Cell-based High-throughput Screening Research Articles

Abstract A004: Broad and synergistic anti-tumor effects of small-molecule inhibitors of hypoxia inducible factors when paired with immune checkpoint blockade

Abstract Motivation: Hypoxia inducible factors (HIFs) are critical transcriptional regulators that play fundamental roles in adaptive responses to low oxygen, both in normal and disease conditions. Dysregulated activation of HIFs has been implicated in a variety of cancers due to their role in mediating a broad range of key processes including angiogenesis, glycolysis, cell survival, cancer stem cell specification, and metastasis. Methods: In this study, we utilized virtual and cell-based high-throughput screening approaches to identify compounds that disrupt the binding of HIF-1α and HIF-2α to HIF-1β, leading to the suppression of HIF target gene expression. Results: In syngeneic animal models, we observed significant tumor regression when HIF inhibitors are combined with immunotherapy agents in multiple tumor models. The combined treatment enhanced the infiltration of natural killer (NK) cells into the tumor microenvironment. Conclusion: This study highlights the anti-tumor effects of HIF inhibitors and the immune response resulting in sustained tumor regression. The promising outcomes warrant further exploration of these inhibitors as potential cancer treatment agents. Citation Format: Shaima Salman, Tina Y. Huang, Yousang Hwang, Anmol Kumar, Dominic Dordai, Alexander D. MacKerell, Gregg L. Semenza. Broad and synergistic anti-tumor effects of small-molecule inhibitors of hypoxia inducible factors when paired with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A004.

A High-Throughput Drug Repurposing Strategy to Treat TBX2 and/or TBX3 Dependent Cancers.

The highly homologous T-box transcription factors TBX2 and TBX3 are critical for embryonic development, and their overexpression in postnatal tissues contributes to a wide range of malignancies, including melanoma and rhabdomyosarcoma. Importantly, when TBX2 and TBX3 are depleted in cancers where they are overexpressed, the malignant phenotype is inhibited, and they have therefore been regarded as druggable targets. However, the time and costs associated with de novo drug development are challenging and result in drugs that are costly, especially for patients in low- and middle-income countries. In the current study, we therefore combined a targeted and drug repurposing approach to identify drugs that are expected to be more efficacious and cost-effective with significantly reduced side effects. A high-throughput cell-based immunofluorescence screen was performed to identify drugs in the Pharmakon 1600 drug library that can negatively regulate TBX2 and/or TBX3 levels. "Hit" drugs were validated for their effect on TBX2/TBX3 levels and cytotoxicity in TBX2/TBX3-dependent melanoma and rhabdomyosarcoma cells. To this end, immunofluorescence, western blotting, quantitative real-time PCR, and MTT cell viability assays were performed. Niclosamide, piroctone olamine, and pyrvinium pamoate, were identified as TBX2 and/or TBX3-targeting drugs, and they exhibited cytotoxicity in a TBX2/TBX3-dependent manner. Furthermore, these "Hit" drugs were shown to induce senescence and/or apoptosis. Niclosamide, piroctone olamine, and pyrvinium pamoate are promising, cost-effective therapeutic agents for the treatment of TBX2/TBX3-dependent cancers.

Multienzymatic Orthogonal Activation of DNA Codec Enables Tumor-Specific Imaging of Base Excision Repair Activity.

Accurate monitoring of base excision repair (BER) activity in cancer cells is critical for advancing the comprehension of DNA repair processes, gaining insights into cancer development, and guiding treatment strategies. However, current assay techniques for assessing BER activity in cancer cells face challenges due to the heterogeneous origins and diversity of BER enzymes. In this work, we present a highly reliable triple loop-interlocked DNA codec (GATED) that enables precise assessment of BER activity in cancer cells through signal amplification mediated by multienzyme orthogonal activation. The GATED device features a dumbbell-shaped DNA probe to encode two BER enzymes for BER-related signal conversion as well as two bound circular DNA to decode the apurinic/apyrimidinic sites for apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification. Importantly, GATED is orthogonally activated by multiple target BER enzymes (i.e., uracil DNA glycosylase, thymine DNA glycosylase, and APE1), resulting in a unified fluorescent signal that significantly improves the detection specificity and sensitivity to BER enzymes. Additionally, we demonstrate that the GATED has exceptional biostability within complex biological systems, where it was successfully employed to monitor BER activity in cancer cells with high specificity and enabled cell-based high-throughput screening for BER inhibitors. The GATED provides a much-needed tool for the real-time monitoring of BER activity and the screening of BER inhibitors in cancer cells, potentially advancing both the investigation and clinical application of BER biology.

