Screening Of Small-molecule Inhibitors Research Articles

Abstract PO3-25-08: Artificial Intelligence-based screening of small molecule inhibitors of phosphoenolpyruvate carboxykinase 2 (PCK2), a novel cancer therapeutic target

Abstract Background: Metabolic rewiring is a hallmark of cancer that is often mediated by alterations in metabolic isozyme expression. Isozymes that are overexpressed in cancer and catalyze rate limiting steps in key metabolic processes are potential therapeutic targets. We previously identified phosphoenolpyruvate carboxykinase 2 (PCK2) as cancer dominant isoform, required for maintaining high metabolic activity and proliferation of lung, prostate, and breast cancer (BC) cells in vitro and in vivo. No PCK2 specific small molecule inhibitors exist. Methods: mRNA expression levels were assessed in the TCGA data. PCK2 MISSION shRNAs (Millipore) were used to knock-down expression of PCK2 in BC cells. Alterations in metabolic flux caused by PCK2 down-regulation were examined via 13C-labeling and mass spectrometry using a SCIEX 5500 QTRAP and SelexION instrument for mobility separation of metabolites. The AtomNetTM (Atomwise Inc.) deep convolutional neural network structure-based drug design software was used to identify candidate small molecule PCK2 inhibitors which were tested in PCK1 and PCK2 in vitro enzyme activity assays. The lead compound was assessed for drug-like properties by QickPropV4.0.001w, and its growth inhibitory activity was tested in MDA-MB-321 and BT-549 cells. Results: Analysis of mRNA levels in TNBC samples revealed a significant decrease in PCK1 expression compared to normal tissues, while PCK2 expression remained elevated or even increased. Quantification of mRNA levels in TNBC cell lines confirmed the higher expression of PCK2 compared to PCK1. To assess functional significance of PCK2 expression, we downregulated PCK2 using shRNA constructs in TNBC cell lines. Western blot analysis confirmed efficient knockdown of PCK2 protein without affecting PCK1 expression. Clonogenic assays demonstrated that PCK2-depleted BT-549 and MDA-MB-231 cells exhibited impaired proliferation, with reduced colony number and size compared to control cells. Metabolic analysis of PCK2-depleted TNBC cells demonstrated decreased levels of glycolytic and TCA intermediates and reduced flux through pyruvate carboxylase, indicating altered metabolic pathways upon PCK2 depletion. To identify potential inhibitors of PCK2, virtual screening was conducted using compound structures. We used a PCK2 protein structure homology model and designated the 3-mercaptopicolinic acid-binding site of the PCK1 structure as the site of interest to perform virtual drug screening of 7 million diverse compounds. The first screen yielded 86 structures to be tested in vitro, of which 5 demonstrated > 16% PCK2 inhibition. The top hit (IC50=2.4µM) was used to refine a second round of in silico screen that yielded 87 second-round candidates that were assessed for PCK2 inhibition in vitro. 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate emerged as the lead compound. QickProp predicted favorable absorption, distribution, metabolism, and excretion properties, but relatively low cell permeability (apparent Caco-2 and MDCK2 cell permeabilities 18 nm/sec and 8 nm/sec, respectively). The specificity of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was tested in PCK1 and PCK2 enzymatic assays and revealed IC50 of 500nM on PCK1 and an IC50 of 3 nM on PCK2. We next tested the cell growth inhibitory effect of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on parental MDA-MB-231 and BT-549 cells and their PCK2 knockdown clones. Cell growth and viability significantly decreased in the MDA-MD-231 and BT-549 parental cells (the IC50 for MDA-MB-231 was 173 μM) and in shRNA non-targeting control cells, but not in the PCK2 depleted clones, demonstrating PCK2-dependent inhibitory effect. Conclusion: We identified 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate as a high affinity selective PCK2 enzyme inhibitor. This compound also has significant growth inhibitory activity in BC cell lines in vitro and represents a novel therapeutic lead compound. Citation Format: Alejandro Rios Hoyo, Naing Lin Shan, Hao-Kuen Lin, Gerson Espinoza Campos, Mostafa Ahmed, Sheila Umlauf, Rebecca Cardone, Yulia V. Surovtseva, Vignesh Gunasekharan, Lajos pusztai. Artificial Intelligence-based screening of small molecule inhibitors of phosphoenolpyruvate carboxykinase 2 (PCK2), a novel cancer therapeutic target [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 PO3-25-08.

