Homogeneous Time-resolved Fluorescence Assay Research Articles

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<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= < 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.

8-Aminoguanine and Its Actions on Renal Excretory Function.

The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Development of a robust HTRF assay with USP7 full length protein expressed in E. coli prokaryotic system for the identification of USP7 inhibitors

Deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) is a promising therapeutic target. Several USP7 inhibitors accommodated in the catalytic triad of USP7 have been reported with the aid of high-throughput screening (HTS) methods using USP7 catalytic domain truncation. However, the drawbacks of previously reported biochemical cleavage assays, including poor stability, fluorescence interference, time-consuming, expensive, more importantly the selectivity issue, have challenged the USP7-targeted drug discovery. In this work, we demonstrated the functional heterogeneity and essential role of different structural elements in the USP7 full activation, highlighting the necessity of USP7 full length in drug discovery. Apart from reported two pockets in the catalytic triad, five additional ligandable pockets were predicted based on the proposed USP7 full length models by AlphaFold and homology modelling. A reliable homogeneous time-resolved fluorescence (HTRF) HTS method was established based on the cleavage mechanism of USP7 towards the ubiquitin precursor UBA10. The USP7 full length protein was successfully expressed in the relatively cost-effective E. coli prokaryotic system and used to simulate the auto-activated USP7 in nature. Via screening our in-house library (∼ 1500 compounds), 19 hit compounds with >20% of inhibition rate were identified for further optimization. This assay will enrich the toolbox for the identification of highly potent and selective USP7 inhibitors for clinical use.

Study on endogenous inhibitors against PD-L1: cAMP as a potential candidate

The discovery of new anti-cancer drugs targeting the PD-1/PD-L1 pathway has been a research hotspot in recent years. In this study, biological affinity ultrafiltration (BAU), UPLC-HRMS, molecular dynamic (MD) simulations and molecular docking methods were applied to search for endogenous active compounds that can inhibit the binding of PD-L1 to PD-1. We screened dozens of potential cancer related endogenous compounds. Surprisingly, cyclic adenosine monophosphate (cAMP) was found to have a direct inhibitory effect on the PD-1/PD-L1 binding with an in vitro IC50 value of about 36.4 ± 9.3 μM determined by homogeneous time-resolved fluorescence (HTRF) assay. cAMP could recover the proliferation of Jurkat T cells co-cultured with DU-145 cells and may suppress PD-L1 expression of DU-145 cells. cAMP was demonstrated to bind and induce PD-L1 dimerization by FRET assay, and also predicted by MD simulations and molecular docking. The finding of cAMP as a potential inhibitor directly targeting the PD-1/PD-L1 interaction could advance our understanding of the activity of endogenous compounds regulating PD-L1.

Early detection of exon 1 huntingtin aggregation in zQ175 brains by molecular and histological approaches.

Huntingtin-lowering approaches that target huntingtin expression are a major focus for therapeutic intervention for Huntington's disease. When the cytosine, adenine and guanine repeat is expanded, the huntingtin pre-mRNA is alternatively processed to generate the full-length huntingtin and HTT1a transcripts. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 huntingtin protein. In evaluating huntingtin-lowering approaches, understanding how the targeting strategy modulates levels of both transcripts and the huntingtin protein isoforms that they encode will be essential. Given the aggregation-propensity of exon 1 huntingtin, the impact of a given strategy on the levels and subcellular location of aggregated huntingtin will need to be determined. We have developed and applied sensitive molecular approaches to monitor the levels of aggregated and soluble huntingtin isoforms in tissue lysates. We have used these, in combination with immunohistochemistry, to map the appearance and accumulation of aggregated huntingtin throughout the CNS of zQ175 mice, a model of Huntington's disease frequently chosen for preclinical studies. Aggregation analyses were performed on tissues from zQ175 and wild-type mice at monthly intervals from 1 to 6 months of age. We developed three homogeneous time-resolved fluorescence assays to track the accumulation of aggregated huntingtin and showed that two of these were specific for the exon 1 huntingtin protein. Collectively, the homogeneous time-resolved fluorescence assays detected huntingtin aggregation in the 10 zQ175 CNS regions by 1-2 months of age. Immunohistochemistry with the polyclonal S830 anti-huntingtin antibody showed that nuclear huntingtin aggregation, in the form of a diffuse nuclear immunostain, could be visualized in the striatum, hippocampal CA1 region and layer IV of the somatosensory cortex by 2 months. That this diffuse nuclear immunostain represented aggregated huntingtin was confirmed by immunohistochemistry with a polyglutamine-specific antibody, which required formic acid antigen retrieval to expose its epitope. By 6 months of age, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. Homogeneous time-resolved fluorescence analysis showed that the comparative levels of soluble exon 1 huntingtin between CNS regions correlated with those for huntingtin aggregation. We found that soluble exon 1 huntingtin levels decreased over the 6-month period, whilst those of soluble full-length mutant huntingtin remained unchanged, data that were confirmed for the cortex by immunoprecipitation and western blotting. These data support the hypothesis that exon 1 huntingtin initiates the aggregation process in knock-in mouse models and pave the way for a detailed analysis of huntingtin aggregation in response to huntingtin-lowering treatments.

