Homogeneous Time-resolved Fluorescence Assay Research Articles

N-methylmorpholine incorporation into the structure of biphenyl leads to the bioactive inhibitor of PD-1/PD-L1 interaction

We present new small-molecular probes targeting the human PD-L1 protein. The molecules were designed by incorporating a newly discovered N-methylmorpholine substituent into a known biphenyl-based structure. Four prototype derivatives of 4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carbonitrile (STD4), comprising a morpholine substituent fused with a biphenyl core at different orientations were first verified for their potential binding to PD-L1 using the molecular docking method. A more favorable 7-phenyl derivative of STD4 was then equipped with an amide bond, pyridine, and either a tris(hydroxymethyl)aminomethane or serinol tail leading to two final molecules. Among them, compound 1c showed activity in three bioassays, i.e., the homogenous time-resolved fluorescence (HTRF) assay, immune checkpoint blockade (ICB) assay, and T-cell activation (TCA) assay. Our work shows that morpholine can substitute for dioxane and becomes a promising component in PD-L1-targeting molecules. This finding unlocks new avenues for optimizing PD-L1-targeting compounds, presenting exciting prospects for future developments in this field.

Design, synthesis, pharmacological evaluation, and computational study of benzo[d] isothiazol-based small molecule inhibitors targeting PD-1/PD-l1 interaction

Blockade of the programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is an attractive strategy for immunotherapy, but the clinical application of small molecule PD-1/PD-L1 inhibitors remains unclear. In this work, based on BMS-202 and our previous work YLW-106, a series of compounds with benzo[d]isothiazol structure as scaffold were designed and synthesized. Their inhibitory activity against PD-1/PD-L1 interaction was evaluated by a homogeneous time-resolved fluorescence (HTRF) assay. Among them, LLW-018 (27c) exhibited the most potent inhibitory activity with an IC50 value of 2.61 nM. The cellular level assays demonstrated that LLW-018 exhibited low cytotoxicity against Jurkat T and MDA-MB-231. Further cell-based PD-1/PD-L1 blockade bioassays based on PD-1 NFAT-Luc Jurkat cells and PD-L1 TCR Activator CHO cells indicated that LLW-018 could interrupt PD-1/PD-L1 interaction with an IC50 value of 0.88 μM. Multi-computational methods, including molecular docking, molecular dynamics, MM/GBSA, MM/PBSA, Metadynamics, and QM/MM MD were utilized on PD-L1 dimer complexes, which revealed the binding modes and dissociation process of LLW-018 and C2-symmetric small molecule inhibitor LCH1307. These results suggested that LLW-018 exhibited promising potency as a PD-1/PD-L1 inhibitor for further investigation.

Design, Synthesis, and Evaluation of 8-(o-Tolyl)quinazoline Derivatives as Small-Molecule PD-1/PD-L1 Antagonists.

Small-molecule inhibitors targeting programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interactions can compensate for the shortcomings of antibody-based inhibitors and have attracted considerable attention, some of which have already entered clinical trials. Herein, based on our previous study on small-molecule PD-L1 inhibitors, we reported a series of 8-(o-tolyl)quinazoline derivatives by the skeleton merging strategy. Homogenous time-resolved fluorescence (HTRF) assay against PD-1/PD-L1 interaction identified compound A5, which showed the most potent inhibition with an IC50 value of 23.78 nM. Meanwhile, based on the results of HTRF assay, the structure-activity relationships (SARs) of the tail were focused on. Cell-based PD-1/PD-L1 blockade assay further revealed that A5 significantly blocked the PD-1/PD-L1 interaction at 1.1 μM in the co-culture system of Jurkat-NFAT-PD-1 cells and Hep3B-OS8-hPD-L1 cells with no significant cytotoxicity on Jurkat cells. Moreover, the proposed binding mode of A5 was investigated by a docking analysis. These results indicate that compound A5 is a promising lead compound that deserves further investigation.

Use of in vitro methods combined with in silico analysis to identify potential skin sensitizers in the Tox21 10K compound library.

