High-throughput Screening Assay Research Articles

Engineering the Activity of a Template-Independent DNA Polymerase.

Enzymatic DNA writing technologies based on the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) have the potential to advance DNA information storage. TdT is unique in its ability to synthesize single-stranded DNA de novo but has limitations, including catalytic inhibition by ribonucleotide presence and slower incorporation rates compared to replicative polymerases. We anticipate that protein engineering can improve, modulate, and tailor the enzyme's properties, but there is limited information on TdT sequence-structure-function relationships to facilitate rational approaches. Therefore, we developed an easily modifiable screening assay that can measure the TdT activity in high-throughput to evaluate large TdT mutant libraries. We demonstrated the assay's capabilities by engineering TdT mutants that exhibit both improved catalytic efficiency and improved activity in the presence of an inhibitor. We screened for and identified TdT variants with greater catalytic efficiency in both selectively incorporating deoxyribonucleotides and in the presence of deoxyribonucleotide/ribonucleotide mixes. Using this information from the screening assay, we rationally engineered other TdT homologues with the same properties. The emulsion-based assay we developed is, to the best of our knowledge, the first high-throughput screening assay that can measure TdT activity quantitatively and without the need for protein purification.

High-Throughput Screening Assay for Convalescent Sera in COVID-19: Efficacy, Donor Selection, and Variant Neutralization

Convalescent sera, rich in pathogen-specific antibodies, offers passive immunity to patients with infectious diseases. Screening assays using convalescent sera are crucial for evaluating therapeutic efficacy, selecting suitable serum donors, and standardizing assays. They measure antibody levels, neutralizing potential, and specificity against viruses like SARS-CoV-2, ensuring therapeutic serum contains potent antibodies. Standardized procedures enable reliable results and wider adoption of serum therapy for COVID-19. We have developed a high-content image-based assay for screening convalescent sera against SARS-CoV-2 variants. Using various cell lines, we identified optimal candidates, employed immunofluorescence to visualize infected cells, and assessed neutralizing antibody efficacy. Screening convalescent sera for therapeutic potential identified neutralizing activity against SARS-CoV-2 variants. Dose–response analysis showed variable neutralizing activity, with some sera exhibiting broad neutralization. Additionally, we explored the synergy between neutralizing sera and β-d-N4-hydroxycytidine (NHC), an initial metabolite of molnupiravir. These assays enhance serum therapy’s benefits for COVID-19 treatment and aid in understanding neutralizing activity against SARS-CoV-2 variants, addressing viral challenges.

Binder-Responsive DNA Strand Displacement Enables High-Throughput and High-Fidelity Discovering of Small Molecular DNA Binders.

High-throughput screening (HTS) is pivotal in the discovery of small molecules that bind to DNA, yet there are limited sensing mechanisms available for designing HTS assays for DNA binders. Herein, we introduce a binder-responsive toehold-mediated DNA strand displacement (BR-TMSD) technique featuring programmable reaction kinetics in response to DNA-binder interactions. When two DNA binders are used, BR-TMSD is initiated through a rapid binder displacement, followed by the DNA strand displacement. The orthogonal displacement reactions of BR-TMSD enables a high-fidelity, dual-channel HTS assay, returning 19 new DNA binders from a library of 1,170 compounds.

