Fluorescence Resonance Energy Transfer Assay Research Articles

High-Throughput Screening of G-Quadruplex Ligands by FRET Assay.

Fluorescence resonance energy transfer (FRET) is a distance-dependent process by which energy is transferred from an excited donor fluorophore to an acceptor molecule when the donor and acceptor are in close proximity to each other. Depending on the assay design, FRET can provide a real-time measurement of structural integrity and dynamics of biomacromolecules in solution and is particularly suitable for studying G-quadruplex (G4) nucleic acids and their ligand interactions. FRET-based assays are ideally suited for high throughput screening (HTS) methodology because they are simple, sensitive, and easily automated. G4s are stable nucleic acid structures involved in important regulatory roles in gene replication, transcription, and genomic instability. Four-stranded G4s are promising drug targets as these non-canonical structures areenriched in oncogene promoters, 5' UTRs, and telomeres, and have been linked to regulation of gene expression in cancer and other diseases. Although molecules that bind to G4s, with subsequent influence on gene expression, have been well documented, the identification of new chemical scaffolds that potently and selectively bind to G4s and control specific gene expression are still much less common. Here, we describe a detailed protocol of a FRET-based HTS methodology to identify novel G4 ligands.

In vitro lipid transfer assays of phosphatidylinositol transfer proteins provide insight into the in vivo mechanism of ligand transfer

Fluorescence resonance energy transfer (FRET) assays and membrane binding determinations were performed using three phosphatidylinositol transfer proteins, including the yeast Sec14 and two mammalian proteins PITPα and PITPβ. These proteins were able to specifically bind the fluorescent phosphatidylcholine analogue NBD-PC ((2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine)) and to transfer it to small unilamellar vesicles (SUVs). Rate constants for transfer to vesicles comprising 100% PC were slower for all proteins than when increasing percentages of phosphatidylinositol were incorporated into the same SUVs. The rates of ligand transfer by Sec14 were insensitive to the inclusion of equimolar amounts of another anionic phospholipid phosphatidylserine (PS), but the rates of ligand transfer by both mammalian PITPs were strikingly enhanced by the inclusion of phosphatidic acid (PA) in the receptor SUV. Binding of Sec14 to immobilized bilayers was substantial, while that of PITPα and PITPβ was 3–7 times weaker than Sec14 depending on phospholipid composition. When small proportions of the phosphoinositide PI(4)P were included in receptor SUVs (either with PI or not), Sec14 showed substantially increased rates of NBD-PC pick-up, whereas the PITPs were unaffected. The data are supportive of a role for PITPβ as functional PI transfer protein in vivo, but that Sec14 likely has a more elaborate function.

Molecular Mechanism of Action of RORγt Agonists and Inverse Agonists: Insights from Molecular Dynamics Simulation.

As an attractive drug-target, retinoic acid receptor-related orphan receptor-gamma-t (RORγt) has been employed widely to develop clinically relevant small molecular modulators as potent therapy for autoimmune disease and cancer, but its molecular mechanism of action (MOA) remains unclear. In the present study, we designed and discovered two novel RORγt ligands that are similar in structure, but different in efficacy. Using fluorescence resonance energy transfer (FRET) assay, compound 1 was identified as an agonist with an EC50 of 3.7 μM (max. act.: 78%), while compound 2 as an inverse agonist with an IC50 value of 2.0 μM (max. inh.: 61%). We performed molecular dynamics (MD) simulations, and elucidated the MOA of RORγt agonist and inverse agonist. Through the analyses of our MD results, we found that, after RORγt is bound with the agonist 1, the side chain of Trp317 stays in the gauche- conformation, and thus helps to form the hydrogen bond, His479-Trp502, and a large hydrophobic network among H11, H11′, and H12. All these interactions stabilize the H12, and helps the receptor recruit the coactivator. When the RORγt is bound with the inverse agonist 2, the side chain of Trp317 is forced to adopt the trans conformation, and these presumed interactions are partially destroyed. Taken together, the critical role of residue Trp317 could be viewed as the driving force for the activation of RORγt.