Introduction: Skin sensitization, which leads to allergic contact dermatitis, is a key toxicological endpoint with high occupational and consumer prevalence. This study optimized several in vitro assays listed in OECD skin sensitization test guidelines for use on a quantitative high-throughput screening (qHTS) platform and performed in silico model predictions to assess the skin sensitization potential of prioritized compounds from the Tox21 10K compound library. Methods: First, we screened the entire Tox21 10K compound library using a qHTS KeratinoSensTM (KS) assay and built a quantitative structure-activity relationship (QSAR) model based on the KS results. From the qHTS KS screening results, we prioritized 288 compounds to cover a wide range of structural chemotypes and tested them in the solid phase extraction-tandem mass spectrometry (SPE-MS/MS) direct peptide reactivity assay (DPRA), IL-8 homogeneous time-resolved fluorescence (HTRF) assay, CD86 and CD54 surface expression in THP1 cells, and predicted in silico sensitization potential using the OECD QSAR Toolbox (v4.5). Results: Interpreting tiered qHTS datasets using a defined approach showed the effectiveness and efficiency of in vitro methods. We selected structural chemotypes to present this diverse chemical collection and to explore previously unidentified structural contributions to sensitization potential. Discussion: Here, we provide a skin sensitization dataset of unprecedented size, along with associated tools, and analysis designed to support chemical assessments.

Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

A combined approach of structure-based virtual screening and NMR to interrupt the PD-1/PD-L1 axis: Biphenyl-benzimidazole containing compounds as novel PD-L1 inhibitors.

Immunotherapy has emerged as a game-changing approach for cancer treatment. Althoughmonoclonal antibodies (mAbs) targeting the programmed cell death protein 1/programmed cell death protein 1 ligand 1 (PD-1/PD-L1) axis have entered the market revolutionizing the treatment landscape of many cancer types, small molecules, although presenting several advantages including the possibility of oral administration and/or reduced costs, struggled to enter in clinical trials, suffering of water insolubility and/or inadequate potency compared with mAbs. Thus, the search for novel scaffolds for both the design of effective small molecules and possible synergistic strategies is an ongoing field of interest. In an attempt to find novel chemotypes, a virtual screening approach was employed, resulting in the identification of new chemical entities with a certain binding capability, the most versatile of which was the benzimidazole-containing compound 10. Through rational design, a small library of its derivatives was synthesized and evaluated. The homogeneous time-resolved fluorescence (HTRF) assay revealed that compound 17 shows the most potent inhibitory activity (IC50 ) in the submicromolar range and notably, differently from the major part of PD-L1 inhibitors, exhibits satisfactory water solubility properties. These findings highlight the potential of benzimidazole-based compounds as novel promising candidates for PD-L1 inhibition.

Solubilizer Tag Effect on PD-L1/Inhibitor Binding Properties for m-Terphenyl Derivatives.

Although heavily studied, the subject of anti-PD-L1 small-molecule inhibitors is still elusive. Here we present a systematic overview of the principles behind successful anti-PD-L1 small-molecule inhibitor design on the example of the m-terphenyl scaffold, with a particular focus on the neglected influence of the solubilizer tag on the overall affinity toward PD-L1. The inhibitor developed according to the proposed guidelines was characterized through its potency in blocking PD-1/PD-L1 complex formation in homogeneous time-resolved fluorescence and cell-based assays. The affinity is also explained based on the crystal structure of the inhibitor itself and its costructure with PD-L1 as well as a molecular modeling study. Our results structuralize the knowledge related to the strong pharmacophore feature of the m-terphenyl scaffold preferential geometry and the more complex role of the solubilizer tag in PD-L1 homodimer stabilization.

Small-Molecule U2 Auxiliary Factor Homology Motif (UHM) Domain Inhibitors Cause Splicing Pattern Changes in U2AF1 Mutant Leukemia Cells and Induce Sub-G1 Cell Cycle Arrest

