High-throughput Screening Of Small Molecules Research Articles

Abstract 4990: Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis

Abstract Monoclonal antibodies such as rituximab exert targeted tumor cell lysis via complement dependent cytotoxicity (CDC). These antibodies are often combined with chemotherapy in clinical treatment protocols. However, the impact of chemotherapy on cell-intrinsic pathways that regulate antibody-mediated CDC is not known. Through a phage display screen, we identified that genotoxic stress caused upregulation of a membrane complement regulatory protein (mCRP), CD46, prompting further investigation into its implications and mechanism. Using cell lines from diffuse large B cell lymphoma (DLBCL) as our model, we confirmed that genotoxic chemotherapy upregulates CD46, as well as other mCRPs such as CD55 and CD59. We hypothesized that upregulated mCRPs could impede Rituximab-mediated CDC. We assessed this in-vitro using rituximab and complement human serum. Aligned with our hypothesis, chemotherapy treatment of DLBCL cells significantly impeded rituximab-induced CDC. Using blocking antibodies to each mCRP, reversal of the effect of chemotherapy on CDC only occurred after co-treatment with a CD59 blocking antibody, suggesting that CD59 is a key player for chemotherapy-invoked resistance to CDC. To further elucidate the underlying cellular pathway, we performed a high-throughput small molecule screen using rituximab-induced CDC as a readout, with or without cotreatment of a genotoxic chemotherapeutic (etoposide). We identified Chk1 inhibitors as consistent top rescuers. qPCR and flow cytometry revealed that Chk1 inhibition prevented the upregulation of mCRPs transcriptionally. Sp1 is a known transcription factor driving constitutive CD59 expression. Inhibiting Sp1 similarly prevented chemotherapy-induced upregulation of CD59 and resistance to CDC. However, CHIP-qPCR showed no increased binding of total Sp1 protein to the CD59 promoter after chemotherapy. Instead, positive co-immunoprecipitation of pATM and p300 with Sp1 only with etoposide and not with co-treatment of Chk1 inhibitor suggests Chk1-mediated activation of Sp1. Mechanistically, we have found that chemotherapy-activated Chk1 modulates Sp1-mediated CD59 transcription and that the induced upregulation of CD59 is able to prevent antibody-mediated CDC. Overall, these results indicate novel immune regulation by DNA damage response, particularly in the context of complement regulatory proteins. Our results advocate consideration when using simultaneous administration of chemotherapy with CDC-inducing monoclonal antibodies, and suggest Chk1 blockade as a possible therapeutic addition. Citation Format: Allison S. Chan, Patrick W. Jaynes, Nurulhuda B. Mustafa, Akshaya A. Anbuselvan, Charmaine Z. Ong, Anand D. Jeyasekharan. Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis [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 4990.

Abiotic Small Molecule Inhibitors and Activators of the LasR Quorum Sensing Receptor in Pseudomonas aeruginosa with Potencies Comparable or Surpassing N-Acyl Homoserine Lactones.

The opportunistic pathogen Pseudomonas aeruginosa controls almost 10% of its genome, including myriad virulence genes, via a cell-to-cell chemical communication system called quorum sensing (QS). Small molecules that either inhibit or activate QS in P. aeruginosa represent useful research tools to study the role of this signaling pathway in infection and interrogate its viability as an antivirulence target. However, despite active research in this area over the past 20+ years, there are relatively few synthetic compounds known to strongly inhibit or activate QS in P. aeruginosa. Most reported QS modulators in this pathogen are of low potency or have structural liabilities that limit their application in biologically relevant environments such as mimics of the native N-acyl l-homoserine lactone (AHL) signals. Here, we report the results of a high-throughput screen for abiotic small molecules that target LasR, a key QS regulator in P. aeruginosa. We screened a 25,000-compound library and discovered four new structural classes of abiotic LasR modulators. These compounds include antagonists that surpass the potency of all known AHL-type compounds and mimetics thereof, along with an agonist with potency approaching that of LasR's native ligand. The novel structures of this compound set, along with their anticipated robust physicochemical profiles, underscore their potential value as probe molecules to interrogate the roles of QS in this formidable pathogen.