Thieno[2,3-b]thiophene Derivatives as Potential EGFRWT and EGFRT790M Inhibitors with Antioxidant Activities: Microwave-Assisted Synthesis and Quantitative In Vitro and In Silico Studies.

Microwave-assisted synthesis and spectral analysis of certain novel derivatives of 3,4-diaminothieno[2,3-b]thiophene-2,5-dicarbonitrile 1-7 were carried out. Compounds 1-7 were examined for cytotoxicity against MCF-7 and A549 cell lines using the quantitative MTT method, and gefitinib and erlotinib were used as reference standards. Compounds 1-7 were shown to be more active than erlotinib against the two cell lines tested. Compound 2 outperformed regular erlotinib by 4.42- and 4.12-fold in MCF-7 and A549 cells, respectively. The most cytotoxic compounds were subsequently studied for their suppression of kinase activity using the homogeneous time-resolved fluorescence assay versus epidermal growth factor receptor (EGFRWT) and EGFR790M. With IC50 values of 0.28 ± 0.03 and 5.02 ± 0.19, compound 2 was demonstrated to be the most effective against both forms of EGFR. Furthermore, compound 2 also had the best antioxidant property, decreasing the radical scavenging activity by 78%. Molecular docking research, on the other hand, was carried out for the analyzed candidates (1-7) to study their mechanism of action as EGFR inhibitors. In silico absorption, distribution, metabolism, excretion, and toxicity tests were also performed to explain the physicochemical features of the examined derivatives.

Discovery of anticancer agents with c-Met inhibitory potential by virtual and experimental screening of a chemical library

c-Met receptor tyrosine kinase has recently emerged as an important target with therapeutic implications in pancreatic cancer. In this study, we carried out a docking virtual screening on an in-house library of 441 synthesized compounds and selected the compounds with the best interactions with the c-Met protein to be subjected to experimental tests. Ten compounds belonging to 3 different classes of chemical structures were selected for this purpose and their antiproliferative effects were studied against 4 pancreatic ductal adenocarcinoma (PDAC) cell lines including AsPC-1, Suit-2, Panc-1 and Mia-Paca-2 cells, primary PDAC cells and also c-Met amplified EBC-1 cell line by sulforhodamine-B assay. Apoptosis induction was examined by Hoechst 33258 staining and annexin V-FITC/propidium iodide flow cytometric assay. The best compound was also assayed in three-dimensional cultures of AsPC-1 cells and its c-Met inhibitory potential was studied by immunoblotting and a homogenous time resolved fluorescence (HTRF) assay. The compound with a phenanthrotriazine hydrazinyl scaffold bearing nitrophenyl pendant (PhTH) was the most active derivative, with IC50 values in the range of 5–8 μM. This compound exerted antiproliferative effect against AsPC-1 cells also in the presence of hepatocyte growth factor (HGF). PhTH induced apoptosis, dose-dependently inhibited spheroid growth, inhibited c-Met activity in cell-free HTRF assay and also inhibited the phosphorylation of c-Met and its downstream effector ERK1/2 in AsPC-1 cells. Molecular docking and dynamics simulation and MM-PBSA analysis confirmed close interactions of PhTH with c-Met kinase domain. Some of the tested compounds in this study seem to be potential c-Met inhibitors with promising activities against PDAC cells.