Mutations in factors involved in the spliceosomal pathway have emerged to be an important contributor to the development of myeloid neoplasms including MDS and secondary AML (sAML). A set of frequently mutated genes encoding RNA splicing factors including SF3B1, U2AF1, SRSF2, and ZRSR2 or haploinsufficiency of LUC7L2 have beenreported, but pharmacological intervention targeting these genes remains limited and under-developed. Combination of mutations of these splicing factors are rarely found in patients indicating that multiple defects in a spliceosome pathway may be deleterious. Heterozygous mutation in U2AF1 found in MDS leads to the acquisition of the neomorphic mutant U2AF1 (U2AF1 wt) that relies on wild-type U2AF1 (U2AF1 wt) to survive and proliferate. To support our hypothesis, we undertook a discovery campaign to identify small-molecule compounds targeting U2AF1. By recognizing the challenge of discovering compounds selectively targeting the mutant zinc-finger domains in U2AF1 found clinically, we devised a tractable intervention strategy to inhibit the UHM domain of U2AF1 (U2AF1-UHM). U2AF1-UHM binds with U2AF2 to form a protein complex (U2AF1/U2AF2) that recognizes the 3' splice site in mRNA processing. U2AF1-UHM inhibitors may decrease the binding between U2AF1 and U2AF2 and abrogate U2AF1/U2AF2 functions to change expression patterns of protein isoforms. To identify hit compounds for development, we performed screening of ~3000 fragment molecules against U2AF1-UHM using the thermal shift assay. Two of the top hits were derivatives of the same chemical scaffold. We verified that both hits inhibited the binding of U2AF1-UHM and U2AF2-ULM (U2AF ligand motifs) in our Homogenous Time-Resolved Fluorescence (HTRF) assay. We next evaluated the top hit, SF-1-8, in our K562-U2AF1 S34F mutant cell line and obtained an IC50 at the micromolar range. In contrast, SF-1-8 had no activity in K562-U2AF1 wt cell line and bone marrow cells obtained from healthy individuals. Based on SF-1-8, we performed chemical modifications to develop structure-activity relationship (SAR) of SF-1-8 and obtained SF-1-50 that was two-fold more potent than SF-1-8. To assess the selectivity of SF-1-8 and analogs to other UHM containing proteins, we further determined the IC50 values of our SF-1-8 analogs to RBM39-UHM, SPF45-UHM, and PUF60-UHM to construct the selectivity profiles of our compounds. The selectivity profiles of SF-1-8 and SF-1-50 showed they were selectively more effective to U2AF1-UHM than RBM39-UHM, SPF45-UHM, and PUF60-UHM. We then studied pathways impacted by these inhibitors in K562-U2AF1 S34F cells by performing RNA-seq in K562-U2AF1 S34F cells treated with SF-1-8 at 5 uM and control. We found that 36 and 63 genes were significantly (p <0.002) up- and down-regulated respectively by SF-1-8 in K562-U2AF1 S34F cells. Downregulated genes included CBL, CBLL1, and a subset of collagen genes ( COL1, COL3, COL5) and upregulated genes included ATF3 and BCL2. When analyzing the transcript changes affected by SF-1-8, we identified substantial protein isoform changes in genes involved with proteosome, endocytosis, apoptosis, extracellular matrix/cell adhesion, histones, and stress response. Primary effects from changes of protein isoform patterns ameliorated the restoration of the trans-Golgi network ( ERGIC3, COPB2), secretory pathway ( RUSC1, AP4E1) and impairment of clathrin mediated endocytosis and endosome-lysosome transport ( CBL, PICALM, VAMP7, ASAP1). NKM-1 cell line was previously characterized to carry U2AF1 mutation. We also found SF-1-50 caused accumulation of sub-G1 cells in NKM-1 cells in a dose-dependent manner. In summary, SF-1-8 caused disruption of extra- and intracellular protein transport in K562-U2AF1 S34F cells and represented a new class of small-molecule inhibitors to target U2AF1-UHM. Further optimization of this class of compounds will allow us to develop effective chemical probes for study in the U2af1 murine models and assess the potential of U2AF1 as a therapeutic target in MDS and sAML.

A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

Discovery of conformationally constrained c-Abl inhibitors with potential neuroprotective effects against Parkinson’s disease

Abelson tyrosine kinase (c-Abl) is involved in various biological processes in neurodegenerative diseases and is an attractive target for anti-PD (Parkinson's disease) drug discovery. Based on our previous work, we designed several novel c-Abl inhibitors through a conformational constrained strategy and evaluated their pharmacological activities. Among them, compound A6 exhibited superior inhibitory activity against c-Abl than nilotinib in the homogenous time-resolved fluorescence (HTRF) assay. Furthermore, A6 displayed higher neuroprotective effects against SH-SY5Y cell death induced by MPP+ and lower cytotoxicity than that of nilotinib. Molecular modeling revealed that the 1H-pyrrolo[2,3-B]pyridine ring may contribute to the high affinity of A6 for binding to c-Abl. Collectively, these results suggest that A6 deserves further investigation as a c-Abl inhibitor for neurodegenerative disorders.

Target immobilization on glass microfiber membranes as a label-free strategy for hit identification.