Discovery of a novel Xanthone derivative P24 for anti-AD via targeting sTGFBR3

Soluble transforming growth factor beta receptor 3 (sTGFBR3) antagonist is a new focus in the research and development of Alzheimer's disease (AD) drugs. Our previous studies have identified sTGFBR3 as a promising new target for AD, with few targeted antagonists identified. In this study, we performed structural modeling of sTGFBR3 using AlphaFold2, followed by high-throughput virtual screening and surface plasmon resonance assays. which collectively identified Xanthone as potential compounds for targeting sTGFBR3. After optimizing the sTGFBR3-Xanthone complex using molecular dynamics (MD) simulations, we prepared a series of novel Xanthone derivatives and evaluated their anti-inflammatory activity, toxicity, and structure-activity relationship in BV2 cell model induced by lipopolysaccharides (LPS) or APP/PS1/tau mouse brain extract (BE). Several derivatives with the most potent anti-inflammatory activity were tested for blood-brain barrier permeability and sTGFBR3 affinity. Derivative P24, selected for its superior properties, was further evaluated in vitro. The results indicated that P24 increased the activation of TGF-β signaling and decreased the activation of IκBα/NF-κB signaling by targeting sTGFBR3, thereby regulating the inflammation-phagocytosis balance in microglia. Moreover, the low acute toxicity, long half-life, and low plasma clearance of P24 suggest that it can be sustained in vivo. This property may render P24 a more effective treatment modality for chronic diseases, particularly AD. The study demonstrates P24 serve as potential novel candidates for the treatment of AD via antagonizing sTGFBR3.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1 (AP1), we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of DMSO, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery—it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket, and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of DMSO and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

ETMR-18. ADVANCING ETMR RESEARCH: NOVELIN VITRO ANDIN VIVO PRECLINICAL MODELS FOR DEVELOPING MORE EFFECTIVE TREATMENTS

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a rare and aggressive tumor that occurs mainly in infants under three years of age. Genetically, ETMRs are characterized by amplification and fusion of a microRNA cluster on chromosome 19 (C19MC) with TTYH1, present in ~90% of the tumors. ETMR patients still have a poor prognosis without any recent advances in treatment. A major limitation to develop more effective treatment strategies is the lack of sufficient preclinical models. METHODS Establishment of ETMR patient-derived xenograft (PDX) models and tumoroid lines, molecular characterization, high throughput in vitro drug screens, targeted drug treatments in vitro and in vivo. RESULTS We have established four new tumoroid and five new PDX ETMR models. These models faithfully recapitulate ETMR patients’ tumors based on their histological, transcriptomic, and methylome signatures. The in vitro models were derived from two patients and two PDX models and included three C19MC-amplified cell lines, of which two were a primary-relapse pair, and one a primary C19MC non-amplified model. In vivo we established the same models as PDX models subcutaneously plus an additional C19MC-amplified relapse model. High-throughput drug screening assays, using drug libraries with 225 clinically approved drugs and other small molecules, were conducted across the panel of in vitro models. Potential therapeutic candidates with promising activity in ETMR tumoroid lines identified from these screens include FGFR, XPO1, MTOR, MDM2 and topoisomerase inhibitors. CONCLUSIONS The establishment of novel preclinical models represents a significant advancement in ETMR research, offering a platform that captures the molecular complexity of the disease. The identification of several inhibitors shows the potential of these models for drug discovery and preclinical testing. Ongoing in vitro and in vivo experiments with these drugs and combinations aim to further validate their activity, moving towards the development of more effective therapies for ETMR patients.

Discovery and validation of a novel inhibitor of HYPE-mediated AMPylation.

Adenosyl monophosphate (AMP)ylation (the covalent transfer of an AMP from Adenosine Triphosphate (ATP) onto a target protein) is catalyzed by the human enzyme Huntingtin Yeast Interacting Partner E (HYPE)/FicD to regulate its substrate, the heat shock chaperone binding immunoglobulin protein (BiP). HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the endoplasmic reticulum and mounting a unfolded protein response in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy towardthe treatment of various protein misfolding diseases, including neuropathy and early-onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE's AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE's enzymatic activity at scale, and provided the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.

Predicting Chemical Immunotoxicity through Data-Driven QSAR Modeling of Aryl Hydrocarbon Receptor Agonism and Related Toxicity Mechanisms.