Development of small-molecule Tau-SH3 interaction inhibitors that prevent amyloid-β toxicity and network hyperexcitability

Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-β–induced dysfunction in preclinical models of AD and also prevents amyloid-β–independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain–containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain–containing proteins ameliorates amyloid-β–induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-β–induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

ObjectiveThioesterase superfamily member 1 (Them1) is a long chain acyl-CoA thioesterase comprising two N-terminal HotDog fold enzymatic domains linked to a C-terminal lipid-sensing steroidogenic acute regulatory transfer-related (START) domain, which allosterically modulates enzymatic activity. Them1 is highly expressed in thermogenic adipose tissue, where it functions to suppress energy expenditure by limiting rates of fatty acid oxidation, and is induced markedly in liver in response to high fat feeding, where it suppresses fatty acid oxidation and promotes glucose production. Them1−/− mice are protected against non-alcoholic fatty liver disease (NAFLD), suggesting Them1 as a therapeutic target. MethodsA high-throughput small molecule screen was performed to identify promising inhibitors targeting the fatty acyl-CoA thioesterase activity of purified recombinant Them1.Counter screening was used to determine specificity for Them1 relative to other acyl-CoA thioesterase isoforms. Inhibitor binding and enzyme inhibition were quantified by biophysical and biochemical approaches, respectively. Following structure-based optimization, lead compounds were tested in cell culture. ResultsTwo lead allosteric inhibitors were identified that selectively inhibited Them1 by binding the START domain. In mouse brown adipocytes, these inhibitors promoted fatty acid oxidation, as evidenced by increased oxygen consumption rates. In mouse hepatocytes, they promoted fatty acid oxidation, but also reduced glucose production. ConclusionThem1 inhibitors could prove attractive for the pharmacologic management of NAFLD.

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

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

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

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

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

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

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

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

Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and Crohn's disease. The biological effects of TNF are mediated by binding to TNF receptors, TNF receptor 1 (TNFR1), or TNF receptor 2 (TNFR2), and this coupling makes TNFR1-specific inhibition by small-molecule therapies essential to avoid deleterious side effects. Recently, we engineered a time-resolved fluorescence resonance energy transfer biosensor for high-throughput screening of small molecules that modulate TNFR1 conformational states and identified zafirlukast as a compound that inhibits receptor activation, albeit at low potency. Here, we synthesized 16 analogues of zafirlukast and tested their potency and specificity for TNFR1 signaling. Using cell-based functional assays, we identified three analogues with significantly improved efficacy and potency, each of which induces a conformational change in the receptor (as measured by fluorescence resonance energy transfer (FRET) in cells). The best analogue decreased NF-κB activation by 2.2-fold, IκBα efficiency by 3.3-fold, and relative potency by two orders of magnitude. Importantly, we showed that the analogues do not block TNF binding to TNFR1 and that binding to the receptor's extracellular domain is strongly cooperative. Despite these improvements, the best candidate's maximum inhibition of NF-κB is only 63%, leaving room for further improvements to the zafirlukast scaffold to achieve full inhibition and prove its potential as a therapeutic lead. Interestingly, while we find that the analogues also bind to TNFR2 in vitro, they do not inhibit TNFR2 function in cells or cause any conformational changes upon binding. Thus, these lead compounds should also be used as reagents to study conformational-dependent activation of TNF receptors.

Abstract 6416: Statins inhibit onco-dimerization of 4Ig-B7-H3

Abstract B7-H3 (CD276) is a member of the B7 family of immune regulators, but unlike the immune checkpoint targets that have revolutionized cancer therapy, blockade has resulted in mixed outcomes. B7-H3 has 2Ig and 4Ig isoforms, has no known direct ligand, and many of the physiological functions remain elusive. While protein expression of B7-H3 is differentially expressed on many solid tumors and correlates with poor survival, mechanisms of how B7-H3 elicits a pro-tumorigenic phenotype are still poorly understood, significantly hampering therapeutic efforts for targeting. Using a facile, recombinase-enhanced split-luciferase reporter, we visualized 4Ig-B7-H3 homodimerization in live U2OS cells by bioluminescence imaging and confirmed 4Ig-B7-H3 dimerization using a second non-invasive imaging technique: fluorescence lifetime imaging microscopy (FLIM) where 4Ig-B7-H3 was exogenously re-expressed following knockout of CD276 in HeLa cervical cancer cells. Homodimerization of 4Ig-B7-H3 correlated with enhanced clonogenic cell growth in HeLa, SKOv3, MDA-MB-231, and U2OS cells and tumor growth in vivo for several models. This phenotype was reversed following knockout of B7-H3 and rescued upon lentiviral re-expression. By RPPA and phospho-kinase array analysis, increases in PI3K/AKT signaling, Jak/Stat pathway, and modulators of HIF1α and NF-κβ pathways were observed with B7-H3 expression and homodimerization (p<0.05), all which are known to support increased oncogenic functions, such as increased glycolysis, increased survival and proliferation, as well as cytokine modulation and immune evasion. Next, capitalizing on our bioluminescence-based reporter of 4Ig-B7-H3 homodimerization, we performed a high-throughput small molecule screen with a 5,362 Bioactive compound library to identify modulators of 4Ig-B7-H3 dimerization. Notably, our HTS identified several HMG-CoA reductase inhibitors (statins) as significant inhibitors of B7-H3 dimerization (p<0.01). Treatment with 1 μM mevastatin or atorvastatin calcium inhibited clonogenic growth of HeLa cervical, SKOv3 ovarian and MDA-MB-231 breast cancer cells, which all endogenously express B7-H3. In vivo, treatment with 10 mg/kg of atorvastatin calcium reduced tumor growth and increased mouse survival (p=0.035) of SKOv3-ip tumors and produced functional cures (15-30%) of HeLa or MDA-MB-231 xenografts in nude mice in a B7-H3 tumor expression-dependent manner, proven through knockout and rescue experiments in live cells and animals. Thus, in the context of studies to dissect the tumor-intrinsic mechanism(s) of B7-H3 biochemical functions and activation, we identified a novel, dimerization-dependent oncogenic role for 4Ig-B7-H3 that increased oncogenic intracellular signaling activation and was modulated by statin treatment. This may provide a molecular mechanism for the statin-mediated immune modulation and enhanced cancer survival clinically reported for several tumor types. Citation Format: Margie Nicole Sutton, Sarah E. Glazer, Riccardo Muzzioli, Ping Yang, Seth T. Gammon, David Piwnica-Worms. Statins inhibit onco-dimerization of 4Ig-B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6416.