Trends in ADAMTS13 Activity Testing over the Last 7 Years in a Single UK TTP Specialist Centre

BackgroundHistorically a diagnosis of thrombotic thrombocytopenic purpura (TTP) was made on autopsy. Following the identification of ADAMTS13 deficiency as central to the pathophysiology of TTP, measurement of the enzyme became a vital diagnostic tool. ADAMTS13 activity can be measured using fluorescence resonance energy transfer (FRET), immunoblotting (IB) or enzyme-linked immunosorbent assay (ELISA). However, such assays have traditionally been performed in only a few selective laboratories nationally. ADAMTS13 testing serves as both a diagnostic investigation for those with suspected TTP, as well as a method of monitoring those at risk of relapse. Due to the diversity in local practice, availability, and reporting of both acute and serial ADAMTS13 measurements, it remains unclear as to what constitutes best practice with regards to frequency of testing and how the test can result best support clinical decision making.AimTo review changing trends and patterns in ADAMTS13 testing before and after the set-up of a regional TTP Specialist Centre. To evaluate how this might inform future practice with regards to regional testing provision, patient monitoring and elective Rituximab.MethodsThis was a retrospective study of ADAMTS13 testing from 2011-18. Data was collected using the Trust electronic laboratory software system, instituted in 2011.The data was reviewed to identify serial trends in testing and in relation to the set-up of a regional TTP Specialist Service provision in 2013.ResultsA total of 2140 ADAMTS13 activity requests for 464 individual patients were performed between January 2011 and January 2018 using the Technoclone ELISA ADAMTS13 activity assay.A review of the number of ADAMTS13 activity tests performed demonstrated a serial annual rise with total tests per year- 2011: 39, 2012: 88, 2013: 137, 2014: 301, 2015: 401, 2016: 494, 2017: 679. This equated to a 17-fold increase in testing from 2011 compared to 2017, and 5-fold increase from 2013 to 2018 since the set-up of the regional Specialist Service.External referrals for testing constituted 14% of all ADAMTS13 activity requests with a significant 600% rise in all external requests between 2013 and 2018. These included external requests for analysis from 23 different hospital trusts, with the furthest requesting trust being 54 miles from the regional centre.65% of external requests represented acute referrals with 35% representing monitoring requests for patients receiving shared care between their local hospital and Specialist Centre.Of all internal requests, 82% were from haematology followed by critical care and nephrology. In contrast to external requesting, of the internal requests only 5% were for acute diagnosis compared with 95% of requests made for monitoring purposes.ADAMTS13 activity for monitoring in this patient cohort represented a combination of assessing response to treatment following an acute diagnosis, as well as long term monitoring in remission to identify potential relapse. Internal requests for both acute and monitoring purposes rose annually, with a 20-fold increase in monitoring requests from 2011 to 2017, and a 3-fold increase in acute requests over the same time period.ConclusionsADAMTS13 testing has continued to significantly increase annually suggesting a combination of improved diagnostic awareness and increased disease monitoring. The setup of a regional clinical service including laboratory diagnostics results in a significant increase in acute testing requests serving a wide geographic area. In an era where agents are available to potentially avoid acute relapse, more patients are being monitored using serial ADAMTS13 measurements during follow-up. Although avoiding acute clinical relapse has significant implications for the health service and patient mortality, the economics of serial testing in a condition where the enzyme activity is so unpredictable, remains unclear. There is wide variation globally in ADAMTS13 testing and hence a wider evaluation of current practice and outcomes would not only be valuable in establishing greater consensus on best practice, but also inform us more of the natural history of this condition. DisclosuresNo relevant conflicts of interest to declare.

Interactions Between Spermine-Derivatized Tentacle Porphyrins and The Human Telomeric DNA G-Quadruplex.