Computational modeling has emerged as a time-saving and cost-effective alternative to traditional animal testing for assessing chemicals for their potential hazards. However, few computational modeling studies for immunotoxicity were reported, with few models available for predicting toxicants due to the lack of training data and the complex mechanisms of immunotoxicity. In this study, we employed a data-driven quantitative structure-activity relationship (QSAR) modeling workflow to extensively enlarge the limited training data by revealing multiple targets involved in immunotoxicity. To this end, a probe data set of 6,341 chemicals was obtained from a high-throughput screening (HTS) assay testing for the activation of the aryl hydrocarbon receptor (AhR) signaling pathway, a key event leading to immunotoxicity. Searching this probe data set against PubChem yielded 3,183 assays with testing results for varying proportions of these 6,341 compounds. 100 assays were selected to develop QSAR models based on their correlations to AhR agonism. Twelve individual QSAR models were built for each assay using combinations of four machine-learning algorithms and three molecular fingerprints. 5-fold cross-validation of the resulting models showed good predictivity (average CCR = 0.73). A total of 20 assays were further selected based on QSAR model performance, and their resulting QSAR models showed good predictivity of potential immunotoxicants from external chemicals. This study provides a computational modeling strategy that can utilize large public toxicity data sets for modeling immunotoxicity and other toxicity endpoints, which have limited training data and complicated toxicity mechanisms.

Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 μM) and CaV3.3 (IC50 = 0.49 μM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Identification of Leishmania donovani PEX5-PTS1 Interaction Inhibitors through Fluorescence Polarization-Based High-Throughput Screening.

Leishmaniasis, an infectious disease caused by pathogenic Leishmania parasites, affects millions of people in developing countries, and its re-emergence in developed countries, particularly in Europe, poses a growing public health concern. The limitations of current treatments and the absence of effective vaccines necessitate the development of novel therapeutics. In this study, we focused on identifying small molecule inhibitors which prevents the interaction between peroxin 5 (PEX5) and peroxisomal targeting signal 1 (PTS1), pivotal for kinetoplastid parasite survival. The Leishmania donovani PEX5, containing a C-terminal tetratricopeptide repeat (TPR) domain, was expressed and purified, followed by the quantification of kinetic parameters of PEX5-PTS1 interactions. A fluorescence polarization-based high-throughput screening assay was developed and small molecules inhibiting the LdPEX5-PTS1 interaction were discovered through the screening of a library of 51,406 compounds. Based on the confirmatory assay, nine compounds showed half maximal inhibitory concentration (IC50) values ranging from 3.89 to 24.50 µM. In silico docking using a homology model of LdPEX5 elucidated that the molecular interactions between LdPEX5 and the inhibitors share amino acids critical for PTS1 binding. Notably, compound P20 showed potent activity against the growth of L. donovani promastigotes, L. major promastigotes, and Trypanosoma brucei blood stream form, with IC50 values of 12.16, 19.21, and 3.06 μM, respectively. The findings underscore the potential of targeting LdPEX5-PTS1 interactions with small molecule inhibitors as a promising strategy for the discovery of new anti-parasitic compounds.

Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a β-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious β-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

Abstract LB082: A high-throughput immune-oncology screen identifies immunostimulatory properties of low dose chemotherapy in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous subtype broadly characterized by the lack of hormone receptor (HR) and HER2 expression. Compared to HR+ breast cancers, TNBCs typically have a greater immune cell infiltrate and are more likely to respond to immune checkpoint inhibition (ICI). The results from KEYNOTE-355 and KEYNOTE-522 trials led to the approvals of anti-PD-1 in combination with physician’s choice chemotherapy for the treatment of TNBC in adjuvant and neoadjuvant settings. However, few patients achieve a durable response, and there remains a need to optimize combination strategies to enhance the benefits of ICI in TNBC. Therefore, we developed a high-throughput co-culture screening assay to identify compounds enhancing T-cell-mediated cytotoxicity. Co-cultures included three cell types: mKate2-labeled PyMT tumor cells expressing the OVA antigen, BPF-labeled PyMT cells, and CD8+ OT-1 T-cells. The screening library included over 400 FDA-approved compounds or agents under investigation for oncology indications. Each compound was evaluated at five concentrations (3µM-12nM) and across three-time points (24hr, 48hr, and 72hr) to provide insight into the kinetics. Cell viability was assessed via image analysis at each time point, and enhanced cell death in the PyMT-OVA cells compared to the non-OVA PyMT cells was considered evidence of enhanced T-cell mediated cytotoxicity. Five chemotherapy agents were chosen as priority hits for mechanistic follow-up due to their ability to enhance T-cell mediated cytotoxicity at multiple doses and multiple timepoints: paclitaxel, bleomycin sulfate, kinesin-inhibitor, ABT-751, and etoposide. We are exploring the dose dependency of MHC-I and MHC-II induction, as well as the minimum dose required to cause immunogenic cell death. We further found that low doses of these agents do not negativity affect CD8+ T-cell proliferation, whereas high doses may have deleterious effects on CD8+ T-cell functioning. The results of these studies provide mechanistic insight into potential new chemotherapy partners to enhance anti-PD-1 efficacy in TNBC patients and suggest benefit to further investigating the immunostimulatory potential of low dose chemotherapy. Citation Format: Kennady K. Bullock, Thomas Hasaka, Emily Days, Joshua Bauer, Ann Richmond. A high-throughput immune-oncology screen identifies immunostimulatory properties of low dose chemotherapy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB082.