Pharmacological Characterization of JNJ-75276617, a Menin-KMT2A Inhibitor, As Targeted Treatment for KMT2A-Altered and NPM1-Mutant Acute Leukemia

Background: AML is characterized by a differentiation block leading to the accumulation of immature cells. Therefore therapies capable of inducing differentiation of these immature cells, particularly if also associated with antiproliferative activity, are considered an attractive therapeutic approach in acute leukemias. Histone-lysine N-methyltransferase-2A (KMT2A; also known as Mixed lineage leukemia 1 (MLL1))-fusion proteins require direct interaction with the nuclear scaffolding protein menin, a key component of the menin-KMT2A complex, for transcription of multiple leukemogenic target genes. Genetic or pharmacologic disruption of the interaction between menin-KMT2A has been shown to induce differentiation and block progression of KMT2A-rearranged (KMT2A-r) AML and B-cell ALL in vivo. Similarly, disruption of the crucial menin-KMT2A interaction inhibits leukemias driven by mutations in nucleophosmin 1 (NPM1) which constitute ~30% of AML and rely on the expression of menin-KMT2A target genes, e.g. MEIS1. Blocking the menin-KMT2A interaction by small-molecule inhibitors is being explored clinically as a treatment for KMT2A-altered and NPM1-mutant AML which represent areas of high unmet medical need. Results and Conclusions: JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between KMT2A (wild-type [WT] and fusion) and menin. JNJ-75276617 binds with high affinity to the KMT2A binding pocket on human, mouse, and dog menin. JNJ-75276617 has a high biochemical potency across species evaluated by a homogenous time-resolved fluorescence (HTRF) assay. In KMT2A-r and cytoplasmic NPM1 (NPM1c)-mutant AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with the chromatin of KMT2A-target gene promoters, resulting in reduced expression of menin-KMT2A target genes such as MEIS1 and FLT3. Moreover, JNJ-75276617 increased expression of differentiation genes such as ITGAM and MNDA. JNJ-75276617 displayed potent antiproliferative activity across a range of AML cell lines and patient samples harboring KMT2A alterations or NPM1c mutations in vitro, with >95.5-fold selectivity over WT KMT2A/NPM1 AML and normal peripheral blood mononuclear cells (PBMCs). JNJ-75276617 treatment of KMT2A-r and NPM1c AML cell lines induced differentiation marker expression, followed by induction of apoptotic cell death. Additionally, JNJ-75276617 induced neutrophil-like morphological differentiation in KMT2A-AF9 fusion gene-transduced mouse bone marrow cells. Using in vivo preclinical AML models, JNJ-75276617 resulted in reduction of leukemic burden and provided a significant dose-dependent survival benefit with accompanying decreases in expression of menin-KMT2A target genes. JNJ-75276617 showed good oral bioavailability and favorable pharmacokinetics in preclinical animal species. Our data demonstrate JNJ-75276617 to be a highly selective and potent menin-KMT2A that disrupts the crucial menin-KMT2A protein complex and mediates significant preclinical activity against AML/ALL cell lines and primary AML cells. JNJ-75276617 also demonstrates pharmacodynamic markers to assess target engagement. Collectively, these data support the recently initiated first-in-human trial evaluating JNJ-75276617 as monotherapy for relapsed/ refractory acute leukemia patients harboring either KMT2A or NPM1 alterations (NCT04811560).

Guidelines for HTRF technology in EGFR kinase assay

AbstractEpidermal growth factor receptor (EGFR) is among the first discovered transmembrane receptor tyrosine kinases. Ligand binding to the extracellular domain of EGFR results in the formation of homo‐dimeric or hetero‐dimeric complexes, activation, and auto‐phosphorylation in the intracellular tyrosine kinase domain, then regulates the cell's proliferation, differentiation, migration, and invasion. It has been reported that EGFR is overexpressed, amplified, or mutated in several epithelial tumors. EGFR tyrosine kinase inhibitors (EGFR‐TKIs) specifically bind to the tyrosine kinase domain of EGFR. Homogeneous time‐resolved fluorescence (HTRF) assay is successfully used in kinase assays and is a crucial tool in the drug discovery of various kinases. Moreover, the HTRF assay has been determined to exhibit many advantages, such as being easy to miniaturize, simple to read, and excellent to repeat. In these guidelines, the theory, materials, and reagent preparation of HTRF kinase assay are described and emphasized. Furthermore, the protocol and optimization of the assay are determined to establish an HTRF screening platform, then an optimized HTRF kinase assay is performed to test the compounds for screening potential EGFR‐TKIs. In addition, these guidelines can contribute to verifying the high‐throughput screening results of HTRF and help to reveal the underlying mechanism of the EGFR kinase and tested compounds.