G-rich DNA sequences have the potential to fold into non-canonical G-Quadruplex (GQ) structures implicated in aging and human diseases, notably cancers. Because stabilization of GQs at telomeres and oncogene promoters may prevent cancer, there is an interest in developing small molecules that selectively target GQs. Herein, we investigate the interactions of meso-tetrakis-(4-carboxysperminephenyl)porphyrin (TCPPSpm4) and its Zn(II) derivative (ZnTCPPSpm4) with human telomeric DNA (Tel22) via UV-Vis, circular dichroism (CD), and fluorescence spectroscopies, resonance light scattering (RLS), and fluorescence resonance energy transfer (FRET) assays. UV-Vis titrations reveal binding constants of 4.7 × 106 and 1.4 × 107 M−1 and binding stoichiometry of 2–4:1 and 10–12:1 for TCPPSpm4 and ZnTCPPSpm4, respectively. High stoichiometry is supported by the Job plot data, CD titrations, and RLS data. FRET melting indicates that TCPPSpm4 stabilizes Tel22 by 36 ± 2 °C at 7.5 eq., and that ZnTCPPSpm4 stabilizes Tel22 by 33 ± 2 °C at ~20 eq.; at least 8 eq. of ZnTCPPSpm4 are required to achieve significant stabilization of Tel22, in agreement with its high binding stoichiometry. FRET competition studies show that both porphyrins are mildly selective for human telomeric GQ vs duplex DNA. Spectroscopic studies, combined, point to end-stacking and porphyrin self-association as major binding modes. This work advances our understanding of ligand interactions with GQ DNA.

The sensitive detection of ODAM by using sandwich-type biosensors with a cognate pair of aptamers for the early diagnosis of periodontal disease

This research aims to develop biosensors which could diagnose periodontal diseases in early phases and predict the illness stage of patients, in order to give them adequate treatment timely. Human odontogenic ameloblast-associated protein (ODAM) is considered to be a potential biomarker for periodontal diseases, based on high correlation between the level of ODAM in gingival crevicular fluid (GCF) and the degree of periodontitis. Many aptamers, including a cognate pair of aptamers which can bind to the different sites of ODAM, were successfully screened in a very stringent condition employing saliva as a counter target through the graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX). For the characterization of the aptamer candidates, GO-based fluorescence resonance energy transfer (GO-FRET) and surface plasmon resonance (SPR) assays were conducted. The sandwich-type binding of a cognate pair of aptamers to ODAM was additionally confirmed by employing circular dichroism (CD) and magnetic beads-based fluorescence imaging methods. The resulting cognate pair of aptamers, OD64 and OD35, were found to have their dissociation constant (Kd), 47.71 nM and 51.36 nM, respectively. The minimum detectable concentrations of a sandwich-type SPR biosensor were found to be 0.24 nM and 1.63 nM, respectively, for both buffered and saliva samples. Finally, using this cognate pair of aptamers, a sandwich-type lateral flow strip biosensor was successfully realized. This research shows the potential for implementation of a cognate pair of aptamers on point-of-care biosensors which enables simple, rapid, and non-invasive saliva-based diagnosis of periodontal-related diseases that can overcome current diagnostic methods and improve health care system.

Protease inhibitors broadly effective against feline, ferret and mink coronaviruses

Ferret and mink coronaviruses typically cause catarrhal diarrhea in ferrets and minks, respectively. In recent years, however, systemic fatal coronavirus infection has emerged in ferrets, which resembles feline infectious peritonitis (FIP) in cats. FIP is a highly fatal systemic disease caused by a virulent feline coronavirus infection in cats. Despite the importance of coronavirus infections in these animals, there are no effective commercial vaccines or antiviral drugs available for these infections. We have previously reported the efficacy of a protease inhibitor in cats with FIP, demonstrating that a virally encoded 3C-like protease (3CLpro) is a valid target for antiviral drug development for coronavirus infections. In this study, we extended our previous work on coronavirus inhibitors and investigated the structure-activity relationships of a focused library of protease inhibitors for ferret and mink 3CLpro. Using the fluorescence resonance energy transfer assay, we identified potent inhibitors broadly effective against feline, ferret and mink coronavirus 3CLpro. Multiple amino acid sequence analysis and modelling of 3CLpro of ferret and mink coronaviruses were conducted to probe the structural basis for these findings. The results of this study provide support for further research to develop broad-spectrum antiviral agents for multiple coronavirus infections. To the best of our knowledge, this is the first report on small molecule inhibitors of ferret and mink coronaviruses.

