI-AML3 cell lines, with reliable quality control metrics. Among the epigenetic drugs tested, five compounds were identified as potential hits that synergistically enhanced the efficacy of DSP-5336 across both cell lines.The five identified compounds exhibited enhanced anti-leukemic effects when combined with the menin inhibitor in either one of the cell lines. The targets of these drugs varied, including histone deacetylases, histone acetyltransferases and BRD4 inhibition, suggesting many alternate mechanisms of action involving epigenetic modulation. Remarkably, one of the 5 hits C301-3161, a KAT2A inhibitor, showed limited activity in either cell line alone but increased cell killing with DSP-5336 in both cell lines, highlighting its potential as a specific therapeutic for combining with DSP-5336 in AML patients. Conclusions: Our findings suggest combining menin inhibitors with other epigenetic drugs may improve AML treatment outcomes. The combination drug screen successfully identified four novel compounds that synergistically enhance menin inhibitor efficacy in both KMT2A-rearranged and NPM1-mutant AML cell lines. These compounds hold significant promise as potential therapeutic candidates for further investigation. Future studies should focus on elucidating the underlying mechanisms of action for each hit and evaluating their effects in preclinical animal models. Ultimately, this research may pave the way for the development of innovative combination therapeutic strategies for AML patients with KMT2A-rearragned and NPM1-mutant AML. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Maurizio Affer, Anya K. Sondhi, Alexandra Chirino, Claude-Henry Volmar, Shaun Brothers, Justin Watts, Justin Taylor. Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3218.

Abstract 647: FDA approved library screen revealed Ewing and clear cell sarcomas have increased sensitivity to filanesib over other cancer types

Abstract There is a compelling need for the development of more effective and less toxic therapies for patients with Ewing Sarcoma (ES) and Clear Cell Sarcoma (CCS). ES and CCS are characterized by chromosomal translocations which result in two distinct fusion proteins: EWS-ATF1 (t(12;22)(q13;q12)) in CCS and EWS-FLI1 (t(11;22)(q24;q12)) in ES. There is a strong premise that targeting the fusion proteins or their key downstream signaling pathways will be disease modifying and improve outcomes for patients. Preliminary high throughput screening studies using an FDA approved small molecule compound library identified filanesib, a kinesin spindle protein (KIF11) inhibitor, as having potent anti-proliferative activity in ES and CCS cells over other cancer subtypes. The mechanism linking the fusion proteins with KIF11 and the tumorigenic role of KIF11 in ES and CCS has yet to be described. Previous studies have shown that the EWS-ATF1 fusion protein forms a heterodimer with transcription factors to target the promoters of its downstream genes. In breast cancer, the knockdown of KIF11 has been shown to significantly reduce the phosphorylation of these transcription factors. Additionally, KIF11 has been shown to directly interact with p300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT). HATs are necessary for EWS-FLI1-induced chromatin remodeling. We hypothesize that the fusion proteins, EWS-FLI1 and EWS-ATF1, form a complex with KIF11 that regulates the activity of co-transcription factors during chromatin remodeling. The target of filanesib, KIF11, has essential roles in maintaining cell cycle progression through mitosis and its inhibition results in mitotic arrest and subsequent cell death. We used ImageStream to assess the effects of filanesib on the cell cycle. ImageStream allows both a quantitative and qualitative analysis of the cell cycle by measuring the population of cells that are positive for a given marker as well as imaging each cell by microscopy. Results demonstrate that filanesib promotes an increase in cells arrested in prophase with dramatically altered spindle formation. We are currently addressing the role of KIF11 in regulating the expression levels of the fusion proteins’ associated transcriptional coactivators and downstream target genes. By completing these studies, we aim to, i) outline the mechanism underpinning the relationship between KIF11 and the oncogenic fusion proteins in ES and CCS cells, and ii) gain an understanding of why ES and CCS cells are particularly vulnerable to KIF11 inhibition. This work has been supported in part by the Flow Cytometry and the Molecular Genomics Cores at the H. Lee Moffitt Cancer Center & Research Institute, a comprehensive cancer center designated by the National Cancer Institute and funded in part by Moffitt’s Cancer Center Support Grant (P30-CA076292) Citation Format: Hannah L. Walker-Mimms, Nicole Londono, Yi Liao, Neelkamal Chaudhary, Dessiree Pena Gomez, Yonghong Zhang, Mingxiang Teng, Uwe Rix, Derek R. Duckett. FDA approved library screen revealed Ewing and clear cell sarcomas have increased sensitivity to filanesib over other cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 647.