Evaluation of a flavonoid library for inhibition of interaction of HIV-1 integrase with human LEDGF/p75 towards a structure–activity relationship

Background: Proteinśprotein interaction (PPI) between lens epithelium-derived growth factor (LEDGF/p75) and human immunodeficiency virus (HIV) integrase (IN) becomes an attractive target for anti-HIV drug development. Methods: The blockade of this interaction by small molecules could potentially inhibit HIV-1 replication. In this study, a panel of 99 structurally related flavonoids were was tested, concerning their ability to inhibit IN-LEDGF/p75 interaction, using a homogeneous time time-resolved fluorescence (HTRF) assay. Results: From the obtained results, it was possible to observe that the flavonoid with hydroxyl group in C3-, C4-, C5- and C7-position on the A-ring, C4′- and C5′-position of the B-ring, a carbonyl group of the C-ring, was more active against IN-LEDGF/p75 interaction, through competitive inhibition. Moreover, the binding modes of representative compounds, including myricetin, luteolin, dihydrorobinetin, naringenin, epicatechin, genistein and helichrysetin, were analyzedanalysed by molecular docking. Biolayer interferometry assay confirmed that these representative compounds disrupted the PPI by binding to IN with KD values ranging from 1.0 to 3.6 µM. Conclusion: This study presents the first to quantitative comparation of the effect of flavonoids with different structural subclasses on IN-LEDGF/p75 interaction. Our findings provide new insights into the development of inhibitors targeting IN-LEDGF/p75 interaction using flavonoids. Key Messages HIV-1 integrase (IN)-LEDGF/p75 interaction is an attractive target for antiviral drug development. For the first time, the structure-activity relationship of flavonoids belonging to seven flavonoidic subclasses on IN-LEDGF/p75 interaction was determined. This study comprehends an HTRF-based screening system, biolayer interferometry and an in silico molecular docking analysis.

Computational Cosolvent Mapping Analysis Leads to Identify Salicylic Acid Analogs as Weak Inhibitors of ST2 and IL33 Binding.

Cytokine signaling initiated by the binding of the cytokine receptors to cytokines plays important roles in immune regulation and diseases. Structurally, cytokine receptors interact with cytokines via an extensive, rugged interface that represents a challenge in inhibitor development. Our computational analysis has previously indicated that butyric acid, mimicking acidic residues, preferentially binds to sites in ST2 (Stimulation-2) that interact with acidic residues of IL33, the endogenous cytokine for ST2. To investigate if a charged group in small molecules facilitates ligand binding to ST2, we developed a biochemical homogeneous time resolved fluorescence assay to determine the inhibition of ST2/IL33 binding by five molecules containing an aromatic ring and a charged group. Three molecules, including niacin, salicylic acid, and benzamidine, exhibit inhibition activities at millimolar concentrations. We further employed the computational cosolvent mapping analysis to identify a shared mode of interaction between niacin, salicylic acid, and ST2. The mode of interaction was further confirmed by four analogous compounds that exhibited similar or improved activities. Our study provided the evidence of inhibition of ST2 and IL33 binding by salicylic acid and analogs. The results suggest that biological activity of salicylic acid may be partly mediated through modulating extracellular cytokine receptors and cytokine interaction.