Pharmacological boosting of cGAS activation sensitizes chemotherapy by enhancing antitumor immunity

Enhancing chemosensitivity is one of the largest unmet medical needs in cancer therapy. Cyclic GMP-AMP synthase (cGAS) connects genome instability caused by platinum-based chemotherapeutics to type I interferon (IFN) response. Here, by using a high-throughput small-molecule microarray-based screening of cGAS interacting compounds, we identify brivanib, known as a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor, as a cGAS modulator. Brivanib markedly enhances cGAS-mediated type I IFN response in tumor cells treated with platinum. Mechanistically, brivanib directly targets cGAS and enhances its DNA binding affinity. Importantly, brivanib synergizes with cisplatin in tumor control by boosting CD8+ Tcell response in a tumor-intrinsic cGAS-dependent manner, which is further validated by a patient-derived tumor-like cell clusters model. Taken together, our findings identify cGAS as an unprecedented target of brivanib and provide a rationale for the combination of brivanib with platinum-based chemotherapeutics in cancer treatment.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.

New Insights into the Physiology of Iron Transport: An Interdisciplinary Approach

The TransCure project entitled 'Iron Transporters DMT1 and FPN1' took an interdisciplinary approach combining structural biology, chemistry and physiology to gain new insights into iron transport. Proteins studied included Divalent Metal Transporter 1 (DMT1, SLC11A2), enabling the import of Fe2+ into the cytoplasm, and the iron efflux transporter Ferroportin (FPN1, SLC40A1). The physiology and pathophysiology, and the mechanisms underlying iron transport in the gut, across the placenta and in bone were investigated. Small molecule high-throughput screening was used to identify improved modulators of DMT1. The characterization of DMT1 inhibitors have provided first detailed insights into the pharmacology of a human iron transport protein. In placental physiology, the identification of the expressional and functional alterations and underlying mechanisms in trophoblast cells clarified the association between placental iron transport by DMT1/FPN1 and gestational diabetes mellitus. In bone, iron metabolism was found to differ between cells of the monocyte/ macrophage lineages, including osteoclasts. Osteoclast development and activity depended on exogenous iron, the expression of high levels of the transferrin receptor (TFR) and low levels of FPN1 suggesting the expression of an “iron storage” phenotype by these cells. The principles and main findings of the TransCure studies on transmembrane iron transport physiology are summarized in this review.

Transcriptional profiling of neuronal ion channels in dorsal root ganglion-derived immortal cell line (F-11) under different culture conditions.

Pathological pain is a prevalent condition that affects majority of adults with a variety of underlying disease conditions. Current available pharmacological pain treatments have several negative and potentially life-threatening side effects associated with their long-term use. Due to the heterogeneity of pain perception and the diversity of neuronal mechanisms that contribute to pain, high-throughput screening of small molecules that may have underlying analgesic properties is essential for identifying new analgesic treatments that are both effective and safe. The F-11 hybrid immortalized cell line is one of the currently available dorsal root ganglion (DRG) cell lines used for drug screening. While F-11 cells are commonly used as analogs to primary DRG sensory neurons, they differ significantly in physiological properties. The present study investigated the impact of differentiation protocols on the expression of mature neuron ion channels and receptors in F-11 cells. Using a customized gene array of more than eighty neuronal ion channels and receptors including voltage-gated ion channels, transient receptor potential channels, and cannabinoid receptors, we assessed the following groups: control F-11 cells; F-11 cells cultured under different culture conditions, and murine DRG tissue. The expression profiles of majority of the investigated ion channels and receptors in F-11 cells were found to be lower compared to primary mouse DRG neurons. F-11 cells cultured under low serum (LSM) conditions had increased expression of several investigated targets including voltage-gated ion channels and cannabinoid receptors when compared to control F-11 cells. The study showed that the culture conditions significantly modulated the transcriptional expression of studied ion channels and receptors, and that long-term culture (21 days) may adversely affect the expression of many of the studied targets.