Anticancer agent 5-fluorouracil reverses meropenem resistance in carbapenem-resistant Gram-negative pathogens

The global increasing incidence of clinical infections caused by carbapenem-resistant Gram-negative pathogens requires urgent and effective treatment strategies. Antibiotic adjuvants represent a promising approach to enhance the efficacy of meropenem against carbapenem-resistant bacteria. This study shows that the anticancer agent 5-fluorouracil (5-FU, 50 µM) significantly reduced the minimum inhibitory concentration of meropenem against blaNDM-5 positive Escherichia coli by 32-fold through cell-based high-throughput screening. Further pharmacological studies indicated that 5-FU exhibited potentiation effects on carbapenem antibiotics against 42 Gram-negative bacteria producing either metallo-β-lactamases (MBLs), such as NDM and IMP, or serine β-lactamases (Ser-BLs), like KPC and OXA. These bacteria included E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp., 32 of which were obtained from human clinical samples. Mechanistic investigations revealed that 5-FU inhibited the transcription and expression of the blaNDM-5 gene. In addition, 5-FU combined with meropenem enhanced bacterial metabolism, and stimulated the production of reactive oxygen species (ROS), thereby rendering bacteria more susceptible to meropenem. In a mouse systemic infection model, 5-FU combined with meropenem reduced bacterial loads and effectively elevated the survival rate of 83.3%, compared with 16.7% with meropenem monotherapy. Collectively, these findings indicate the potential of 5-FU as a novel meropenem adjuvant to improve treatment outcomes against infections caused by carbapenem-resistant bacteria.

164 Combining in silico, in vitro, and in vivo approaches for studying taste perception and preferences of domestic cats and dogs

Abstract Developing food products for pet cats and dogs is an important and challenging area of the food industry. The diet offered must be both balanced and palatable to ensure the amount of food eaten meets their nutritional requirements. The taste perception of pets is different from humans in many cases (Li et al., 2005) and therefore species-specific research is necessary. Hence, understanding the flavor preferences of cats and dogs is an important area of research and requires multiple disciplines and scientific approaches. We have developed a range of complimentary methods for studying taste perception and preferences of domestic cats and dogs In silico: homology models of the pet taste receptors have been developed. These models are used to identify candidate compounds and elucidate the mechanisms of taste perception of pets. In vitro: cell-based high throughput screening assays of pet taste receptors have also been developed. These assays are used to identify taste-active compounds and confirm response to those identified via the in silico method to refine the model. In vivo: finally, a two-bottle choice test for cats has been developed. This test is used to confirm hedonic responses and determine optimum concentrations of single compounds and mixtures identified by the in silico and/or in vitro methods. Using this novel approach, we now have a deeper understanding of the taste perception and preferences of pets, with specific examples including Umami (McGrane et al., 2023) and Kokumi (Laffitte et al., 2021) taste perception of cats and bitter taste perception of dogs (Gibbs et al., 2022). This approach has also enabled us to reduce the number of in vivo tests required, so implementing the 3Rs, by prioritizing the most promising tastants using the in silico and in vitro methods. Gibbs, M., Winnig, M., Riva, I., Dunlop, N., Waller, D., Klebansky, B., Logan, D.W., Briddon, S.J., Holliday, N.D., McGrane, S.J. 2022. Bitter taste sensitivity in domestic dogs (Canis familiaris) and its relevance to bitter deterrents of ingestion. PLoS One. 17:e0277607. Laffitte, A., Gibbs, M., Hernangomez de Alvaro, C., Addison, J., Lonsdale, Z.N., Giribaldi, M.G., Rossignoli, A., Vennegeerts, T., Winnig, M., Klebansky, B., Skiles, J., Logan, D.W., McGrane, S.J. 2021. Kokumi taste perception is functional in a model carnivore, the domestic cat (Felis catus). Sci Reports 2021 111. 11:1–17. Li, X., Li, W., Wang, H., Cao, J., Maehashi, K., Huang, L., Bachmanov, A.A., Reed, D.R., Legrand-Defretin, V., Beauchamp, G.K., Brand, J.G. 2005. Pseudogenization of a sweet-receptor gene accounts for cats’ indifference toward sugar. PLoS Genet. 1:0027–0035.McGrane, S.J., Gibbs, M., Hernangomez De Alvaro, C., Dunlop, N., Winnig, M., Klebansky, B., Waller, D. 2023. Umami taste perception and preferences of the domestic cat (Felis catus), an obligate carnivore. Chem Senses. 48.