A184 REPURPOSING DRUGS FOR SPLEEN TYROSINE KINASE (SYK) PEDIATRIC PATIENTS USING HIGH-THROUGHPUT SCREENING

BackgroundSpleen Tyrosine Kinase (SYK) is a cytosolic, non-receptor tyrosine kinase with an imperative role in immune and non-immune processes. Recently, we identified six gain-of-function SYK variants in patients that presented multi-organ inflammation and immune dysregulation. The SYK variants displayed constitutive SYK phosphorylation in human embryonic kidney (HEK) 293T, colonic epithelial cells (SW480), and in knock-in heterozygous SYK mice. These observations mark SYK as a therapeutic target for autoimmune diseases.Phenotype drug discovery accelerates this process and can be done successfully with an appropriate phenotype. A possible phenotype displayed by SYK variants is SYK phosphorylation, as high-throughput screening can identify hit compounds that reduce the constitutive activation of phosphorylated SYK (p-SYK).Aims Aim 1: Determine the screening phenotype with wildtype (WT) and SYK S550Y variant in HEK293T cells. Recently, we observed increased phosphorylation in gain-of-function SYK variants We hypothesize that we can use phosphorylated-SYK (p-SYK) levels to identify hit compounds that can decrease the kinase activity in these variants. With stable transfected SYK WT and SYK S550Y HEK293T cell-line, protein analyses will be completed to characterize the appropriate screening phenotype.Aim 2 Establish an assay for high-throughput drug screening. We will utilize homogenous time-resolved fluorescence (HTRF) assay. The signal measured from HTRF is positively proportional to the level of p-SYK; therefore, we expect that S550Y cells will have a higher signal than the WT.Aim 3 Validate hit compounds in HEK293T and zebrafish. We will create a dose-response curve with the hit compounds in in vitro and in vivo models.MethodsWe will use stable transfection to established overexpressing SYK WT and S550Y HEK293T cells. We will apply homogenous time-resolved fluorescence (HTRF) to quantify p-SYK levels during the drug screening.ResultsProtein analyses have verified high expression of p-SYK in stable transfected HEK293T cells. No stimulation was required, as the cells showed an increased phosphorylation level at baseline. Downstream signaling partners such as p-ERK and p-JNK of the MAPK pathway displayed an upregulation. This suggests that the sustained activation of p-SYK may consequently affect cellular processes and contribute to the clinical manifestations observed in patients.ConclusionsThis research study will identify hit compounds that can produce a safe and effective biological response in pediatric patients with gain-of-function SYK variants. Personalizing medicine throughout high-throughput drug screening can accelerate drug repurposing for pediatric patients with multiple systemic diseases and immune dysregulation.Funding AgenciesNone

Identification of 20(S)-Ginsenoside Rh2 as a Potential EGFR Tyrosine Kinase Inhibitor.

As the main active ingredients of Panax ginseng, ginsenosides possess numerous bioactivities. Epidermal growth factor receptor (EGFR) was widely used as a valid target in anticancer therapy. Herein, the EGFR targeting activities of 20(S)-ginsenoside Rh2 (20(S)-Rh2) and the relationship of their structure-activity were investigated. Homogeneous time-resolved fluorescence assay showed that 20(S)-Rh2 significantly inhibited the activity against EGFR kinase. 20(S)-Rh2 was confirmed to effectively inhibited cell proliferation in a dose-dependent manner by MTT assay. Furthermore, quantitative real-time PCR and western blotting analysis revealed that 20(S)-Rh2 inhibited A549 cells growth via the EGFR-MAPK pathway. Meanwhile, 20(S)-Rh2 could promote cell apoptosis, block cell cycle, and reduce cell migration of A549 cells, respectively. In silico, the result suggested that both hydrophobic interactions and hydrogen-bonding interactions could contribute to stabilize their binding. Molecular dynamics simulation showed that the side chain sugar moiety of 20(S)-Rh2 was too flexible to be fixed at the active site of EGFR. Collectively, these findings suggested that 20(S)-Rh2 might serve as a potential EGFR tyrosine kinase inhibitor.

The PROTAC selectively degrading Bcl-xL represents a novel Hedgehog pathway inhibitor with capacity of combating resistance to Smoothened inhibitors while sparing bone growth.

