Fluorescence Polarization Assay Research Articles

Cytosine-Rich DNA Binding Insulin Stronger than Guanine-Rich Aptamers: Effect of Aggregation of Insulin for Its Detection.

The detection of insulin is an important analytical task. Previously, guanine-rich DNA was believed to bind insulin, and an insulin aptamer was selected based on a few guanine-rich libraries. Insulin is a unique analyte, and it forms different aggregation states as a function of its concentration and buffer conditions, which may affect the detection of insulin. Herein, using fluorescence polarization assays, three insulin preparation methods were evaluated: direct dissolution, ethylenediaminetetraacetic acid (EDTA) treatment to remove Zn2+, and dissolution in acid followed by neutralization. All the insulin samples containing Zn2+ barely bind to the aptamer DNA, whereas monomers and dimers of insulin with Zn2+ removed were able to bind. Compared to the previously reported aptamer, C-rich DNA showed stronger binding affinities and faster binding kinetics. The sigmoidal binding curves and slow binding kinetics showed that multiple DNA strands and insulin molecules gradually bind, and it took approximately 1 h to reach saturation. This insulin binding was nonspecific, and other tested proteins also can bind to C-rich and G-rich DNA with even strong affinities. These results provide important information on the detection of insulin and further insights into the binding mechanisms between oligomeric insulin and DNA.

Architecture and genomic arrangement of the MurE–MurF bacterial cell wall biosynthesis complex

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.

A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 μM) and GS441524 (IC50 15.2 μM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

Seroprevalence and risk factors for brucellosis and leptospirosis in swine from rural communities of Argentina

Objectives. To determine the seroprevalence of brucellosis and leptospirosis in pigs reared on small-scale farms in rural areas of the district of Tandil, Buenos Aires Province, Argentina; to analyse the associated risk factors; to describe the prevalent Leptospira spp. serogroups; and to determine the spatial distribution of both diseases. Materials and methods. A total of 340 serum samples were obtained. Buffer Plate Agglutination Test, Rose Bengal Test, and Fluorescence Polarisation Assay were used to detect antibodies against Brucella spp. Microscopic Agglutination Test was applied for serological diagnosis of leptospirosis. Statistical methods were used to evaluate the factors associated with the infections. Results. Seroprevalence of 0% and 22.6% was observed for brucellosis and leptospirosis, respectively. The most prevalent Leptospira serogroups identified were Canicola, Ballum, Icterohaemorrhagiae and Pomona. The main risk factors associated with leptospirosis infection were farms with a higher number of pigs and the presence of horses and wild boars near or within the farms. The use of milling by-products of cereal grains as animal feed and the presence of weaning cages were also associated. A relevant spatial cluster of seropositivity to Leptospira spp. was identified in a low-altitude area. Conclusions. Our results suggest that brucellosis is probably controlled, but Leptospira spp. are present in the farming systems under study. Knowledge of brucellosis and leptospirosis seroprevalence, spatial distribution and associated risk factors can be useful for the prevention and control of endemic zoonotic diseases in the region.

Disease Progression and Serological Assay Performance in Heritage Breed Pigs following Brucella suis Experimental Challenge as a Model for Naturally Infected Feral Swine.

Invasive feral swine (Sus scrofa) are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. Brucella suis, the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for B. suis infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for B. suis in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following B. suis infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with B. suis and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the Brucella abortus/suis complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for B. suis surveillance among feral swine would improve understanding of spillover risks at the national level.

Development of a Peptide Sensor Derived from Human ACE2 for Fluorescence Polarization Assays of the SARS-CoV-2 Receptor Binding Domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuing emergence of infectious variants have caused a serious pandemic and a global economic slump since 2019. To overcome the situation and prepare for future pandemic-prone diseases, there is a need to establish a convenient diagnostic test that is quickly adaptable to unexpected emergence of virus variants. Here we report a fluorescent peptide sensor 26-Dan and its application to the fluorescence polarization (FP) assay for the highly sensitive and convenient detection of SARS-CoV-2. The 26-Dan sensor was developed by fluorescent labeling of the 26th amino acid of a peptide derived from the N-terminal α-helix of human angiotensin-converting enzyme 2 (hACE2) receptor. The 26-Dan sensor maintained the α-helical structure and showed FP changes in a concentration-dependent manner of the receptor binding domain (RBD) of the virus. The half maximal effective concentrations (EC50's) for RBD of Wuhan-Hu-1 strain, Delta (B.1.617.2), and Omicron (BA.5) variants were 51, 5.2, and 2.2 nM, respectively, demonstrating that the 26-Dan-based FP assay can be adaptable to virus variants that evade standard diagnostic tests. The 26-Dan-based FP assay could also be applied to model screening of a small molecule that inhibits RBD binding to hACE2 and identified glycyrrhizin as a potential inhibitor. The combination of the sensor with a portable microfluidic fluorescence polarization analyzer allowed for the detection of RBD in a femtomolar range within 3 min, demonstrating the assay could be a promising step toward a rapid and convenient test for SARS-CoV-2 and other possible future pandemic-prone diseases.

