Homogeneous Time-resolved Fluorescence Research Articles

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.

Discovery of potent CSK inhibitors through integrated virtual screening and molecular dynamic simulation.

Oncogenic overexpression or activation of C-terminal Src kinase (CSK) has been shown to play an important role in triple-negative breast cancer (TNBC) progression, including tumor initiation, growth, metastasis, drug resistance. This revelation has pivoted the focus toward CSK as a potential target for novel treatments. However, until now, there are few inhibitors designed to target the CSK protein. Responding to this, our research has implemented a comprehensive virtual screening protocol. By integrating energy-based screening methods with AI-driven scoring functions, such as Attentive FP, and employing rigorous rescoring methods like Glide docking and molecular mechanics generalized Born surface area (MM/GBSA), we have systematically sought out inhibitors of CSK. This approach led to the discovery of a compound with a potent CSK inhibitory activity, reflected by an IC50 value of 1.6 nM under a homogeneous time-resolved fluorescence (HTRF) bioassay. Subsequently, molecule 2 exhibits strong growth inhibition of MD anderson - metastatic breast (MDA-MB) -231, Hs578T, and SUM159 cells, showing a level of growth inhibition comparable to that observed with dasatinib. Treatment with molecule 2 also induced significant G1 phase accumulation and cell apoptosis. Furthermore, we have explored the explicit binding interactions of the compound with CSK using molecular dynamics simulations, providing valuable insights into its mechanism of action.

Discovery of 4-phenyl-1H-indazole derivatives as novel small-molecule inhibitors targeting the PD-1/PD-L1 interaction

The inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway with small molecules is a promising approach for cancer immunotherapy. Herein, novel small molecules compounds bearing various scaffolds including thiophene, thiazole, tetrahydroquinoline, benzimidazole and indazole were designed, synthesized and evaluated for their inhibitory activity against the PD-1/PD-L1 interaction. Among them, compound Z13 exhibited the most potent activity with IC50 of 189.6 nM in the homogeneous time-resolved fluorescence (HTRF) binding assay. Surface plasmon resonance (SPR) assay demonstrated that Z13 bound to PD-L1 with high affinity (KD values of 231 nM and 311 nM for hPD-L1 and mPD-L1, respectively). In the HepG2/Jurkat T co-culture cell model, Z13 decreased the viability rate of HepG2 cells in a concentration-dependent manner. In addition, Z13 showed significant in vivo antitumor efficacy (TGI = 52.6 % at 40 mg/kg) without obvious toxicity in the B16-F10 melanoma model. Furthermore, flow cytometry analysis demonstrated that Z13 inhibited tumor growth in vivo by activating the tumor immune microenvironment. These findings indicate that Z13 is a promising PD-1/PD-L1 inhibitor deserving further investigation.

Abstract LB030: Building a platform of validated high throughput screening approaches for molecular glue degrader identification

Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.

Pharmacological and phytochemical insights on the pancreatic β-cell modulation by Angelica L. roots

Ethnopharmacological relevanceAngelica roots are a significant source of traditional medicines for various cultures around the northern hemisphere, from indigenous communities in North America to Japan. Among its many applications, the roots are used to treat type 2 diabetes mellitus; however, this application is not mentioned often. Ethnopharmacological studies have reported the use of A. japonica var. hirsutiflora, A. furcijuga, A. shikokiana, and A. keiskei to treat diabetes symptoms, and further reports have demonstrated the three angelica roots, i.e., A. japonica var. hirsutiflora, A. reflexa, and A. dahurica, exhibit insulin secretagogue activity. Aim of the studyThis study aimed to phytochemically characterize and compare angelica roots monographed in the European Pharmacopeia 11th, isolate major plant metabolites, and assess extracts and isolates' capability to modulate pancreatic β-cell function. Materials and methodsRoot extracts of Angelica archangelica, Angelica dahurica, Angelica biserrata, and Angelica sinensis were phytochemically profiled using liquid chromatography method coupled with mass spectrometry. Based on this analysis, simple and furanocoumarins were isolated using chromatography techniques. Extracts (1.6–50 μg/mL) and isolated compounds (5–40 μmol/L) were studied for their ability to modulate insulin secretion in the rat insulinoma INS-1 pancreatic β-cell model. Insulin was quantified by the homogeneous time-resolved fluorescence method. ResultsForty-one secondary metabolites, mostly coumarins, were identified in angelica root extracts. A. archangelica, A. dahurica, and A. biserrata root extracts at concentration of 12.5–50 μg/mL potentiated glucose-induced insulin secretion, which correlated with their high coumarin content. Subsequently, 23 coumarins were isolated from these roots and screened using the same protocol. Coumarins substituted with the isoprenyl group were found to be responsible for the extracts' insulinotropic effect. ConclusionsInsulinotropic effects of three pharmacopeial angelica roots were found, the metabolite profiles and pharmacological activities of the roots were correlated, and key structures responsible for the modulation of pancreatic β-cell function were identified. These findings may have implications for the traditional use of angelica roots in treating diabetes. Active plant metabolites may also become lead structures in the search for new antidiabetic treatments.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.MethodsSingle-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation.Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.Data availabilityThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.Graphical