The discovery of novel chemical entities targeting G protein-coupled receptors (GPCRs) is usually guided by their receptor affinity. However, traditional affinity assay methods and hit identification procedures are usually laborious and expensive. In this work, the type-2 vasopressin receptor (V2R) was chosen as a prototypical GPCR. Membrane fragments from cells highly expressing SNAP-V2R were immobilized on the surface of a glass microfiber (GMF) coated with O6-benzylguanine (BG). This was achieved by transferring the benzyl group of BG to the active site of the SNAP-tag through a nucleophilic substitution reaction. As a result, a biofilm called SNAP-V2R@GMF-BG was produced that showed good specificity and stability. The adsorption ratio for each V2R ligand treated with SNAP-V2R@GMF-BG was determined by HPLC and exhibited a good linear correlation with the Ki value determined by displacement assays. Furthermore, a Ki prediction assay was performed by comparing the data with that generated by a homogeneous time-resolved fluorescence (HTRF) assay. SNAP-V2R@GMF-BG was also used to screen hit compounds from natural products. After SNAP-V2R@GMF-BG was incubated with the total extract, the ligand that binds to V2R could be separated and subjected to LC‒MS analysis for identification. Baicalein was screened from Clerodendranthus spicatus and verified as a potential V2R antagonist. This V2R-immobilized GMF platform can help determine the affinity of V2R-binding hit compounds and screen the compounds efficiently and accurately.

Novel quinazoline-1,2,3-triazole hybrids with anticancer and MET kinase targeting properties

Oncogenic activation of receptor tyrosine kinases (RTKs) such as MET is associated with cancer initiation and progression. We designed and synthesized a new series of quinazoline derivatives bearing 1,2,3-triazole moiety as targeted anticancer agents. The MET inhibitory effect of synthesized compounds was assessed by homogeneous time-resolved fluorescence (HTRF) assay and western blot analysis. Sulforhodamine B assay was conducted to examine the antiproliferative effects of synthetic compounds against 6 cancer cell lines from different origins including MET-dependent AsPC-1, EBC-1 and MKN-45 cells and also Mia-Paca-2, HT-29 and K562 cells. The growth inhibitory effect of compounds in a three-dimensional spheroid culture was examined by acid phosphatase (APH) assay, while apoptosis induction was evaluated by Annexin V/propidium iodide method. Compound 8c bearing p-methyl benzyl moiety on the triazole ring exhibited the highest MET inhibitory capacity among tested agents that was further confirmed by western blot findings. Derivatives 8c and 8h exhibited considerable antiproliferative effects against all tested cell lines, with more inhibitory effects against MET-positive cells with IC50 values as low as 6.1 μM. These two agents also significantly suppressed cell growth in spheroid cultures and induced apoptosis in MET overexpressing AsPC-1 cells. Moreover, among a panel of 24 major oncogenic kinases, the PDGFRA kinase was identified as a target of 8c and 8h compounds. The docking study results of compounds 8c and 8h were in agreement with experimental findings. The results of the present study suggest that quinazoline derivatives bearing 1,2,3-triazole moiety may represent promising targeted anticancer agents.

Evaluation of a high-throughput H295R homogenous time resolved fluorescence assay for androgen and estrogen steroidogenesis screening

The H295R test guideline assay evaluates the effect of test substances on synthesis of 17β-estradiol (E2) and testosterone (T). The objective of this study was to leverage commercial immunoassay technology to develop a more efficient H295R assay to measure E2 and T levels in 384-well format. The resulting Homogenous Time Resolved Fluorescence assay platform (H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA guideline 890.1200 aromatase assay, and azole fungicides active in the HT-H295R assay. Quality control performance criteria were met for all conditions except E2 synthesis inhibition where low basal hormone synthesis was observed. Five proficiency chemicals were active for both the E2 and T endpoints, consistent with guideline classifications. Of the nine OECD core reference chemicals, 9/9 were concordant with outcomes for E2 and 7/9 for T. Likewise, 9/13 and 11/13 OECD supplemental chemicals were concordant with anticipated effects for E2 and T, respectively. Of the 10 azole fungicides screened, 7/10 for E2 and 8/10 for T exhibited concordant outcomes for inhibition. Generally, all active chemicals in the training set demonstrated equivalent or greater potency in the H295R-HTRF assay, supporting the sensitivity of the platform. The adaptation of HTRF technology to the H295R model provides an efficient way to evaluate E2 and T modulators in accordance with guideline specifications.

Design, Synthesis, and Antitumor Activity Evaluation of 2-Arylmethoxy-4-(2,2'-dihalogen-substituted biphenyl-3-ylmethoxy) Benzylamine Derivatives as Potent PD-1/PD-L1 Inhibitors.