Abstract LB_A20: Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors

Abstract Background: Drug resistance is a major obstacle to successful treatment of cancer and leukemia. The role of RNA epigenetics in cancer drug resistance is largely unknown. Our previous studies demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018). The Cancer Genome Atlas Program (TCGA) data demonstrate that elevated expression levels of NSUN1 and NSUN2 are correlated with significantly shorter patients’ survival in almost all cancer subtypes (https://www.proteinatlas.org/ENSG00000111641-NOP2/pathology). However, due to the lack of NSUN1 and NSUN2 proteins’ crystal structures, no NSUN1/2-targeting therapeutics have been developed yet. Methods: 1. Argonne artificial intelligence (AI)/supercomputer design of NSUN1/2-targeting small-molecule compound libraries, simulation of compound-protein interactions and analysis of structure activity relationship (SAR). 2. Novel RNA epigenetic technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). 3. Functional drug screening, H&E histology and immunohistochemistry (IHC). 4. Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results:1. Identification of the ligand-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of NSUN1/2-targeting small-molecule libraries by AI/supercomputing. 2. Identification of several NSUN1/2 inhibitors that effectively inhibit growth of the venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. 3. Demonstration of the lead inhibitors execute their effects through disruption of NSUN1/2-mediated (ACS) and translational complexes (TLC). 4. Characterization of the lead inhibitors’ binding modules with AI/supercomputing SAR analysis. 5. In vivo validation of the efficacies of the identified lead inhibitors using syngeneic AML mouse models. Conclusion:By leveraging the power of the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in oncogenic bioprocesses and drug (venetoclax) resistance, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancer subtypes. Citation Format: Jason X Cheng, Shaun Wood, Derrick Tang, Linda Degenstein, Andrzej Joachimiak, Rick Stevens. Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A20.

Histone Methyltranferase SETD8 Stimulates Proliferation and Anti-Apoptotic Effect in Acute Myeloid Leukemia through Epigenetically Upregulation of CXCR4

Acute myeloid leukemia (AML) is the most common type of leukemia., characterized by the rapid and uncontrolled growth of abnormal cells. Epigenetic modifications control gene expression without affecting its DNA sequence. H4K20me1 is the mono-methylation occurring on lysine 20 of histone H4, acting as an epigenetic mark that promotes gene expression. SETD8 is responsible for catalyzing the mono-methylation of lysine 20 on histone H4. While SETD8's role and mechanisms have been studied in various types of cancer, its function in AML remains unclear. This study is based on the screening of a small molecule compound library, which identified the inhibitor UNC0379 targeting the histone methyltransferase SETD8, showing clear inhibitory effects on three non-M3 AML cell lines. Bioinformatical analysis revealed elevated SETD8 expression in AML patients compared to normal individuals and higher SETD8 expression corresponded with lower overall survival rates (OS). Further investigations using AML cell lines and normal peripheral blood mononuclear cells (PBMCs) demonstrated that SETD8 knockdown or UNC0379 intervention inhibited cell proliferation and increased apoptosis in AML cells, while PBMC cells remained unaffected. In an AML model constructed with C1498 cell injection in C57BL/6J mice, we observed that SETD8 knockdown and treatment with UNC0379 inhibited tumor formation and infiltration. To elucidate the molecular mechanism of SETD8's impact on AML, we conducted transcriptome sequencing on SETD8 knockdown AML cells and found significantly decreased mRNA expression of CXCR4, which was further validated at the protein level. Also, BeatAML and TCGA datasets demonstrated a positive correlation between SETD8 and CXCR4 mRNA expression. These phenomena motivated us to clarify the regulation relationship between SETD8 and CXCR4. CXCR4 is a crucial membrane receptor. After stimulation, it promotes AML cell proliferation and survival. CHIP experiments in SETD8 knockdown cells revealed reduced H4K20me1 enrichment in the CXCR4 promoter region. Subsequent rescue experiments demonstrated that overexpressing CXCR4 or using its corresponding agonist SDF-1alpha in SETD8 knockdown cells moderately restored cell proliferation and reduced apoptosis. In summary, we discovered that SETD8 promotes CXCR4 transcriptional activation through catalyzing the H4K20me1 mark, thereby influencing AML cell proliferation and apoptosis. SETD8 presents a promising therapeutic target through genetic or pharmacological intervention.