A Sensitive High-Throughput Screening Method for Identifying Small Molecule Stimulators of the Core Particle of the Proteasome.

Fluorescence resonance energy transfer (FRET) technology is a useful tool to monitor protein interactions as well as protease activity. We have recently reported a biochemical assay utilizing a FRET reporter peptide to monitor the activity of the 20S catalytic particle (20S CP) of the proteasome. This assay is designed specifically to have increased sensitivity to identify stimulators of the 20S CP, which may hold therapeutic potential to treat protein-accumulation diseases. The protocol described here details the necessary steps in synthesizing the FRET reporter peptide and performing the FRET assay with the 20S CP. © 2018 by John Wiley & Sons, Inc.

Development of a Broadly Applicable Assay for Measurement of Glycan-Directed Enzymatic Activity

Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degradation. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compound screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can determine the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind concanavalin A (ConA), the assay concept can be adapted to glycans that bind other lectins.

Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z′ score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure–activity relationships.

Use of Protein Kinase–Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors

Inositol hexakisphosphate kinases (IP6Ks) regulate a myriad of cellular processes, not only through their catalytic activity (which synthesizes InsP7, a multifunctional inositol pyrophosphate signaling molecule) but also through protein–protein interactions. To further study the enzymatic function and distinguish between these different mechanisms, specific inhibitors that target IP6K catalytic activity are required. Only one IP6K inhibitor is commonly used: N2-(m-(trifluoromethyl)benzyl) N6-(p-nitrobenzyl)purine (TNP). TNP is, however, compromised by weak potency, inability to distinguish between IP6K isoenzymes, off-target activities, and poor pharmacokinetic properties. Herein, we describe a new inhibitor discovery strategy, based on the high degree of structural conservation of the nucleotide-binding sites of IP6Ks and protein kinases; we screened for novel IP6K2 inhibitors using a focused set of compounds with features known, or computationally predicted, to target nucleotide binding by protein kinases. We developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay of adenosine diphosphate (ADP) formation from adenosine triphosphate (ATP). Novel hit compounds for IP6K2 were identified and validated with dose–response curves and an orthogonal assay. None of these inhibitors affected another inositol pyrophosphate kinase, PPIP5K. Our screening strategy offers multiple IP6K2 inhibitors for future development and optimization. This approach will be applicable to inhibitor discovery campaigns for other inositol phosphate kinases.

7-O-Methylwogonin from Scutellaria baicalensis Disturbs Mitotic Progression by Inhibiting Plk1 Activity in Hep3B Cells.

Polo-like kinase 1, a mitotic Ser/Thr kinase, has emerged as a molecular target for the development of anticancer drugs. In this study, we found that polo-like kinase 1 activity was inhibited by 7-O-methylwogonin and related flavones, including baicalein, dihydrobaicalein, and viscidulin II, isolated from Scutellaria baicalensis. Although dihydrobaicalein exhibited the highest polo-like kinase 1 inhibitory activity among the four compounds, it also inhibited other kinases, such as vaccinia-related kinase 2 and polo-like kinase 2. Baicalein and viscidulin II also showed low selectivity to polo-like kinase 1 since they inhibited polo-like kinase 3 and polo-like kinase 2, respectively. However, 7-O-methylwogonin exhibited selective polo-like kinase 1 inhibitory activity, as evidenced from in vitro kinase assays based on fluorescence resonance energy transfer assays and ADP-Glo kinase assays. In addition, examination of mitotic morphology and immunostaining using specific antibodies for the mitotic markers, p-histone H3 and mitotic protein monoclonal 2, in Hep3B cells showed that 7-O-methylwogonin treatment increased mitotic cell populations due to inhibition of mitotic progression as a result of polo-like kinase 1 inhibition. The pattern of 7-O-methylwogonin-induced mitotic arrest was similar to that of BI 2536, a specific polo-like kinase 1 inhibitor. Thus, it was suggested that 7-O-methylwogonin disturbed mitotic progression by inhibiting polo-like kinase 1 activity. These data suggest that 7-O-methylwogonin, a polo-like kinase 1 inhibitor, may be a useful anticancer agent because of its polo-like kinase 1 selectivity and effectiveness.