Abstract 620: Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups

Abstract Oncogenic mutations in members of the RAS family, HRAS, NRAS, and KRAS, are frequently identified in various cancers and often lead to increased signaling of the mitogen-activated protein kinase (MAPK) pathway. KRAS is the most frequently mutated RAS isoform, with a mutation incidence of >10% across all cancers, and up to 70% and 20% in pancreatic and non-small cell lung cancer (NSCLC), respectively. RAS has long been considered to be undruggable, and direct inhibition of RAS with small molecule inhibitors has only recently been achieved, with the approval of the KRAS G12C-mutant selective inhibitors sotorasib and adagrasib for treatment of NSCLC. Indirect targeting of RAS-mutated cancers by inhibitors of downstream components such as MEK and ERK has shown variable responses. Novel insights into MAPK signaling have renewed interest into targeting the MAPK pathway in RAS-mutant cancer, with pan-RAF dimer inhibitors and dual RAF/MEK inhibitors reaching clinical trials. To improve the chances of success, it is important to determine which patient populations are most likely to respond to these inhibitors. To gain more insight into the cellular targeting of MAPK pathway inhibitors, eighteen inhibitors of the MAPK components KRAS, RAF, MEK, and ERK were profiled on a panel of 130 cell lines in cell viability assays (the Oncolines® panel). The cell lines represented a diverse range of tumor tissues and had different genomic backgrounds. Many of the cell lines had mutations in KRAS, NRAS, or BRAF. Drug responses were determined by exposing the cell lines to nine-point duplicate dose ranges of the inhibitors. Using bioinformatics, cell line responses to the inhibitors were related to genomic and transcriptomic features, to identify determinants of drug response in both tissue-agnostic and tissue-specific contexts. The results of our analyses revealed variable responses of KRAS-mutant cell lines to the inhibitors, reflecting clinical responses. Tissue-focused analyses showed that the different responses could be traced back to the different KRAS amino acid substitutions. For instance, in pancreatic cancer cell lines, the KRAS Q61H mutation predicted increased sensitivity to MAPK pathway inhibitors compared to other KRAS amino acid substitutions. Additionally, in tissue groups with a more heterogenous mutation profile, co-mutations explained the decreased effectivity of MAPK pathway inhibitors in KRAS-mutant cell lines, such as PIK3CA mutations in colorectal cancer cell lines. Lastly, a gene expression-based signature of MAPK activity predicted response to MAPK pathway inhibitors, independent of mutation status. Our findings have uncovered determinants of response to MAPK pathway inhibitors in RAS-mutant cell lines. These determinants can serve as markers to select patients who are most likely to benefit from MAPK inhibitors. Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Guido J. Zaman. Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups [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 620.

High-throughput virtual screening, pharmacophore modelling and antagonist effects of small molecule inhibitors against cytotoxin-induced cytotoxicity

Cobra venom cytotoxins (CTX) cause dermonecrosis in envenomed patients who suffered from limb amputations due to the limitation of serotherapy-based antivenoms. This study aimed to identify small molecule inhibitors against CTX. A structure-based high-throughput virtual screening (HTVS) was conducted based on a conserved CTX, using the Natural Product Activity and Species Source (NPASS) screening library. The hits were valerenic acid, 1-oxo-2H-isoquinoline-4-carboxylic acid, acenaphthene, and 5-bromopyrrole-2-carboxamide, which interacted with contemporary antivenom binding site A and functional loops I–III of CTX, respectively, in molecular docking studies. Furthermore, molecular dynamic simulations were performed along with analysis of ligand fitness through their pharmacophore and pharmacokinetics properties. The antagonist effects of these hits on CTX-induced cytotoxicity were examined in human keratinocytes (HaCaT). Despite having a low binding affinity (KD = 14.45 × 10 − 4 M), acenaphthene demonstrated a significant increase of cell viability at 6 h and 24 h in experimental envenomed HaCaT. It also demonstrated the highest neutralization potency against CTX with a median effective concentration (EC50) of 0.05 mL/mg. Acenaphthene interacted with the functional loop II, which is the crucial cytotoxic site of CTX. It has an aromatic ring as its primary pharmacophoric feature, commonly used for rational drug design. In conclusion, acenaphthene could be a promising lead compound as a small molecule inhibitor. Communicated by Ramaswamy H. Sarma

Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split Luciferase.

Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.

A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4-SMAD3-DNA complex.

The transforming growth factor-beta (TGFβ) signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small-molecule inhibitors specifically targeting SMAD4, the downstream master regulator of the TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGFβ signaling. In this study, we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format. This assay enables simultaneous monitoring of the protein‒protein interaction between SMAD4 and SMAD3, as well as the protein‒DNA interaction between SMADs and their consensus DNA-binding motif. The multiplexed TR-FRET assay exhibits high sensitivity, allowing the dynamic analysis of the SMAD4-SMAD3-DNA complex at single-amino acid resolution. Moreover, the multiplexed uHTS assay demonstrates robustness for screening small-molecule inhibitors. Through a pilot screening of an FDA-approved bioactive compound library, we identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small-molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFβ signaling agents.

Screening for new inhibitors of the human Mitochondrial Pyruvate Carrier and their effects on hepatic glucose production and diabetes

BackgroundThe mitochondrial pyruvate carrier (MPC) is a protein complex composed of two subunits, MPC1 and MPC2. This carrier is at the interface between glycolysis and mitochondrial metabolism and plays an essential role in hepatic glucose production. MethodsHere we describe an in vitro screen for small molecule inhibitors of the MPC using a strain of Lactococcus lactis that has been engineered to co-express the two subunits of the human MPC and is able to import exogenous 14C-pyruvate. We then tested the top candidates for potential antidiabetic effects through the repression of gluconeogenesis. ResultsBy screening the Prestwick compound library of 1′200 drugs approved by the Food and Drug Administration for inhibitors of pyruvate uptake, twelve hit molecules were identified. In a secondary screen, the most potent inhibitors were found to inhibit pyruvate-driven oxygen consumption in mouse C2C12 muscle cells. Assessment of gluconeogenesis showed that Zaprinast, as well as the established MPC inhibitor UK5099, inhibited in vitro and in vivo hepatic glucose production. However, when tested acutely in mice without the administration of gluconeogenic substrates, MPC inhibitors raised blood glucose levels, pointing to liver-independent effects. Furthermore, chronic treatment with Zaprinast failed to correct hyperglycemia in both lean and obese diabetic mouse models. ConclusionsNew MPC inhibitors have been identified, showing inhibitory effects on hepatic glucose production. General significanceFor potential antidiabetic applications, MPC inhibitors should target the liver without undesired inhibition of mitochondrial pyruvate metabolism in the skeletal muscles or pancreatic beta-cells in order to avoid dual effects on glycemia.

High-Content and High-Throughput Clonogenic Survival Assay Using Fluorescence Barcoding.

The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA's utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.

Integrative CRISPR Activation and Small Molecule Inhibitor Screening for lncRNA Mediating BRAF Inhibitor Resistance in Melanoma.

The incidence of melanoma, being one of the most commonly occurring cancers, has been rising since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. The conventional targeted therapy is well defined and effective for advanced-stage melanomas for patients not responding to the standard-of-care immunotherapy. However, targeted therapies do not prove to be as effective as patients inevitably develop V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-inhibitor resistance to the respective drugs. Factors which are driving melanoma drug resistance mainly involve mutations in the mitogen-activated protein kinase (MAPK) pathway, e.g., BRAF splice variants, neuroblastoma RAS viral oncogene homolog (NRAS) amplification or parallel survival pathways. However, those mechanisms do not explain all cases of occurring resistances. Therefore, other factors accounting for BRAFi resistance must be better understood. Among them there are long non-coding RNAs (lncRNAs), but these remain functionally poorly understood. Here, we conduct a comprehensive, unbiased, and integrative study of lncRNA expression, coupled with a Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-mediated activation (CRISPRa) and small molecule inhibitor screening for BRAF inhibitor resistance to expand the knowledge of potentially druggable lncRNAs, their function, and pave the way for eventual combinatorial treatment approaches targeting diverse pathways in melanoma.