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.

Structure-Guided Discovery of PD-1/PD-L1 Interaction Inhibitors: Peptide Design, Screening, and Optimization via Computation-Aided Phage Display Engineering.

Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 μM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 μM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.

A186 INFLAMMATION MODIFIES DOSE-DEPENDENT RESPONSES OF INTESTINAL ANTI-TUMOUR MICRORNAS TO CRANBERRY PROANTHOCYANIDIN AND ITS MICROBIAL METABOLITE 3-(4-HYDROXYPHENYL)-PROPIONIC ACID

Abstract Background Cranberries are a rich source of proanthocyanidins (PAC), a type of polyphenol with anti-cancer properties. We previously found that PAC, along with its microbially-derived metabolite 3-(4-hydroxyphenyl)-propionic acid (HPPA), trigger unique regulatory responses of microRNAs (miRNAs) in intestinal epithelial cells including the upregulation of miR-146a-5p. Both miR-146a-5p and miR363-3p are anti-inflammatory and downregulate anti-tumorigenic signalling pathways such as the interleukin (IL)-17 and wingless-related integration site (Wnt) respectively. miR-146a-5p also attenuates IL-17-promoting cytokines levels (e.g., IL-6). Aims To determine if miR-146a-5p and miR-363-3p respond to different physiologically relevant concentrations of PAC and HPPA in inflammatory and non-inflammatory conditions. Methods Fully differentiated Caco-2BBe1 colonic epithelial cells were treated with two doses of PAC-enriched cranberry extract (50μg/ml, 100μg/ml), HPPA (5μg/ml, 10μg/ml), or with Dulbecco’s Modified Eagle Medium (DMEM; control) for 24 hours, followed by IL-1β (1ng/ml) or mock stimulation for three hours. Human IL-6 homogeneous time-resolved fluorescence (HTRF) kits were used to quantify IL-6 in cell supernatant. RNA was extracted and used for miRNA profiling using Nanostring technology. Statistical analysis was performed in R version 4.2.1 with the R-packages NanostringDiff, NanostringNorm, and Pheatmap. Results At homeostasis, 42 and 2 (miR-146a and miR363-3p) miRNAs, respectively, uniquely responded to increasing PAC or HPPA concentrations. In the inflammatory state, no miRNAs responded to increasing concentrations of PAC and HPPA. However, the expression of miR-363-3p increased (qampersand:003C0.001) in response to 50μg/ml of PAC + IL-1β but decreased in response to 5μg/ml of HPPA + IL-1β , and the expression of miR-146a-5p increased (qampersand:003C0.001) in response to 5μg/ml of HPPA + IL-1β, and 50μg/ml PAC + IL-1β. Predicted miRNA gene-pathway analysis revealed that miR-146a-5p, miR-363-3p, and the other 42 miRNAs commonly target pathways involved in the tumorigenesis of colorectal cancer such as mitogen-activated protein kinases (MAPK), hedgehog, and Wnt pathways. Though, in inflamed cells, only HPPA (5 or 10 μg/ml) attenuated IL-6 secretion (pampersand:003C0.05), which may be driven by increased expression of miR-146a-5p. Conclusions These findings suggest that cranberries proanthocyanidins and their metabolites affect miRNAs involved in cancer related pathways in different manners and concentrations, which may depend on the inflammatory status. The gut microbiota may be partially responsible for unlocking these effects. Funding Agencies Ocean Spray Cranberries, Inc. and the Natural Sciences of Engineering Research Council of Canada (NSERC)

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.