Abstract 3440: Targeting the mutant estrogen receptor in metastatic breast cancer

Abstract Introduction: Estrogen receptor gene (ESR1) drives approximately 70-80% of breast cancer cases, which are primarily treated with endocrine therapies. Mutations in the ESR1 exclusively occur in the ligand binding domain (LBD) and are significantly enriched following endocrine therapy. These mutations retain the estrogen receptor (ER) expression thus conferring ligand-independent growth, which is a paramount contributor to endocrine therapy resistant disease. Worse progression-free survival is consistently associated with ESR1 mutation status; therefore, there is an unmet need for therapies against these mutations. To this end, we recently developed TX-245, a bis-benzamide molecule that hampers the function of the two most common ESR1 mutations - Y537S and D538G. Methods: TX-245 was evaluated in vitro using MTT cell proliferation assay and colony formation assay carried out in multiple ER+ breast cancer cells harboring wild type (WT) ESR1 or mutant (MT) ESR1. Approaches such as tumor spheroid cultures were also implemented to study the effect of TX-245 compared to selected SERDs/SERMs via CellTiter-Glo 3D viability assay. Mechanistic insights were obtained performing TR-FRET assay, fluorescence polarization assay, RNA-seq, ChIP-seq, western blot, and qRT-PCR. The therapeutic efficacy in vivo was evaluated using cell xenografts, patient derived xenografts (PDX), patient derived explants (PDE), and metastatic mouse models of MT-ER breast cancer. Results: TX-245 demonstrated greater anti-proliferative dose-dependent activity in models harboring WT and MT ESR1, including those resistant to fulvestrant and tamoxifen, when compared to other SERDs/SERMs. RNAseq data suggests that TX-245 dramatically alters the transcription of ERα-regulated genes, with repression of canonical estradiol-upregulated and induction of estradiol-repressed genes. TX-245 was able to antagonize ER-chromatin interaction and ER-transcriptional signaling in breast cancer cells driven by ESR1 mutation. TX-245 treatment downregulated ER protein expression and decreased expression of ER targeted genes (i.e., GREB and PGR) in multiple ER+ cell lines. TX-245 treatment as a single agent or in combination with palbociclib resulted in tumor regression in xenografts expressing WT, Y537S or D538G ESR1 mutation. PDX and PDE models consistently exhibited substantial tumor growth inhibition as well as decreased proliferation (Ki67 staining). Metastases studies using intracardiac and intratibial injection demonstrated that TX-245 inhibited the progression of established metastatic niches in mice injected with Y537S or D538G ER+ breast cancer cells. Conclusion: Our results indicate that TX-245 inhibits cell proliferation in cell lines that express WT or mutant ESR1, induce ER degradation, suppress ER-mediated signaling and reduce tumor burden in mouse models. These studies highlight the utility of TX-245 in targeting mutations in the ER-LBD. Citation Format: Karla Parra, Suryavathi Viswanadhapalli, Tanner Reese, Xihui Liu, JunHao Liu, Zexuan Liu, Tae-Kyung Lee, Chia Yuan Chen, Kara Kassees, Jung-Mo Ahn, Ratna Vadlamudi, Ganesh Raj, Carlos Roggero. Targeting the mutant estrogen receptor in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3440.