Effects of Computer Virtual Screening and Treatment Based on Pharmacophore BRD4-Targeted Small-Molecule Inhibitors on NSE and CA19-9 Levels in Patients with Liver Cancer

Objective: Neuron-specific enolase (NSE) and carbohydrate antigen 19-9 (CA19-9) levels were compared before and after treatment with brd4-targeting small-molecule inhibitors based on pharmacophores and computer technology to investigate an efficient virtual screening method for bromodomain protein 4 (brd4)-targeted small-molecule inhibitors. The purpose of this research was to examine the efficacy of pharmacophore screening and BRD4 targeting by small-molecule inhibitors in the management of liver cancer. Methods: Subjects included 106 individuals with hepatocellular cancer who were hospitalised between July 2019 and December 2021. Using a receiver operating characteristic (ROC) curve, we assessed the clinical relevance and utility of pharmacophore-based virtual screening for brd4-targeted small-molecule inhibitors. The levels of the tumour markers NSE and CA19-9 were analysed for changes using computational methods. Results: The pharmacophore model was built using the HipHop technique, and its results were validated and optimised utilising computational technology analysis. Twenty-four small-molecule classes were covered by the pharmacophore, and the number of mismatched pharmacodynamic characteristics was 0. The first eight pharmacophores each had two clusters of hydrophobic atoms, a hydrogen bond receptor, and an aromatic ring at its core. There were no changes to the pharmacodynamic characteristics, although their locations varied. False-positive and true-positive rates are depicted on the horizontal and vertical axes, respectively, in the figure below; the other two pharmacophore groups contained a hydrophobic cluster, a hydrogen bond receptor, and the centre of an aromatic ring. The ROC curve was quite consistent, as indicated by an AUC value close to 1, which was between 0 and 1. For pharmacophores 07 and 06, the area under the curve (AUC) was 0.949 and 0.955, respectively, very near to 1. Both the hydrophobic cluster and the hydrogen bond receptor are required for a pharmacophore to exist. The examination of pharmacodynamic definitions revealed a positive correlation between database reliability and element type (P <0.05). Compared to pre-healing levels, post-healing CA19-9 and NSE levels were statistically considerably reduced (P < 0.05). Conclusions: The assessment approach was highly accurate and specific, and the pharmacophore-based virtual screening method and computer technology for brd4-targeting small-molecule inhibitors were of great benefit in the regulation of the cell cycle and the process of cell proliferation. Effective treatment modalities can be created to increase levels of tumour markers NSE and CA19-9, and pharmacophore and computational-based small-molecule inhibitors of brd4 can be employed as crucial virtual screening criteria for the evaluation of liver cancer.

Grb2-related adaptor protein GRAP is a novel regulator of liver fibrosis

AimsExcessive liver fibrosis is frequently observed in chronic liver diseases and associated with decline of liver functions. Hepatic stellate cells (HSCs) are considered the principal mediator of liver fibrosis by trans-differentiating into myofibroblasts. In the present study we investigated the role of Grb2-related adaptor protein (GRAP) in HSC activation and liver fibrosis. Methods and materialsLiver fibrosis was induced by carbon tetrachloride (CCl4) injection. Gene expression was examined by quantitative PCR. Cell proliferation was evaluated by EdU incorporation. DNA-protein interaction was examined by chromatin immunoprecipitation (ChIP). Key findingsGRAP expression was up-regulated during HSC-myofibroblast transition both in vivo and in vitro. Mechanistically, serum response factor (SRF) and myocardin-related transcription factor A (MRTF-A) formed a complex to bind to the GRAP promoter and activate GRAP transcription. Small interfering RNA (siRNA) mediated GRAP silencing blocked HSC-myofibroblast transition in vitro. Importantly, adeno-associated virus 6 (AAV6) mediated GRAP knockdown in myofibroblasts attenuated liver fibrosis in mice. Of note, inhibition of ERK signaling abrogated enhancement of HSC-myofibroblast transition by GRAP over-expression. SignificanceOur data suggest that GRAP, possibly via ERK activation, regulates HSC-myofibroblast transition and contributes to liver fibrosis. Screening for small-molecule GRAP inhibitors may yield novel therapeutic solutions against liver fibrosis.