Identification of amentoflavone as a potent highly selective PARP-1 inhibitor and its potentiation on carboplatin in human non-small cell lung cancer

Nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme in the repair of DNA and is a promising target in the development of chemosensitizers. This study first investigated the inhibitory effects of amentoflavone (AMF) and its derivatives on PARP-1 and the potentiation of AMF on carboplatin (CBP) in non-small cell lung cancer (NSCLC). This study aims to evaluate the inhibitory effect of AMF against PARP-1 and its potentiation on CBP in lung cancer both in vitro and in vivo. The inhibitory effect of AMF on PARP-1 was investigated using molecular docking and cell-free model of PARP-1 assay. Its potentiation on CBP in lung cancer was also evaluated. Fluorescence resonance energy transfer assay was used to detect the inhibitory effects of AMF and its analogues on PARP-1. Molecular docking was employed to predict the binding mode of AMF and PARP-1. MTT assay, isobologram analysis, Hoechst staining, and Annexin V-PI double staining were used to confirm the potentiation of AMF on CBP in vitro. siRNA (PARP-1)-A549 cells were used to reveal the action target of AMF. Western blot analysis, immunohistochemistry, and Tunnel assay were employed to evaluate the potentiation of AMF on CBP in A549 xenograft mice. AMF and its analogues exerted excellent inhibitory effects on PARP-1 with IC50 values ranging from 0.198 μM to 0.409 μM. Docking experiment showed that AMF can stably bind to PARP-1 with a comparable binding energy to olaparib. AMF can decrease the expression of PAR induced by H2O2in vitro. AMF synergistically increased the CBP anti-proliferative effect in A549. However, its potentiation nearly disappeared when the cells were transfected with siRNAs against PARP-1. Oral administration of AMF (100 mg/kg), combined with CBP, remarkably inhibited A549 tumor growth and ki67 expression, and increased apoptosis compared with CBP-alone group. All results suggest that AMF can be a potential PARP-1 inhibitor and a candidate adjuvant agent to boost the anticancer effect of CBP in NSCLC.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5μg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 μM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9μg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Nonhormonal selective estrogen receptor modulator 1-(2-[4-{(3R,4S)-7-Methoxy-2, 2-dimethyl-3-phenyl-chroman-4yl}phenoxy]ethyl)pyrrolidine hydrochloride (ormeloxifene hydrochloride) for the treatment of breast cancer.