Novel 2-arylmethoxy-4-(2,2'-dihalogen-substituted biphenyl-3-ylmethoxy) benzylamine derivatives were designed, synthesized, and evaluated in vitro and in vivo against cancers as PD-1/PD-L1 inhibitors. Through the computer-aided structural optimization and the homogeneous time-resolved fluorescence (HTRF) assay, compound A56 was found to most strongly block the PD-1/PD-L1 interaction with an IC50 value of 2.4 ± 0.8 nM and showed the most potent activity. 1H NMR titration results indicated that A56 can tightly bind to the PD-L1 protein with KD < 1 μM. The X-ray diffraction data for the cocrystal structure of the A56/PD-L1 complex (3.5 Å) deciphered a novel binding mode in detail, which can account for its most potent inhibitory activity. Cell-based assays further demonstrated the strong ability of A56 as an hPD-1/hPD-L1 blocker. Especially in an hPD-L1 MC38 humanized mouse model, A56 significantly inhibited tumor growth without obvious toxicity, with a TGI rate of 55.20% (50 mg/kg, i.g.). In conclusion, A56 is a promising clinical candidate worthy of further development.

Discovery of novel resorcinol biphenyl ether-based macrocyclic small molecules as PD-1/PD-L1 inhibitors with favorable pharmacokinetics for cancer immunotherapy

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) is one of the most promising immune checkpoints (ICs) in tumor immunology and has been actively pursued as a target for anticancer drug discovery. Based on our previous research in small molecule PD-1/PD-L1 modulators, we designed and synthesized a series of resorcinol biphenyl ether-bearing macrocyclic compounds and evaluated their anti-PD-1/PD-L1 activities. Among them, compound 8d exhibited the highest inhibitory activity against PD-1/PD-L1 interaction with IC50 of 259.7 nM in the homogenous time-resolved fluorescence (HTRF) assay. In addition, 8d displayed in vitro immunomodulatory effects by promoting HepG2 cell death in a HepG2/Jurkat cell co-culture model. Furthermore, 8d effectively inhibited tumor growth (TGI = 74.6% at 40 mg/kg) in a melanoma tumor model in mice without causing obvious toxicity. Moreover, 8d exhibited favorable pharmacokinetics [e.g. high stability, reasonable half-life, and good oral bioavailability (F = 21.5%)]. Finally, molecular modeling studies showed that 8d bound to PD-L1 with high affinity. These results suggest that 8d may serve as a starting point for further development of macrocyclic small molecule-based PD-1/PD-L1 inhibitors for cancer treatment.

Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF)

O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC50 value of 6.56 ± 1.69 μM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (Kd) of approximately 1.7 μM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.

1750-P: Differential Impacts of Two Novel HNF1A Variants Associated with Familial Young-Onset Diabetes on Protein Function and Insulin Secretion In Vitro

Introduction: Mutations in the HNF1A are a cause of monogenic diabetes and are usually dominantly inherited. We report in vitro and clinical studies on two novel HNF1A variants to determine pathogenicity: a recessive (homozygous) HNF1A variant (p.A251T) and a dominant HNF1A variant (p.S19L). We also investigated the mechanisms by which the former variant affects insulin secretion using beta cells derived from induced pluripotent stem cells (iPSCs). Methods: Nuclear localization, DNA binding, and transactivation activity were examined using standard techniques. iPSC lines from proband carrying the p.A251T variant were differentiated into beta-like cells according to Sui et al, CPHG, 2018. Ca2+ dynamics were monitored with Cal-520, and insulin secretion was measured by homogeneous time-resolved fluorescence assay compared to wild-type HNF1A (WT). Results: HNF1A p.A251T showed a mild (10.4% ± 7.3, p= 0.16) decrease in transactivation activity compared with WT, and decreased (48% of WT, p=0.12) DNA binding. These reductions were significant in p.S19L (DNA binding 57% of WT, p=0.049). Significant impaired nuclear uptake (63% vs 90% cells for WT, p&amp;lt;0.0001) was only apparent for p.S19L. Glucose-stimulated insulin secretion was decreased in A251T iPSCs compared with WT (0.29% vs. 0.46% of total insulin). In contrast, glucose-induced Ca2+ increases were increased in mutant cells (arbitrary units: 7.39 ± 1.23, mean +/− SD, n=725 cells vs. 4.58 ± 0.77, n=724, p= &amp;lt; 0.001) Conclusion: We demonstrate the value of combining multiple in vitro functional studies to decipher novel HNF1A variant pathogenicity. The recessive p.A251T variant has relatively mild effects on protein function compared to a dominant p.S19L variant. However, when studying proband iPSC derived beta-cell A251T impairs glucose-stimulated insulin secretion despite preserved Ca2+ entry, suggesting a novel mechanism of action not captured by standard tests of HNF1A function. Disclosure I.Cherkaoui: None. G.A.Rutter: Consultant; Sun Pharmaceutical Industries Ltd. Q.Du: None. C.Dion: None. H.Leitch: None. P.L.Chabosseau: None. D.M.Egli: None. D.Sachedina: None. J.Wastin: None. S.Misra: Research Support; Dexcom, Inc. Funding Diabetes UK