ATRT-10. SINGLE-CELL TRANSCRIPTOME ANALYSIS REVEALS CELLULAR HIERARCHIES AND THERAPEUTIC VULNERABILITIES OF ETMR

Abstract Embryonal tumors with multilayered rosettes (ETMR) are malignant brain tumors that occur predominantly in infants and young children. Most patients die within two years of diagnosis, and more effective, targeted therapies are urgently needed. To better characterize the oncogenic mechanisms of key driver alterations and to identify novel therapeutic targets, we set out to study the cellular heterogeneity of ETMR using single-cell RNA sequencing. Analyses conducted on >4,000 high-quality cells collected from eleven primary and relapse specimens revealed a common cellular hierarchy across all tumors: A highly proliferative neural stem cell-like population (SOX2+) that gives rise to intermediate progenitors (NEUROD1/NEUROG1+) and more differentiated neuron-like cells (STMN2/4+). These malignant cell populations closely overlap with histological patterns of ETMRs, as confirmed by multiplexed immunofluorescence microscopy on patients’ tumors. Comparison to single-cell datasets from human fetuses indicated high resemblance to normal cortical neurogenesis but also revealed key tumor-specific differences. These include expression of the chromosome 19 miRNA cluster (C19MC, the presumed driver in ~90% of ETMRs), which was restricted to the malignant stem cell-like population. Investigating oncogenic mechanisms of C19MC (comprising 46 miRNA genes) through transcriptome-wide RNA immunoprecipitation analysis, we identified extensive target gene regulation for most C19MC members, including distinct regulators of cell cycle, pluripotency, and neuronal differentiation. Silencing of C19MC families with antisense oligonucleotides resulted in pronounced reduction of ETMR cell line growth, indicating potential avenues for therapeutic targeting in the future. To identify more immediately actionable targets, we investigated inter-cellular signaling between malignant cell populations of ETMRs. Interestingly, we identified marked FGFR and NOTCH receptor-ligand interactions common to all tumors. An in vitro screen of experimental and approved small molecule inhibitors designed to target these interactions nominated several promising candidates for clinical evaluation. Our unpublished results provide much needed insight into targeting ETMR cellular states using multiple modes of action.

Screening and effect analysis of specific small molecule inhibitors of programmed death moleculesin plant

objective to conduct a screen for small molecule inhibitors that can inhibit programmed death of plant molecules and test their inhibitory effects. Methods small molecule drugs with inhibitory or alleviating effects on programmed death of plant cells were selected by screening a large number of literatures, and the indexes of plant root length and fresh weight, Results H2S could reduce the inhibition of root length and fresh weight of Cucumber Seedlings by Cd in a concentration dependent effect. CD induced the accumulation of endogenous H2S, indicating that H2S is involved in the response to CD. H2S reduces ROS accumulation and membrane lipid peroxidation by activating antioxidant systems. H2S reduces apical cell death by decreasing oxidative damage. Conclusions elevated endogenous H2S levels negatively regulate programmed death molecules in plants.

Discovery of potential PD-L1 small molecule inhibitors as novel cancer therapeutics using machine learning-based QSAR models: A virtual drug repurposing study.

Binding of programmed death 1 (PD-1) to programmed death ligand-1 (PD-L1) triggers downstream signaling pathways and results in inhibition of new cytotoxic T-cell activation. Cancer cells can “trick” the immune system with the T-cell suppression ability of the PD-1/PD-L1 signaling mechanism. Blocking the PD-1/PD-L1 interaction through target-specific small molecules can recruit cytotoxic T cells and reactivate the immune response, is a potential therapy in many types of cancer. In spite of that, there are currently no approved small molecule inhibitors targeting the PD-1/PD-L1 pathway. In this study, we aim to discover target-specific and effective small molecules with anti-cancer activity. With this objective, we performed a virtual drug repurposing study on PD1 protein, PD1-PDL1 structure and PDL1-PDL1 homodimer complexes. The results revealed that PDL1-dimer structure is the most suitable target model for virtual screening of small molecule inhibitors. Three different approaches with different algorithms were used in virtual screening to discover the best hit molecules: (i) structure-based, (ii) structure-based pharmacophore (hybrid) and (iii) machine learning-based QSAR. At the end of the study, which included three different approaches, promising PDL1 small molecule inhibitors such as anidulafungin, bemotrizinol, deferoxamine, dequalinium, neratinib, ubiquinol and vilanterol were discovered.