High-Throughput Cell-Based Screening of Small Molecule KRAS Signaling Inhibitors Using a Homogeneous Time-Resolved Fluorescence (HTRF) Assay.

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.

KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery.

Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.

Paxillin family proteins Hic-5 and LPXN promote lipid storage by regulating the ubiquitination degradation of CIDEC

Many metabolic diseases are caused by disorders of lipid homeostasis. CIDEC, a lipid droplet (LD)-associated protein, plays a critical role in controlling LD fusion and lipid storage. However, regulators of CIDEC remain largely unknown. Here, we established a homogeneous time resolved fluorescence (HTRF)-based high-throughput screening method and identified LPXN as a positive regulatory candidate for CIDEC. LPXN and Hic-5, the members of Paxillin family, are focal adhesion adaptor proteins that contribute to the recruitment of specific kinases and phosphatases, cofactors, and structural proteins, participating in the transduction of extracellular signals into intracellular responses. Our data showed that Hic-5 and LPXN significantly increased the protein level of CIDEC and enhanced CIDEC stability not through TAG synthesis and FAK signaling pathways. Hic-5 and LPXN reduced the ubiquitination of CIDEC and inhibited its proteasome degradation pathway. Furthermore, Hic-5 and LPXN enlarged lipid droplets and promoted lipid storage in adipocytes. Therefore, we identified Hic-5 and LPXN as novel regulators of CIDEC. Our current findings also suggest intervention with Hic-5 and LPXN might ameliorate ectopic fat storage by enhancing the lipid storage capacity of white adipose tissues.

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.

Abstract 12379: AT1-AA (Angiotensin II Type 1 Receptor Autoantibody) Aggravates Abdominal Aortic Aneurysm Through Activating AT1R/Gq Allosterically

Background: Angiotensin II type 1 receptor (AT1R) is a key mediator of the rennin-angiotensin-aldosterone system (RAAS). Autoantibodies against AT1R (AT1-AA), as an activator of AT1R, was found in abdominal aortic aneurysm (AAA) related diseases, such as hypertension and atherosclerosis. However, the role of AT1-AA in AAA is still unclear. Methods: We used angiotensin II (AngII)-infused male ApoE -/- mice model of AAA. Ultrasonography measurement, histological assessment, western blot, qRT-PCR and RNA sequencing were used to assess the severity of AAA. Nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay, radio assay of receptors and homogeneous time resolved fluorescence (HTRF) were used to measure the activation feature of AT1R and the affinity of AT1R with AT1-AA or AngII. Results: AT1-AA increased the incidence of AAA (vehicle vs. AT1-AA: 47% vs. 81%; P<0.05), which can be attenuated by AT1R blocker Candesartan. Furthermore, AT1-AA greatly increased severity of AAA, due to augmenting the diameter of abdominal aorta and the degradation of elastin by enhancing the expression of MMP2/9 (matrix metalloproteinase) and the accumulation of inflammatory cells. And AT1-AA could induce phenotypic switch of smooth muscle cells. But AT1-AA does not increase the plasma level of AngII. Mechanically, AT1-AA enhanced AngII-induced recruitment of Gq, but not β-arrestin1/2. AT1-AA potentiated the AngII-mediated Gq-dependent inositol phosphate (EC50 shift=3.829 nM) and the activation of PKC and ERK1/2. The responses were abolished by AT1R blocker and Gq inhibitor. The affinity of AT1-AA for the AT1R in cells was calculated to be 75.49 ± 4.7 nM. AT1-AA cannot compete for the binding of AngII to AT1R. However, AT1-AA significantly potentiated the binding of AngII with AT1R allosterically (mean K D ± SEM: 34.11 ± 0.9765 nM at 0 nM AT1-AA vs. 25.63 ± 1.826 nM at 100 nM AT1-AA; p<0.05), without altering the maximum binding. Conclusion: AT1-AA, as an allosteric activator of AT1R, promotes AAA through increasing the affinity of AngII with AT1R and activating Gq biasedly.