Unsaturated fatty acid perturbation combats emerging triazole antifungal resistance in the human fungal pathogen Aspergillus fumigatus.

Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.

Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

Discovery of small molecule guanylyl cyclase B receptor positive allosteric modulators.

Myocardial fibrosis is a pathological hallmark of cardiovascular disease (CVD), and excessive fibrosis can lead to new-onset heart failure and increased mortality. Currently, pharmacological therapies for myocardial fibrosis are limited, highlighting the need for novel therapeutic approaches. The particulate guanylyl cyclase B (GC-B) receptor possesses beneficial antifibrotic actions through the binding of its natural ligand C-type natriuretic peptide (CNP) and the generation of the intracellular second messenger, cyclic guanosine 3',5'-monophosphate (cGMP). These actions include the suppression of fibroblast proliferation and reduction in collagen synthesis. With its abundant expression on fibroblasts, the GC-B receptor has emerged as a key molecular target for innovative CVD therapeutics. However, small molecules that can bind and potentiate the GC-B/cGMP pathway have yet to be discovered. From a cell-based high-throughput screening initiative of the NIH Molecular Libraries Small Molecule Repository and hit-to-lead evolution based on a series of structure-activity relationships, we report the successful discovery of MCUF-42, a GC-B-targeted small molecule that acts as a positive allosteric modulator (PAM). Studies herein support MCUF-42's ability to enhance the binding affinity between GC-B and CNP. Moreover, MCUF-42 potentiated cGMP levels induced by CNP in human cardiac fibroblasts (HCFs) and notably also enhanced the inhibitory effect of CNP on HCF proliferation. Together, our findings highlight that MCUF-42 is a small molecule that can modulate the GC-B/cGMP signaling pathway, potentially enhancing the antifibrotic actions of CNP. Thus, these data underscore the continued development of GC-B small molecule PAMs as a novel therapeutic strategy for targeting cardiac fibrosis and CVD.

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Structural and thermodynamic framework for PIEZO1 modulation by small molecules.

Mechanosensitive PIEZO channels constitute potential pharmacological targets for multiple clinical conditions, spurring the search for potent chemical PIEZO modulators. Among them is Yoda1, a widely used synthetic small molecule PIEZO1 activator discovered through cell-based high-throughput screening. Yoda1 is thought to bind to PIEZO1's mechanosensory arm domain, sandwiched between two transmembrane regions near the channel pore. However, how the binding of Yoda1 to this region promotes channel activation remains elusive. Here, we first demonstrate that cross-linking PIEZO1 repeats A and B with disulfide bridges reduces the effects of Yoda1 in a redox-dependent manner, suggesting that Yoda1 acts by perturbing the contact between these repeats. Using molecular dynamics-based absolute binding free energy simulations, we next show that Yoda1 preferentially occupies a deeper, amphipathic binding site with higher affinity in PIEZO1 open state. Using Yoda1's binding poses in open and closed states, relative binding free energy simulations were conducted in the membrane environment, recapitulating structure-activity relationships of known Yoda1 analogs. Through virtual screening of an 8 million-compound library using computed fragment maps of the Yoda1 binding site, we subsequently identified two chemical scaffolds with agonist activity toward PIEZO1. This study supports a pharmacological model in which Yoda1 activates PIEZO1 by wedging repeats A and B, providing a structural and thermodynamic framework for the rational design of PIEZO1 modulators. Beyond PIEZO channels, the three orthogonal computational approaches employed here represent a promising path toward drug discovery in highly heterogeneous membrane protein systems.