Screening for small molecule inhibitors of SAH nucleosidase using an SAH riboswitch

Due to the emergence of multidrug resistant pathogens, it is imperative to identify new targets for antibiotic drug discovery. The S-adenosylhomocysteine (SAH) nucleosidase enzyme is a promising target for antimicrobial drug development due to its critical functions in multiple bacterial processes including recycling of toxic byproducts of S-adenosylmethionine (SAM)-mediated reactions and producing the precursor of the universal quorum sensing signal, autoinducer-2 (AI-2). Riboswitches are structured RNA elements typically used by bacteria to precisely monitor and respond to changes in essential bacterial processes, including metabolism. Natural riboswitches fused to a reporter gene can be exploited to detect changes in metabolism or in physiological signaling. We performed a high-throughput screen (HTS) using an SAH-riboswitch controlled β-galactosidase reporter gene in Escherichia coli to discover small molecules that inhibit SAH recycling. We demonstrate that the assay strategy using SAH riboswitches to detect the effects of SAH nucleosidase inhibitors can quickly identify compounds that penetrate the barriers of Gram-negative bacterial cells and perturb pathways involving SAH.

Abstract A024: A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases

Abstract Background: Effective treatment of metastatic disease remains a major challenge in the improvement of patient outcomes in osteosarcoma (OS). Novel anti-metastatic therapies are needed to treat distant metastases. To this end, the current research evaluates whether targeting the dysregulated mRNA translation machinery in OS can inhibit metastases. Hypothesis: We hypothesize that mRNA translation factors present at an abnormally high abundance in OS cells support the rapid synthesis of cytoprotective proteins that are needed to survive in the oxidative stress-rich microenvironment of the lung. Experimental Approach: Databases of OS cell lines and patient tumor data (A. Sweet-Cordero, UCSF) were queried to identify mRNA translation factors with abnormal transcript levels. A limited drug screen of small molecule inhibitors (SMIs) against identified candidates was carried out to evaluate IC50 values in metastatic OS cells. A candidate inhibitor identified from these data was further characterized for synergy with chemical inducers of oxidative stress (e.g. tert-butylhydroquinone [tBHQ]) that mimics conditions encountered in the lung. Drug combination studies examined 2D and 3D tumor spheroid growth, cellular oxidative stress, and PARP-cleavage, under +/- inhibitor and +/- oxidative stress conditions. Metastatic OS cells were engineered to express an antioxidant response element (ARE)-mCherry fluorescent reporter to directly monitor the antioxidant response by fluorescence microscopy. Polysome profiling was used to assess inhibitor-mediated changes in global mRNA translation. The anti-metastatic activity of the inhibitor was tested in the ex vivo pulmonary metastasis assay (PuMA) and in in vivo metastasis models. Results: From cell and patient sample screening, eIF4A1/2 was identified as being abnormally regulated in metastatic OS cells. The SMI, CR-1-31B, specifically targets eIF4A1/2 and was found to have an IC50 of just 8 nM. CR-1-31B was found to inhibit tumor cell growth in 2D and 3D, increase cellular oxidative stress, and enhance PARP-cleavage, but only under oxidative stress conditions. Western analysis of tBHQ-treated metastatic OS cells with the ARE-mCherry reporter confirmed that the temporal expression of mCherry correlated with the upregulation of Nuclear factor erythroid 2-related factor-2 (Nrf2), a key transcriptional regulator of the antioxidant response. CR-1-31B blunted the upregulation of the antioxidant response in 2D and 3D tumor growth conditions with oxidative stress. CR-1-31B, in a dose-dependent manner, decreased the amount of polysomal mRNAs. CR-1-31B reduced the lung tumor burden in the ex vivo PuMA model, delayed primary tumor growth, and reduced lung metastases in in vivo xenograft OS models. Conclusions: Our data demonstrates that dysregulated mRNA translation is a metastatic vulnerability that can be exploited with SMIs. Altogether, these data support the inhibition of metastatic OS by CR-1-31B, highlighting the potential therapeutic utility of this selective translation inhibitor. Citation Format: Michael M. Lizardo, Christopher Hughes, Yue Zhou Huang, Alberto Delaidelli, Taras Shyp, Haifeng Zhang, Sol Snir Shaool, Poul H. Sorensen. A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A024.

A high-throughput MALDI-TOF MS biochemical screen for small molecule inhibitors of the antigen aminopeptidase ERAP1.

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.

Plate-Based Screening for DUB Inhibitors.