Small molecule regulators of microRNAs identified by high-throughput screen coupled with high-throughput sequencing

MicroRNAs (miRNAs) regulate fundamental biological processes by silencing mRNA targets and are dysregulated in many diseases. Therefore, miRNA replacement or inhibition can be harnessed as potential therapeutics. However, existing strategies for miRNA modulation using oligonucleotides and gene therapies are challenging, especially for neurological diseases, and none have yet gained clinical approval. We explore a different approach by screening a biodiverse library of small molecule compounds for their ability to modulate hundreds of miRNAs in human induced pluripotent stem cell-derived neurons. We demonstrate the utility of the screen by identifying cardiac glycosides as potent inducers of miR-132, a key neuroprotective miRNA downregulated in Alzheimer’s disease and other tauopathies. Coordinately, cardiac glycosides downregulate known miR-132 targets, including Tau, and protect rodent and human neurons against various toxic insults. More generally, our dataset of 1370 drug-like compounds and their effects on the miRNome provides a valuable resource for further miRNA-based drug discovery.

Pharmacological Targeting of Interferon-Related DNA Damage Resistant Signature (IRDS) and XRCC4-Mediated DNA Repair Pathways ss a Novel Therapeutic Approach to DIPG Radio-Sensitization

Despite significant efforts to improve the outcomes of pediatric diffuse midline gliomas, such as diffuse intrinsic pontine glioma (DIPG), prognosis remains dismal with a 5-year survival of <1%. The standard of care treatment for DIPG is fractionated radiation, but the disease inevitably progresses in 6 months or less. There is a significant unmet need to improve the clinical outcomes for pediatric DIPG patients. Approximately 70-80% of DIPG tumors contain mutations in TP53 tumor suppressor protein. These TP53 mutations are associated with resistance to radiation treatments in DIPG patients. The mechanisms of increased radio-resistance of p53-mutant DIPG are poorly understood. The objective of this study was to identify novel pharmacological agents that would augment radiation sensitivity of p53 mutant DIPG cell lines, and to establish their molecular mechanism of action. SF8628 pediatric DIPG cell line harboring p53 mutation was obtained from MilliporeSigma. CellRad benchtop X-ray irradiator (Precision X-Ray) was used for radiation sensitization experiments. Vi-CELL BLU cell viability analyzer was used for high-throughput screening of small molecule compound libraries with and without radiation treatments. Proteomics Core facility was utilized for mass spec analysis of protein targets of Compound-X. To identify novel drug candidates that would sensitize DIPG to therapeutic radiation, we carried out an unbiased screen of curated libraries of small molecules with diverse scaffolds in p53 mutant DIPG cells. This radio-sensitization screen yielded a single molecule, Compound-X, that was found to have profound growth-inhibitory and radiation-sensitizing effects in DIPG cells. Compound-X was found to induce a robust cell cycle arrest of DIPG cells in G2/M, the most radio-sensitive phase of the cell cycle. Furthermore, Compound-X elicited a massive apoptotic cell death of DIPG cells. An unbiased RNA sequencing approach revealed that Compound-X inhibits expression of the Interferon-related DNA damage Resistant Signature (IRDS), a sub-group of interferon-stimulated genes (ISGs) known to promote radiation and chemotherapy resistance in high-grade gliomas. To identify the target of Compound-X, we carried out affinity purification of Compound-X associated complexes from p53 mutant DIPG cell lysates. Mass spectrometry analysis of Compound-X-purified protein complexes identified XRCC4 as a protein that uniquely associated with Compound-X. RNAi knock-down experiments revealed that XRCC4 is required for cytotoxic effects of Compound-X. Importantly, Compound-X-mediated XRCC4 targeting caused a delay in DNA DSBs repair after radiation treatment. An unbiased screen of small molecule drug candidates identified a novel XRCC4-targeting agent, Compound-X, as potent radiation sensitizer in p53 mutant DIPG cells. This work may lead to clinical trials investigating novel XRCC4-targeting agent in pediatric patients with DIPG.

Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

ObjectiveNRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DesignWe performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. ResultsUsing high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. ConclusionWe have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic for NRF2high ESCC.

Comprehensive Search for GPCR Compounds which Can Enhance MafA and/or PDX-1 Expression Levels Using a Small Molecule Compound Library.

It has been shown that chronic hyperglycemia gradually decreases insulin biosynthesis and secretion which is accompanied by reduced expression of very important insulin gene transcription factors MafA and PDX-1. Such phenomena are well known as β-cell glucose toxicity. It has been shown that the downregulation of MafA and/or PDX-1 expression considerably explains the molecular mechanism for glucose toxicity. However, it remained unknown which molecules can enhance MafA and/or PDX-1 expression levels. In this study, we comprehensively searched for G protein-coupled receptor (GPCR) compounds which can enhance MafA and/or PDX-1 expression levels using a small molecule compound library in pancreatic β-cell line MIN6 cells and islets isolated from nondiabetic C57BL/6 J mice and obese type 2 diabetic C57BL/KsJ-db/db mice. We found that fulvestrant and dexmedetomidine hydrochloride increased MafA, PDX-1, or insulin expression levels in MIN6 cells. We confirmed that fulvestrant and dexmedetomidine hydrochloride increased MafA, PDX-1, or insulin expression levels in islets from nondiabetic mice as well. Furthermore, these reagents more clearly enhanced MafA, PDX-1, or insulin expression levels in islets from obese type 2 diabetic db/db mice in which MafA and PDX-1 expression levels are reduced due to glucose toxicity. In conclusion, fulvestrant and dexmedetomidine hydrochloride increased MafA, PDX-1, or insulin expression levels in MIN6 cells and islets from nondiabetic mice and obese type 2 diabetic db/db mice. To the best of our knowledge, this is the first report showing some molecule which can enhance MafA and/or PDX-1 expression levels. Therefore, although further extensive study is necessary, we think that the information in this study could be, at least in part, useful at some point such as in the development of new antidiabetes medicine based on the molecular mechanism of β-cell glucose toxicity in the future.

Discovery of 5-sulfonyltetrazoles as neuroblastoma differentiation agents

Previously, we developed an innovative high-content screening (HCS) approach to quantify neuroblastoma cell differentiation based on neurite outgrowth, a morphological differentiation marker of neuroblastoma cells. Here, we report the utilization of this platform to identify 1-methyl-5-(ethylsulfonyl)-1H-tetrazole (3a) as a new neuroblastoma differentiation agent using the ChemBridge DiversetTM commercial synthetic small molecule compound library. We show that this activity can be extended to a group of analogues, which can be accessed via a short two-step synthetic sequence. A new analogue, 5-(allylsulfonyl)-1-methyl-1H-tetrazole (3c) was identified in this synthetic effort as a compound that has even more pronounced differentiation and cytotoxic activities than the original hit compound 3a.

Thioxothiazolo[3,4-a]quinazoline derivatives inhibit the human cytomegalovirus alkaline nuclease

Human cytomegalovirus (HCMV, human herpesvirus 5) is an opportunistic pathogen responsible for serious disease in immunocompromised patients. Current antiviral therapies rely predominantly on drugs interfering with viral DNA replication and packaging. However, the serious side effects of existing drugs and the emergence of drug resistance indicate the need for new targets for anti-HCMV therapy. One such target is the viral alkaline nuclease (AN), an enzyme highly conserved among the Herpesviridae. In this study, we validated the HCMV AN, encoded by the viral UL98 open reading frame, as a drug target by demonstrating that a UL98-deficient HCMV mutant is severely attenuated and shows a reduced ability to spread in cell culture. We established a fluorescence-based enzyme assay suitable for high-throughput screening and used it on a small-molecule compound library. The most promising hit, a thioxothiazolo[3,4-a]quinazoline derivative, blocked AN activity in vitro and inhibited HCMV replication in plaque reduction (PRA) and fluorescence reduction assays (FRA). Several derivatives of the hit compound were tested, some of which had similar or better inhibitory activities. The most potent derivative of hit scaffold A, compound AD-51, inhibited HCMV replication with a 50% effective concentrations (EC50) of 0.9 μM in the FRA and 1.1 μM in the PRA. AD-51 was also active against ganciclovir, foscarnet, and letermovir-resistant HCMVs. Moreover, it inhibited herpes simplex virus, Kaposi's sarcoma-associated herpesvirus, and murine CMV, a mouse virus serving as a model for HCMV. These results suggest that thioxothiazolo[3,4-a]quinazoline derivatives are a new class of herpesvirus inhibitors targeting the viral AN.