Structure-Based Design of Inhibitors of the m6A-RNA Writer Enzyme METTL3.

Methyltransferase-like 3 (METTL3) and METTL14 form a heterodimeric complex that catalyzes the most abundant internal mRNA modification, N6-methyladenosine (m6A). METTL3 is the catalytic subunit that binds the co-substrate S-adenosyl methionine (SAM), while METTL14 is involved in mRNA binding. The m6A modification provides post-transcriptional level control over gene expression as it affects almost all stages of the mRNA life cycle, including splicing, nuclear export, translation, and decay. There is increasing evidence for an oncogenic role of METTL3 in acute myeloid leukemia. Here, we use structural and dynamic details of the catalytic subunit METTL3 for developing small-molecule inhibitors that compete with SAM. Starting from a hit identified by high-throughput docking, protein crystallography and molecular dynamics simulations were employed to guide the optimization of inhibitory activity. The potency was successfully improved by 8000-fold as measured by a homogeneous time-resolved fluorescence assay. The optimized compound is selective against the off-targets RNA methyltransferases METTL1 and METTL16.

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

Abstract P6-10-12: Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening

Abstract Background: Activating somatic ESR1 mutations Y537S and D538G occur more frequently in endocrine therapy-resistant metastatic breast cancer, which is associated with an aggressive phenotype and poor survival in breast cancer patients. These gain of function mutant receptors are constitutively active and allow resistance to first-line endocrine therapies. Therefore, the development of next-generation small molecule drugs targeting mutant estrogen receptor (ER) is an important priority. Here, we searched the small molecule inhibitors for Y537S and D538D ER mutants using DNA-encoded chemical library screening. Methods: Wild type (WT) and mutant ER ligand binding domain (LBD) proteins were expressed in E. coli. The soluble proteins were purified by Ni-NTA chromatography followed by anion exchange and size exclusion chromatography. Homogeneous time-resolved fluorescence (HTRF) and fluorescent polarization (FP) assays were performed in these purified proteins. We employed a DNA-encoded chemical library affinity selection using our in-house collection of 6 billion compounds. Hit compounds were resynthesized and validated in biochemical assays. Finally, we have performed functional studies in CRISPR-Cas9 knock-in of Y537S and D538G mutant MCF-7 breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD of WT, Y537S, and D538G proteins. To test whether the purified WT and mutant proteins are active, HTRF and FP assays were performed in the presence and absence of estradiol and 4OH tamoxifen. Steroid receptor coactivator 3 (SRC3) peptide binding to the WT ER protein occurred only in the presence of estradiol. However, Y537S and D538G proteins are recruited by the SRC3 peptide in the absence of estradiol, indicating that these mutants are constitutively active and bind to SRC3. Furthermore, an in vitro biochemical FP assay was also established for WT and mutants in the presence of estradiol and 4OH tamoxifen. The screen of our multibillion small molecule collection of DNA-encoded chemical libraries identified several hits in WT and mutant ER. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. We have identified that the compounds, CDD-1272 and CDD-1274, are active in HTRF and FP assays. Furthermore, these compounds inhibit WT and mutant cell growth in the presence of estradiol. More importantly, CDD-1274 degrades ER mutant and cyclin D1 proteins. In addition, CDD-1274 induced p21 protein expression in WT and mutant cells. Conclusions: We have identified potent novel ER mutant binders by using our DNA-encoded chemical library platform. Our compounds are active in biochemical and ER mutant cell lines, suggesting these molecules are potential chemical probes to explore in in vivo models of breast cancer. Support: NIH/NCI R03 CA259664 and CPRIT RP220524 to MP. Citation Format: Murugesan Palaniappan, Kurt M. Bohren, Yong Wang, Damian W. Young, Suzanne A. Fuqua, Martin M. Matzuk. Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-12.