High-throughput screening of novel TFEB agonists in protecting against acetaminophen-induced liver injury in mice

Macroautophagy (referred to as autophagy hereafter) is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles. Previous studies showed that autophagy protects against acetaminophen (APAP)-induced injury (AILI) via selective removal of damaged mitochondria and APAP protein adducts. The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes. In the present study, we showed that transcription factor EB (TFEB), a master transcription factor for lysosomal biogenesis, was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers. Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI, respectively. Mechanistically, overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2 (NRF2) activation to protect against AILI. We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists. Among these agonists, salinomycin, an anticoccidial and antibacterial agent, activated TFEB and protected against AILI in mice. In conclusion, genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.

Acoustic droplet ejection facilitates cell-based high-throughput screenings using natural products

Natural Products (NPs) are one of the main sources for drug discovery. Many clinical drugs are NPs or NP-inspired compounds, and recently discovered New Chemical Entities (NCEs) of NPs are emerging as promising new drugs. High-Throughput Screening (HTS) of large sample sets or libraries has grown to be vital for the drug discovery field. Industrial-scale HTS of NP libraries can be limited due to the difficulties entailed in working with tiny extract volumes and the variability in viscosity of NP extracts. For these reasons, the implementation of new technologies to miniaturize different reagent volumes grows to be fundamental. Since Acoustic Droplet Ejection (ADE) emerged as a helpful tool in HTS campaigns for the transference of compound libraries. The aim of this work was to test the effectiveness of ADE for the dispensation of NP extract libraries in cell-based HTS assays.

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death

We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both invitro and invivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.

Quizartinib inhibits necroptosis by targeting receptor-interacting serine/threonine protein kinase 1.

Systemic inflammatory response syndrome (SIRS), at least in part driven by necroptosis, is characterized by life-threatening multiple organ failure. Blocking the progression of SIRS and consequent multiple organ dysfunction is challenging. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important cell death and inflammatory mediator, making it a potential treatment target in several diseases. Here, using a drug repurposing approach, we show that inhibiting RIPK1 is also an effective treatment for SIRS. We performed cell-based high-throughput drug screening of an US Food and Drug Administration (FDA)-approved drug library that contains 1953 drugs to identify effective inhibitors of necroptotic cell death by SYTOX green staining. Dose-response validation of the top candidate, quizartinib, was conducted in two cell lines of HT-22 and MEFs. The effect of quizartinib on necroptosis-related proteins was evaluated using western blotting, immunoprecipitation, and an in vitro RIPK1 kinase assay. The in vivo effects of quizartinib were assessed in a murine tumor necrosis factor α (TNFα)-induced SIRS model. High-throughput screening identified quizartinib as the top "hit" in the compound library that rescued cells from necroptosis in vitro. Quizartinib inhibited necroptosis by directly inhibiting RIPK1 kinase activity and blocking downstream complex IIb formation. Furthermore, quizartinib protected mice against TNFα-induced SIRS. Quizartinib, as an FDA-approved drug with proven safety and efficacy, was repurposed for targeted inhibition of RIPK1. This work provides essential preclinical data for transferring quizartinib to the treatment of RIPK1-dependent necroptosis-induced inflammatory diseases, including SIRS.

A phase 2 study of dasatinib in recurrent clear cell carcinoma of the ovary, fallopian tube, peritoneum or endometrium: NRG oncology/gynecologic oncology group study 0283

ObjectiveGynecologic cancers are traditionally managed according to their presumed site of origin, without regard to the underlying histologic subtype. Clear cell histology is associated with chemotherapy refractoriness and poor survival. Mutations in SWI/SNF chromatin remodeling complex member ARID1A, which encodes for BAF250a protein, are common in clear cell and endometriosis-associated endometrioid carcinomas. High-throughput cell-based drug screening predicted activity of dasatinib, a tyrosine kinase inhibitor, in ARID1A-mutant clear cell carcinoma. MethodsWe conducted a phase 2 clinical trial of dasatinib 140 mg once daily by mouth in patients with recurrent or persistent ovarian and endometrial clear cell carcinoma. Patients with measurable disease were enrolled and then assigned to biomarker-defined populations based on BAF250a immunohistochemistry. The translational endpoints included broad next-generation sequencing to assess concordance of protein expression and treatment outcomes. ResultsTwenty-eight patients, 15 of whom had tumors with retained BAF250a and 13 with loss of BAF250a were evaluable for treatment response and safety. The most common grade 3 adverse events were anemia, fatigue, dyspnea, hyponatremia, pleural effusion, and vomiting. One patient had a partial response, eight (28%) had stable disease, and 15 (53.6%) had disease progression. Twenty-three patients had next-generation sequencing results; 13 had a pathogenic ARID1A alteration. PIK3CA mutations were more prevalent in ARID1A-mutant tumors, while TP53 mutations were more prevalent in ARID1A wild-type tumors. ConclusionsDasatinib was not an effective single-agent treatment for recurrent or persistent ovarian and endometrial clear cell carcinoma. Studies are urgently needed for this rare gynecologic subtype.