Breast cancer is the most common type of diagnosed cancers in women, difficult to treat, and has received international attention because of its aggressive nature and inherent drug resistance mechanisms. Development of a better selective estrogen receptor modulator with good therapeutic profile and less toxicity is very crucial in this scenario. This study was undertaken to evaluate and compare the in vitro and in vivo antitumor activities of ormeloxifene with other clinically used breast cancer drugs. Cytotoxic activity of ormeloxifene was compared with standard drugs, 4-hydroxytamoxifene and Adriamycin. Ormeloxifene (50 μM) concentration showed cytotoxicity of 75% and 82% in MDAMB-231 and 24% and 80% in MCF-7 cells, respectively, after 72 and 144 hr of incubation as displayed by cell viability assay. The same concentration of ormeloxifene was shown to exert 74% caspase-7 activation in MCF-7 cells after 24 hr of incubation by fluorescence resonance energy transfer assay. Cell cycle analysis proved that there was an increase in sub-G1 peak to 64.4% and 33.9% in MDAMB-231 and MCF-7 cells, respectively, after treatment using ormeloxifene (50 μM) for 48 hr. The nonobese diabetic-severe combined immunodeficiency mice bearing tumor xenografts of triple negative MDAMB-231 cells treated with ormeloxifene (3 mg/kg bw) showed significant regression in relative tumor volume compared to control. From the results obtained and as evidenced from prior literature, ormeloxifene in addition to contraceptive use, can be repositioned for the development of an efficacious anticancer drug. These data present the preclinical part of a well concerted effort to place ormeloxifene into further clinical trials.

Abstract P256: Development of an Automated High Throughput Screen for GRK4 as Potential Novel Therapeutics for Treatment of Hypertension and/or Salt Sensitivity

G protein coupled kinase type 4 (GRK4) reduces renal sodium excretion by deactivating the renal dopamine type 1 receptor (D 1 R) through serine phosphorylation. At least 3 polymorphisms in GRK4 have been shown to be associated with the expression of hypertension and/or salt sensitivity in humans and in mice. GRK4 is an ideal therapeutic target for treating hypertension since putting human GRK4 variants in mice results in hypertension which can be reversed by reducing the expression of GRK4. Inhibitors of GRK4 could serve as potent and selective agents to promote sodium excretion in salt sensitive and/or hypertensive individuals. Therefore, we developed an automated high throughput homogeneous time resolved fluorescent resonance energy transfer assay (TR-FRET) to identify small molecule inhibitors of GRK4 isoenzymes. The assay relies on the close proximity (90 A) transfer of 337 nm energy from a europium anti-phosphoserine antibody (Eu-pSer) to an allophycocyanin labeled streptavidin acceptor (streptavidin-APC), producing FRET at 665 nm. The assay was optimized through serial dilutions of streptavidin-APC, Eu-pSer, GRK4, and a peptide consisting of serine sites in the D 1 R, serving as a substrate. To validate the assay and determine its suitability for a large scale high throughput screen, three inhibitors, GRKA, GRKB, and GRKC were selected and concentration response assays were performed to determine IC50 values and variability analysis. The IC50 and (R 2 ) values of GRKA, GRKB, and GRKC were 4.99 μM (0.99), 26.52 μM (0.99), and 179.8 μM (0.82), respectively. GRKA is the most efficient GRK4 inhibitor, while GRKC is the least. This assay will be used to test congeners of compounds A, B, and C to identify more selective candidates, as well as for screening available small molecule compound libraries.

Abstract 2863: Dissecting the structural basis for inhibitors of RNA-binding proteins