Rationale: Primary and acquired resistance to Smoothened (Smo) inhibitors largely hampered their clinical efficacy. Given the important functions of hedgehog (Hh) pathway in bone formation and development, the permanent defects in bone growth caused by Smo inhibitors further restrict the use of Smo inhibitors for pediatric tumor patients. Anti-apoptotic Bcl-2 proteins regulate Hh activity by engaging a Bcl-2 homology (BH) domain sequence found in suppressor of fused (Sufu). In this study, we tested the effect of SIAIS361034, a Proteolysis Targeting Chimera (PROTAC) specifically targeting B-cell lymphoma extra large (Bcl-xL) to the celeblon (CRBN) E3 ligase for degradation, on combating the resistance and reducing the toxicity of bone growth caused by Hh inhibition. Methods: Fluorescence polarization, homogeneous time-resolved fluorescence (HTRF) assay, immunoblot, and immunoprecipitation (IP) were used to evaluate whether SIAIS361034 is an appropriate Bcl-xL PROTAC. Dual luciferase reporter assay, real-time quantitative PCR (RT-qPCR), depilatory model, and SmoA1 model were established to assess the effect of SIAIS361034 on the activity of Hh signaling pathway and its ability to overcome drug resistance in vitro and in vivo. Molecular mechanisms of SIAIS361034 for inhibiting Hh activity were demonstrated by dual luciferase reporter assay, immunoblot, and immunofluorescence staining. PET-CT and histopathology of bone tissues were used to assess the effects of SIAIS361034 on bone growth. Results: We observed that SIAIS361034 efficiently and selectively inhibits the activity of the Hh pathway in vitro and in vivo, by interrupting Bcl-xL/Sufu interaction, therefore, promoting the interaction of Sufu with Gli1. Moreover, SIAIS361034 possesses the ability of combating resistance to current Smo inhibitors caused by Smo mutations and Gli2 amplification and remarkably inhibits the growth of SmoA1 tumors in vivo. In contrast to von Hippel-Lindau (VHL) E3 ligase, our result further reveals little detectable expression of CRBN in two types of cells critical for bone development, human articular chondrocytes and human fetal osteoblastic cells. Moreover, treatment with SIAIS361034 results in no impairment on the bone growth of young mice, accompanying no alteration of the expression of Bcl-xL and Gli1 proteins. Conclusion: Our findings demonstrate that selectively targeting Bcl-xL by PROTAC is a promising strategy for combating resistance to Smo inhibitors without causing on-target drug toxicities of bone growth.

Berbamine Suppresses the Growth of Gastric Cancer Cells by Inactivating the BRD4/c-MYC Signaling Pathway.

PurposeBerbamine (Ber), a bioactive constituent extracted from a traditional Chinese medicinal herb, has been shown to exhibit broad inhibitory activity on a panel of cancer cell types. However, its effects and the underlying molecular mechanisms on gastric cancer (GC) remain poorly understood.MethodsThe anti-growth activity of Ber on two GC cell lines and normal gastric epithelial cell line were evaluated using MTS and clone formation assay. Flow cytometry analysis was employed to evaluate the cell cycle distribution and apoptosis of GC cells. Western blot and quantitative PCR (qPCR) analysis were employed to investigate the anti-GC mechanism of Ber. The inhibitory activity and binding affinity of Ber against BRD4 were evaluated by homogeneous time-resolved fluorescence (HTRF) and surface plasmon resonance (SPR) assay, respectively. Molecular docking and molecular simulations were conducted to predict the interaction mode between BRD4 and Ber.ResultsThe results demonstrated that Ber reduced the proliferation of GC cell lines SGC-7901 and BGC-823 and induced cell cycle arrest and apoptosis. Mechanistically, Ber was identified as a novel natural-derived BRD4 inhibitor through multiple experimental assay, and its anti-GC activity was probably mediated by BRD4 inhibition. Molecular modeling studies suggested that Ber might bind to BRD4 primarily through hydrophobic interactions.ConclusionOur study uncovered the underlying anti-GC activity of Ber in vitro and suggested that Ber holds promise as a potential lead compound in the discovery of novel BRD4 inhibitors.

Discovery of quinazoline derivatives as novel small-molecule inhibitors targeting the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction

Development of small molecule PD-1/PD-L1 inhibitors as a novel immunotherapy strategy exhibits great promise. Herein, a novel series of quinazoline derivatives were designed, synthesized and their inhibitory activity against the PD-1/PD-L1 interaction was evaluated through a homogenous time-resolved fluorescence (HTRF) assay. Among them, the compound 39 exhibited the most potent inhibitory activity with an IC50 value of 1.57 nM. Furthermore, the cellular level assays revealed that 39 could inhibit the PD-1/PD-L1 interaction and restore T-cell function, and showed low toxicity on the PBMCs. In addition, the structure-activity relationships (SARs) of the novel quinazoline derivatives were explored and the binding mode of 39 with dimeric PD-L1 was analyzed by molecular docking. This work demonstrates that incorporation of pyrimidine group between the 2 and 3-positions of the biphenyl structure is an effective strategy for designing novel and more potent small molecule PD-1/PD-L1 inhibitors, and 39 can be regarded as a promising lead compound for further investigation.