Abstract LB027: Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository

Abstract Second-generation androgen ablation therapies (ADT), enzalutamide, and abiraterone are initially effective on metastatic castration-resistant prostate cancer (mCRPC). However, nearly 30-40% of patients eventually become resistant to these treatments due to the presence of androgen-receptor variants (AR-V), which lack a ligand-binding domain. The grim reality that metastatic CRPC is virtually incurable for most patients poses a significant challenge for clinicians striving to enhance these individuals' quality of life and lifespan. Considering drug discovery efforts, we partnered with the National Cancer Institute's (NCI) Natural Product Repository, which possesses a diverse collection of natural product extracts derived from plant, marine, and microbial organisms. We developed high-throughput phenotypic screening assays to screen the crude natural product extract libraries on AR and AR-variants CRPC cell lines (C4-2B and 22Rv1). Several extracts inhibited the growth of both CRPC cell lines and were further confirmed for dose-dependent cell viability inhibition assays. Once we determined the IC50 dose, we performed a Western blot analysis that confirmed the inhibition of AR and AR-V7 expression in CRPC cells.In subsequent studies, we narrowed down by fractionating the most potent extracts and confirmed their ability to inhibit AR expression and signaling on CRPC cell lines. Currently, we are characterizing the potent compounds and their inhibitory activity on AR and AR-variants, in vitro and in vivo CRPC models. Many nutraceuticals have been shown to target AR signaling, synthesis, or degradation which effectively inhibited the growth of CRPC. Our collaborative efforts with NCI will permit us find N-terminal AR degrading molecules (AF-1), which are essential for the AR transcriptional activities of CRPC. In sum, discovery of ovel small molecules from our drug development program have the potential to translate these effective natural products into drugs with clinical benefits to treat CRPC. Citation Format: Neha Tyagi, Sepideh Hosseiniporgham, Ashish Tyagi, Balaji Chandrasekaran, Katherine Bonilla, Inna Kriger, James Saccchettini, Chendil Damodaran. Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB027.

CYP46A1 inhibition and activation: an in vitro high throughput screening assay to assess possible drug interactions using FDA approved chemical library

CYP46A1 inhibition and activation: an in vitro high throughput screening assay to assess possible drug interactions using FDA approved chemical library

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [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 3102.