Leveraging patient derived models of FGFR2 fusion positive intrahepatic cholangiocarcinoma to identify synergistic therapies

Intrahepatic cholangiocarcinoma (ICC) remains a deadly malignancy lacking systemic therapies for advanced disease. Recent advancements include selective FGFR1–3 inhibitors for the 15% of ICC patients harboring fusions, although survival is limited by poor response and resistance. Herein we report generation of a patient-derived FGFR2 fusion-positive ICC model system consisting of a cell line, organoid, and xenograft, which have undergone complete histologic, genomic, and phenotypic characterization, including testing standard-of-care systemic therapies. Using these FGFR2 fusion-positive ICC models, we conducted an unbiased high-throughput small molecule screen to prioritize combination strategies with FGFR inhibition, from which HDAC inhibition together with pemigatinib was validated in vitro and in vivo as a synergistic therapy for ICC. Additionally, we demonstrate broad utility of the FGFR/HDAC combination for other FGFR fusion-positive solid tumors. These data are directly translatable and justify early phase trials to establish dosing, safety, and therapeutic efficacy of this synergistic combination.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

Identification of small-molecule inhibitors of the DNA repair proteins RuvAB from Pseudomonas aeruginosa

The Holliday junction (HJ) branch migrator RuvAB complex plays a fundamental role during homologous recombination and DNA damage repair, and therefore, is an attractive target for the treatment of bacterial pathogens. Pseudomonas aeruginosa (P. aeruginosa, Pa) is one of the most common clinical opportunistic bacterial pathogens, which can cause a series of life-threatening acute or chronic infections. Here, we performed a high throughput small-molecule screening targeting PaRuvAB using the FRET-based HJ branch migration assay. We identified that corilagin, bardoxolone methyl (BM) and 10-(6ˊ-plastoquinonyl) decyltriphenylphosphonium (SKQ1) could efficiently inhibit the branch migration activity of PaRuvAB, with IC50 values of 0.40 ± 0.04 μM, 0.38 ± 0.05 μM and 4.64 ± 0.27 μM, respectively. Further biochemical and molecular docking analyses demonstrated that corilagin directly bound to PaRuvB at the ATPase domain, and thus prevented ATP hydrolysis. In contrast, BM and SKQ1 acted through blocking the interactions between PaRuvA and HJ DNA. Finally, these compounds were shown to increase the susceptibility of P. aeruginosa to UV-C irradiation. Our work, for the first time, reports the small-molecule inhibitors of RuvA and RuvB from any species, providing valuable chemical tools to dissect the functional role of each individual protein in vivo.

A novel high-throughput screening strategy for targeting alpha-synuclein and other long-lived proteins.

Small-molecule high-throughput screening (HTS) campaigns have frequently been used to identify lead molecules that can alter expression of disease-relevant proteins in cell-based assays. However, most cell-based HTS assays require short compound exposure periods to avoid toxicity and ensure that compounds are stable in media for the duration of the exposure. This limits the ability of HTS assays to detect inhibitors of the synthesis of target proteins with long half-lives, which can often exceed the exposure times utilized in most HTS campaigns. One such target is alpha-synuclein (α-syn)-a protein well-known for its pathological aggregation in Parkinson's Disease (PD) and other forms of neurodegeneration known collectively as synucleinopathies. Here, we report the development of an HTS assay using a CRISPR-engineered neuroblastoma cell line expressing a destabilized luciferase reporter inserted at the end of the coding region of the SNCA locus. The resultant destabilized fusion protein exhibited a significant reduction in half-life compared to the endogenous, unmodified α-syn protein, and accurately reported reductions in α-syn levels due to known protein translation inhibitors and specific α-syn siRNAs. The robustness and utility of this approach was shown by using the resulting cell line (dsLuc-Syn) to screen a focused library of 3,192 compounds for reduction of α-syn. These data demonstrate the general utility of converting endogenous loci into destabilized reporter genes capable of identifying inhibitors of gene expression of highly stable proteins even in short-term assays.

Nonretinoid chaperones improve rhodopsin homeostasis in a mouse model of retinitis pigmentosa.

Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.