Identification of signaling pathways modifying human dopaminergic neuron development using a pluripotent stem cell-based high-throughput screening automated system: purinergic pathways as a proof-of-principle.

Introduction: Alteration in the development, maturation, and projection of dopaminergic neurons has been proposed to be associated with several neurological and psychiatric disorders. Therefore, understanding the signals modulating the genesis of human dopaminergic neurons is crucial to elucidate disease etiology and develop effective countermeasures. Methods: In this study, we developed a screening model using human pluripotent stem cells to identify the modulators of dopaminergic neuron genesis. We set up a differentiation protocol to obtained floorplate midbrain progenitors competent to produce dopaminergic neurons and seeded them in a 384-well screening plate in a fully automated manner. Results and Discussion: These progenitors were treated with a collection of small molecules to identify the compounds increasing dopaminergic neuron production. As a proof-of-principle, we screened a library of compounds targeting purine- and adenosine-dependent pathways and identified an adenosine receptor 3 agonist as a candidate molecule to increase dopaminergic neuron production under physiological conditions and in cells invalidated for the HPRT1 gene. This screening model can provide important insights into the etiology of various diseases affecting the dopaminergic circuit development and plasticity and be used to identify therapeutic molecules for these diseases.

Development of a next-generation endogenous OCT4 inducer and its anti-aging effect in vivo

The identification of small molecules capable of replacing transcription factors has been a longstanding challenge in the generation of human chemically induced pluripotent stem cells (iPSCs). Recent studies have shown that ectopic expression of OCT4, one of the master pluripotency regulators, compromised the developmental potential of resulting iPSCs, This highlights the importance of finding endogenous OCT4 inducers for the generation of clinical-grade human iPSCs. Through a cell-based high throughput screen, we have discovered several new OCT4-inducing compounds (O4Is). In this work, we prepared metabolically stable analogues, including O4I4, which activate endogenous OCT4 and associated signaling pathways in various cell lines. By combining these with a transcription factor cocktail consisting of SOX2, KLF4, MYC, and LIN28 (referred to as "CSKML") we achieved to reprogram human fibroblasts into a stable and authentic pluripotent state without the need for exogenous OCT4. In Caenorhabditis elegans and Drosophila, O4I4 extends lifespan, suggesting the potential application of OCT4-inducing compounds in regenerative medicine and rejuvenation therapy.

A novel cell-based screen identifies chemical entities that reverse the immune-escape phenotype of metastatic tumours.

Genetic and epigenetic events have been implicated in the downregulation of the cellular antigen processing and presentation machinery (APM), which in turn, has been associated with cancer evasion of the immune system. When these essential components are lacking, cancers develop the ability to subvert host immune surveillance allowing cancer cells to become invisible to the immune system and, in turn, promote cancer metastasis. Here we describe and validate the first high-throughput cell-based screening assay to identify chemical extracts and unique chemical entities that reverse the downregulation of APM components in cell lines derived from metastatic tumours. Through the screening of a library of 480 marine invertebrate extracts followed by bioassay-guided fractionation, curcuphenol, a common sesquiterpene phenol derived from turmeric, was identified as the active compound of one of the extracts. We demonstrate that curcuphenol induces the expression of the APM components, TAP-1 and MHC-I molecules, in cell lines derived from both metastatic prostate and lung carcinomas. Turmeric and curcumins that contain curcuphenol have long been utilized not only as a spice in the preparation of food, but also in traditional medicines for treating cancers. The remarkable discovery that a common component of spices can increase the expression of APM components in metastatic tumour cells and, therefore reverse immune-escape mechanisms, provides a rationale for the development of foods and advanced nutraceuticals as therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells

Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate d-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses d-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.