Discovery of Small-Molecule Antagonists of Orexin 1/2 Receptors from Traditional Chinese Medicinal Plants with a Hypnotic Effect.

Insomnia is an important public health problem. The currently available treatments for insomnia can cause some adverse effects. Orexin receptors 1 (OX1R) and 2 (OX2R) are burgeoning targets for insomnia treatment. It is an effective approach to screening OX1R and OX2R antagonists from traditional Chinese medicine, which contains abundant and diverse chemical components. This study established an in-home ligand library of small-molecule compounds from medicinal plants with a definite hypnotic effect, as described in the Chinese Pharmacopoeia. Molecular docking was applied to virtually screen potential orexin receptor antagonists using molecular operating environment software, and surface plasmon resonance (SPR) technology was used to detect the binding affinity between potential active compounds and orexin receptors. Finally, the results of virtual screening and SPR analysis were verified through in vitro assays. We successfully screened one potential lead compound (neferine) as an orexin receptor antagonist from the in-home ligand library, which contained more than 1000 compounds. The screened compound was validated as a potential agent for insomnia treatment through comprehensive biological assays. This research enabled the discovery of a potential small-molecule antagonist of orexin receptors for the treatment of insomnia, providing a novel screening approach for the detection of potential candidate compounds for corresponding targets.

Mechanism of skin whitening through San-Bai decoction-induced tyrosinase inhibition and discovery of natural products targeting tyrosinase.

Melanin deposition is the main cause of skin darkening, which can lead to severe physical and psychological distress, necessitating the development of approaches for preserving skin health and fairness. Tyrosinase (TYR) is the rate-limiting enzyme in melanin synthesis, and its activity directly determines the degree of melanin accumulation in the skin, which in turn affects skin color. Currently, TYR inhibitors derived from natural products are widely used for skin whitening. San-Bai decoction (SBD) is effective for skin whitening and softening, but its mechanism of action, efficacy and high efficiency TYR inhibitors for skin whitening remain poorly understood. Here, we employed systems biology and network pharmacology to analyze the active compounds and targets of SBD, using the follow databases: TCMIP, TCMID, and BATMAN-TCM. Construct a molecular network centered on the regulation of TYR by SBD in skin whitening, using STRING database and cytoscape. Enrichment analysis using KOBAS database and ClusterProfiler. Virtual screening of candidate TYR inhibitors using Molecular Operating Environment software and Amber 18 software. SBD may act through tyrosine metabolism, melanogenesis, and other signaling pathways to regulate TYR activity and inhibit melanogenesis. We identified TYR and ESR1 as possible key targets for the whitening effect of SBD and screened out pentagalloylglucose, 1,3,6-tri-O-galloyl-beta-D-glucose, 1,2,4,6-tetragalloylglucose, and liquiritigenin 4',7-diglucoside as inhibitors of TYR, in addition to glycyrrhizic acid, pachymic acid methyl ester, nicotiflorin, gamma-sitosterol, and isoliensinine as inhibitors of ESR1. We also performed virtual drug screening of a library of natural small-molecule compounds (19,505 in total) and screened out lycopsamine, 2-phenylethyl b-D-glucopyranoside, and 6-beta-hydroxyhyoscyamine as inhibitors of TYR. We identified natural compounds with the potential for skin whitening through inhibition of TYR, thus advancing research on SBD and its applications.

Identification of global inhibitors of cellular glycosylation

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.