Abstract RNA-binding proteins (RBPs) are key regulators of cellular functions, especially in post-transcriptional regulations. Dysregulation of RBPs is implicated in many diseases including cancer. One of the RBPs that is overexpressed in a variety of human cancer is Musashi-2 (MSI2). Elevated MSI2 expression is associated with ectopic oncogenic pathways, including but not limited to NUMB/Notch, PTEN/mTOR, TGF-β/SMAD3, making MSI2 a promising therapeutic target for cancer. In the case of NUMB, MSI2 binds to and negatively regulates translation of NUMB, a negative regulator of Notch signaling. Protein structure is critical for drug discovery and structure-based rational design. However, so far there is no structure available for MSI2 protein. Recently, using nuclear magnetic resonance (NMR) we solved the first solution structure of MSI2 RNA-recognition Motif 1 (MSI2-RRM1) that forms the major long narrow RNA-binding pocket. Using a fluorescence polarization (FP) chemical libraries screening, we identified several groups of hit compounds that disrupt the binding of MSI2-NUMB RNA potently. These compounds induced apoptosis, inhibited cancer cell proliferation, invasion and metastasis in multiple tumor models tested in vitro and in vivo. They also interfered with cancer stem cell functions with reduced tumorsphere formation. Several lead compounds showed promising efficacy in animal tumor models of human breast, prostate and colon cancer. To investigate the specificity of the compounds towards other RBPs, the compounds were tested in biophysical binding assays such as FP, time resolved Fluorescence Resonance Energy Transfer assay (TR-FRET), NMR spectroscopy and cell-based assays. We compared the affinity of these MSI2 inhibitors towards other RBPs such as MSI1 and HuR. Using docking and molecular dynamics simulation, we analyzed the structure-activity relationship of these inhibitors towards different RBP targets. This work adds significant information to the structure of MSI2-RRM1 and the structural basis for designing more potent and specific inhibitors of RBPs. Citation Format: Lan Lan, Minli Xing, Xiaoqing Wu, Philip Gao, Justin T. Douglas, Yu Zhan, Gulhumay Gardashova, Jiajun Liu, Robert P. Hanzlik, Jeffrey Aubé, Kristi L. Neufeld, Berl R. Oakley, Roberto N. De Guzman, Liang Xu. Dissecting the structural basis for inhibitors of RNA-binding proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2863.

Abstract 2662: Indenoisoquinoline topoisomerase I inhibitors target the DNA G-quadruplex formed in the c-Myc promoter and cause MYC downregulation

Abstract The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach toward the design of anticancer agents. Indenoisoquinoline derivatives have been designed and developed as Top1 inhibitors and can overcome some shortcomings associated with the camptothecin Top1 inhibitors, resulting in three indenoisoquinoline compounds, indotecan (LMP 400), indimitecan (LMP776), and LMP744 entering Phase I clinical trials at the National Cancer Institute. However, some active indenoisoquinolines did not show potent Top1 inhibition, suggesting a separate mechanism of action. c-Myc is one of the most commonly deregulated genes in human cancers; a DNA G-quadruplex (G4) formed in the proximal promoter region of c-Myc has been found to function as a transcriptional silencer and be amenable to small molecule targeting. As indenoisoquinolines share structure similarity to our c-Myc G-quadruplex (MycG4)-interactive compounds, we hypothesized that indenoisoquinolines could stabilize MycG4 and downregulate MYC transcription. Using a fluorescence resonance energy transfer (FRET) assay, we examined the MycG4 stabilization activity of 56 indenoisoquinoline compounds that we previously developed as Top1 inhibitors. Notably, 26 of the 56 compounds decreased the fluorescence intensity by at least 50%, and the 10 most potent compounds reduced fluorescence intensities by over 70%, indicating that these indenoisoquinolines are effective MycG4 stabilizers. The identified compounds were further characterized by fluorescence anisotropy, circular dichroism, and nuclear magnetic resonance for MycG4 interactions, followed by western blot assays for MYC downregulation. Significantly, the compounds with good MycG4 binding activity and affinity exhibited significant dose-dependent reduction of MYC expression, indicating that targeting the c-Myc promoter G-quadruplex is a likely mechanism of action for these active anticancer compounds. Furthermore, some active indenoisoquinolines exhibited both MYC downregulation and Top1 inhibition, suggesting that dual targeting c-Myc G-quadruplex and Top1 may represent a novel mechanism of action for anticancer drug design. Citation Format: Kaibo Wang, Mohamed S. A. Elsayed, Guanhui Wu, Mark Cushman, Danzhou Yang. Indenoisoquinoline topoisomerase I inhibitors target the DNA G-quadruplex formed in the c-Myc promoter and cause MYC downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2662.

Animal Hen1 2'-O-methyltransferases as tools for 3'-terminal functionalization and labelling of single-stranded RNAs.

S-adenosyl-L-methionine-dependent 2′-O-methylati-on of the 3′-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22–80 nt) of single-stranded RNAs permitting their 3′-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3′-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody-Drug Conjugates.

Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.