Abstract 4674: Reserpine analog modulates transport function of ABCG2

Abstract Backgrounds: Reserpine is an agent that is used for the treatment of hypertension. ABCG2 also known as breast cancer resistance protein (BCRP) is a member of the ABC transporters associated with multidrug resistance (MDR). Our high-throughput screening assay has revealed that reserpine analog inhibits transport function of ABCG2. Aim: To investigate the effect of reserpine analog on ABCG2 function. Results: Flow cytometry assay showed that reserpine analog blocked the efflux of Pheophorbide a (PhA: fluorescent substrate of ABCG2) from ABCG2-expressing HCT-116/BCRP colon cancer cells in a concentration-dependent manner. Furthermore, cell proliferation assay (MTS assay) revealed that 5 and 10 μM reserpine analog sensitized HCT-116/BCRP cells to SN38 with IC50 value 0.61 + 0.12 μM and 0.22 + 0.036 μM, respectively. Finally, reserpine analog did not stimulate ABCG2-mediated ATP hydrolysis, but rather inhibited it (concentration required for 50% inhibition = 2.7 μM), suggesting that reserpine analog might prevent conformational changes required for ATP hydrolysis. Conclusion: Reserpine analog could be developed as a potent modulator to overcome ABCG2-mediated MDR in cancer cells. Citation Format: Shinobu Ohnuma, Shoji Kokubo, Norihiko Sugisawa, Megumi Murakami, Yuuri Hatsuzawa, Minoru Kobayashi, Taiki Kajiwara, Hideyuki Suzuki, Hideaki Karasawa, Kazuhiro Watanabe, Takayuki Doi, Suresh V. Ambudkar, Takashi Kamei, Michiaki Unno. Reserpine analog modulates transport function of ABCG2 [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 4674.

Abstract 3905: Targeting RNA demethylase ALKBH5 blocks growth and improves therapy response in osteosarcoma

Abstract Background: Osteosarcoma (OS) arises primarily in children and adolescents, with a second peak in incidence in those over the age of 50. Unfortunately, outcomes of patients with metastatic or relapsed osteosarcoma have remained abysmal. Moreover, patients who survive have a highly compromised quality of life due to debilitating side effects associated with chemotherapy drugs. Virtually no new drug has been approved for treating OS in the last three decades. Recently our group showed that ALKBH5 is critical for promoting OS growth and metastasis and may thus serve as a unique driver of OS. We therefore hypothesized that epigenetic changes, specifically mRNA demethylation mediated by the RNA demethylase ALKBH5, play a pivotal role in OS growth and metastasis, and hence approaches aimed at inhibiting ALKBH5 activity could prove clinical utility for patients with OS. Methods: shRNA and CRISPR-Cas9-based knockout were used to study the effect of depleting ALKBH5 in osteosarcoma and the effect it has on growth and metastasis. Fluorescence-based high-throughput screening (HTS) assay was used to identify targets of ALKBH5. FDA-approved and LOPAC compound libraries were used. SPR analysis was used to determine the Mefloquine-ALKBH5 interaction. In vitro and In vivo intratibial orthotopic models were used to test the efficacy of Mefloquine for blocking OS growth. Experiments are currently underway to test the effect of combining Mefloquine and doxorubicin in inhibiting OS growth and metastasis. Results: We showed that upregulation of the ALKBH5 attenuates histone H2A monoubiquitination to result in the induction of key pro-tumorigenic genes and consequently unchecked cell cycle progression, incessant DNA replication, and upregulation of DSB repair. Unbiased high throughput screening using a LOPAC library identified mefloquine as a potential ALKBH5 inhibitor. SPR analysis showed the interaction of Mefloquine with ALKBH5 catalytic domain. M6A dot blot confirmed that mefloquine inhibits the catalytic activity of ALKBH5. Cell-based assays showed reduced short-term and long-term growth with the treatment of mefloquine. In vivo models showed a reduction in tumor volume upon mefloquine oral treatment. Conclusion: Our results establish RNA demethylase ALKBH5 as a novel driver of OS. This project has a direct translational relevance as an FDA-approved drug can be repurposed for treating OS patients as a monotherapy or in combination with doxorubicin and be formally tested in the clinic without much delay. Citation Format: Daisy Medina, Santosh Timilsina, Panneerdoss Subbarayalu, Prabhakar Pitta Venkata, Deepika Singh, Trong Phat Do, Shuo Zhou, Yogesh K. Gupta, Daohong Zhou, Manjeet K. Rao. Targeting RNA demethylase ALKBH5 blocks growth and improves therapy response in osteosarcoma [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 3905.

Establishment, optimization and validation of a fluorescence polarization-based high-throughput screening assay targeting cathepsin L inhibitors

Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC50 of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.