Abstract 514: Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice

Abstract Unlike classical Signal transducer and activator of transcription (Stat) signaling, which is transient in non-transformed cells, the aberrant activation of the family member, Stat3, occurs in malignant transformation and is implicated in breast, ovarian and many other human cancers. Thus, Stat3 remains a validated and important target for the discovery of novel anticancer drugs. Despite this, the discovery and development of potent Stat3 small molecule inhibitors has proven to be a significant challenge, and no drug is available yet in the market. We have discovered novel Stat3 irreversible azetidine inhibitors with unprecedent submicromolar potency through systematic medicinal chemistry structure activity relationship (SAR). Mechanism of action of most potent azetidine inhibitors depend on whether they are salicylic acids or not, i.e., salicylic acids preferably bind irreversibly to Stat3 Cys426 site in the DNA-binding domain (DBD), and non-salicylic acids, e.g., current lead H182, bind to Stat3 Cys468 DBD site. As expected, the inhibition of Stat3 DNA-binding activity was time dependent, with IC50 in the range of 0.27-0.87 µM at one hour incubation with active Stat3. On the other hand, azetidine salicylic acids also bind to the SH2 domain, although reversibly and at much weaker affinity, as determined in fluorescent polarization (FP) assay, with IC50 of 10-16 µM at one hour incubation, while non-salicylic acids, e.g., lead H182, present no binding affinity up to 600 µM for the SH2 domain. Despite that H182 does not bind to SH2 domain, it still inhibits its phosphorylation. Though lead compound H182 presents significant mouse in vivo efficacy, mouse in vivo pharmacokinetics shows very low plasma AUC, which correlates with quite high mouse in vitro hepatocyte CLint of 138 µL/min/106 cells; however, human in vitro hepatocyte assay gives much better results and is in the middle stability range (CLint of 14.6 µL/min/106 cells). The in vivo PK in other species, e.g., rat, is being determined. H182 represents a plausible molecule for further development. Citation Format: Yue Chen, Ning Zhai, Yinsong Zhu, Peibin Yue, Nagendra Verma, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco J. Lopez-Tapia, James Turkson. Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 514.

Abstract 3101: Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells

Abstract Introduction: Autophagy pathway activation and upregulation is an important mechanism for cancer cell survival upon exposure to chemotherapeutic agents. LC3, a ubiquitin-like protein, plays a key role in the regulation of cargo selection and autophagosome formation. Given small molecule inhibitors have not been well developed to target LC3, we aim to design and synthesize an LC3 binder and evaluate its activity in the inhibition of autophagy pathway in cancer cells. Methods: The crystal structure of LC3 and p62 complex (PDB: 2ZJD) was used to assist our design of helical sulfonyl-γ-AApeptides to the LC3 interacting region of p62. The peptidomimetics were synthesized on the solid phase and purified by HPLC. We used fluorescent polarization assay to assess the binding affinity of the peptidomimetics to LC3. We then utilized an LC3 HiBiT reporter cell line (U2OS) from Promega, which was engineered to have a fused human LC3-HiBiT with a HaloTag in between to study the effect of the lead LC3 binder on autophagic flux. In addition, confocal microscopy enabled direction visualization of the colocalization of FITC-labeled LC3 binder with LC3 and lysosome, respectively. Cytotoxicity of LC3 binder was evaluated in colorectal cancer cell line HCT116. Results: We designed and synthesized 6 helical sulfonyl-γ-AApeptides (JC1-6) to target the binding interface of LC3 and p62. These peptidomimetic compounds were designed to retain essential LC3 and p62 interactions at the W and L regions and yet have different functional groups on the helical scaffold. Compounds JC1 and JC6 have binding affinity of Kd 280 nM and 150 nM with LC3, respectively. FITC-labelled JC6 was able to penetrate U2OS and HCT116 cells. It had intracytoplasmic colocalization with not only lysosomes but also LC3 aggregates. Direct visualization of intracytoplasmic LC3 aggregates showed that JC6 at 50 µM significantly inhibited the autophagic degradation of LC3 aggregates. This effect was augmented by the presence of 2 µM autophagy activator PP242. Lastly, JC6 demonstrated cytotoxicity with IC50 approximately 50 µM in HCT116 cells bearing baseline autophagy activation compared to lack of cytotoxicity in U2OS cells with no baseline autophagy activity. Conclusion: We designed and synthesized novel helical sulfonyl-γ-AApeptide LC3 binders. With the ability of cellular penetration, they demonstrated inhibition of autophagic flux and preliminary cytotoxicity in cancer cells. Future studies may focus on structural activity relationship and further evaluation of its mechanisms of action in cancer cells. Citation Format: Mentalla Mahmoud, Jianyu Chen, Jianfeng Cai, Hao Xie. Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3101.