The evolutionally conserved and abundant post-translational modifier ubiquitin (Ub) is involved in a vast number of cellular processes. Imbalanced ubiquitination is associated with a range of diseases. Consequently, components of the ubiquitylation machinery, such as deubiquitinating enzymes (DUBs) that control the removal of Ub, are emerging as therapeutic targets. Here, we describe a robust assay suitable for small-molecule inhibitor screening. This assay has the potential to drive the development of small-molecule compounds that can selectively target DUBs.

The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota.

Fusobacterium nucleatum (Fn) is a dominant bacterial species in colorectal cancer (CRC) tissue that is associated with cancer progression and poorer patient prognosis. Following a small-molecule inhibitor screen of 1,846 bioactive compounds against a Fn CRC isolate, we find that 15% of inhibitors are antineoplastic agents including fluoropyrimidines. Validation of these findings reveals that 5-fluorouracil (5-FU), a first-line CRC chemotherapeutic, is a potent inhibitor of Fn CRC isolates. We also identify members of the intratumoral microbiota, includingEscherichia coli, that are resistant to 5-FU. Further, CRC E.coli isolates can modify 5-FU and relieve 5-FU toxicity toward otherwise-sensitive Fn and human CRC epithelial cells. Lastly, we demonstrate that exvivo patient CRC tumor microbiota undergo community disruption after 5-FU exposure and have the potential to deplete 5-FU levels, reducing local drug efficacy. Together, these observations argue for further investigation into the role of the CRC intratumoral microbiota in patient response to chemotherapy.

Screening of small molecule inhibitors against RhoA protein from Alisma using an online detection system

Targeted high-throughput screening of inhibitors from natural products is an effective approach in the treatment of cancer progression. RhoA protein is essential for many signaling pathways. It is closely related to the occurrence and development of tumor. So far, there are only a few reports on the screening of small molecule inhibitors of RhoA protein from natural products. In this study, an online UHPLC-PDA-MS2-RhoA-FLD screening system was established for the first time to identify RhoA inhibitors from medicinal Alisma. Using this online system by adding fluorescent probes protopine [LZ1] to proteins, 17 active components were identified from Alisma, including 14 terpenoids. Their binding abilities were evaluated by Surface Plasmon Resonance experiments. The activities of representative compounds were tested and showed anti-proliferative effect in cancer cells. Mechanistic studies showed that these compounds were able to downregulate the cellular expressions of RhoA associated proteins. This study provides potential lead compounds as small molecule inhibitors of RhoA protein for cancer therapy. This reported method can be used for targeted screening of small molecule inhibitors against tumors, and provides an approach for screening tumor inhibitors from natural products.

Functional Drug Screening of Small Molecule Inhibitors of Epigenetic Modifiers in Refractory AML Patients.

Simple SummaryAcute myeloid leukemia is a cancer originating in the bone marrow and peripheral blood. Genetic mutations observed with the disease are difficult to target therapeutically and patients often have different treatment responses to the standard of care. By finding epigenetic compounds that work in a variety of patients, we could discover better therapies to treat the disease apart from the current standard of care. Here we present an unbiased drug screen of a variety of epigenetic compounds, with some showing effective responses in all or most patient samples. The use of inhibitors of epigenetic modifiers in the treatment of acute myeloid leukemia (AML) has become increasingly appealing due to the highly epigenetic nature of the disease. We evaluated a library of 164 epigenetic compounds in a cohort of 9 heterogeneous AML patients using an ex vivo drug screen. AML blasts were isolated from bone marrow biopsies according to established protocols and treatment response to the epigenetic library was evaluated. We find that 11 histone deacetylase (HDAC) inhibitors, which act upon mechanisms of cell cycle arrest and apoptotic pathways through inhibition of zinc-dependent classes of HDACs, showed efficacy in all patient-derived samples. Other compounds, including bromodomain and extraterminal domain (BET) protein inhibitors, showed efficacy in most samples. Specifically, HDAC inhibitors are already clinically available and can be repurposed for use in AML. Results in this cohort of AML patient-derived samples reveal several epigenetic compounds with high anti-blast activity in all samples, despite the molecular diversity of the disease. These results further enforce the notion that AML is a predominantly epigenetic disease and that similar epigenetic mechanisms may underlie disease development and progression in all patients, despite differences in genetic mutations.