Abstract 6321: HMPL-453, a highly selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays potent activity in FGFR-altered tumor models

Abstract Background: Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate numerous cellular processes. Dysregulation of FGFR signaling due to receptor fusion, mutation or amplification is observed across multiple cancer types, making activated FGFRs an important therapeutic target. Herein, we present the preclinical characterization of HMPL-453, a highly potent and selective inhibitor of FGFR1, 2, and 3, discovered and being currently developed in phase II clinical trial (NCT04353375) by HUTCHMED. Methods: Kinase activity was measured by Transcreener™ Fluorescence Polarization assay or Z’-LYTE kinase assay. In vitro anti-proliferation activity was measured by CellTiter-Glo luminescent or CCK-8 assay. The effect of HMPL-453 on FGFR signaling pathway was detected by western blot. Multiple tumor models with FGFR alteration were applied in Nu/Nu nude mice to determine anti-tumor efficacy of 453 as a single agent. A model in immune-competent BALB/c mice inoculated with the constructed NIH/3T3 cells carrying FGFR2-AHCYL1 fusion was chosen to investigate the combination effect of HMPL-453 with anti-PD-1 antibody. Results: HMPL-453 potently inhibited the tyrosine kinase activities of recombinant FGFR 1, 2, and 3 in vitro (IC50 values of 6, 4, and 6 nM, respectively) with weaker activity against FGFR4 (IC50 = 425 nM). HMPL-453 selectively inhibited proliferation of tumor cell lines with dysregulated FGFR signaling (GI50s: 3~105 nM) compared with cell lines lacking FGFR aberrations (GI50s > 1.5 µM). HMPL-453 demonstrated strong inhibition of phosphorylation of FGFR and downstream protein in tumor cell lines harboring FGFR2 fusion. Oral administration of HMPL-453 could induce time- and dose-dependent inhibition of phosphorylation of FGFR and resulted in remarkable and dose-dependent anti-tumor activity in multiple FGFR-altered tumor models. HMPL-453 at the dose of 50 mg/kg/day could induce tumor regression in most tumor models tested. Moreover, HMPL-453 significantly improved anti-tumor activity of anti-PD-1 antibody in a FGFR2 fusion model by priming the immune environment. Conclusion: HMPL-453 is a highly potent and selective inhibitor of FGFR 1, 2, and 3 with strong activity against FGFR-deregulated tumors in preclinical models, supporting continued investigation in patients with FGFR alterations (such as fusion and mutation) either as a single agent or in combination with PD-1 blockade. Citation Format: Jia Hu, Jun Ni, Longxian Jiao, Jinghong Zhou, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Qihang Zhang, Juntao Yu, Ying Yu, Dongxia Shi, Min Cheng, Weifang Xue, Sumei Xia, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-453, a highly selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays potent activity in FGFR-altered tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6321.

Synthesis of Amino Acids Bearing Halodifluoromethyl Moieties and Their Application to p53-Derived Peptides Binding to Mdm2/Mdm4.

Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF2X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein-protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis. Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(εNHCOCF2X) exhibits an improved inhibition constant Ki compared to the unmodified peptide. Decreasing Ki values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(εNHCOCF2X) to Abu(γNHCOCF2X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4.

Optimization of the C2 substituents on the 1,4-bis(arylsulfonamido)naphthalene-N,N′-diacetic acid scaffold for better inhibition of Keap1-Nrf2 protein-protein interaction

Direct inhibition of the protein-protein interaction (PPI) between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) reduces the ubiquitination and subsequent degradation of Nrf2, leading to Nrf2 accumulation in the cytosol and the nuclear translocation of Nrf2. Once inside the nucleus, Nrf2 binds to and activates the expression of antioxidant response element (ARE) genes involved in redox homeostasis and detoxification. Herein, we report a series of 1,4-bis(arylsulfonamido)naphthalene-N,N′-diacetic acid analogs with varying C2 substituents to explore the structure-activity relationships at this position of the central naphthalene core. The Keap1-binding activities were first screened with a fluorescence polarization (FP) assay followed by further evaluation of the more potent compounds using a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay. It was found that compound 24a with C2-phthalimidopropyl group was the most potent in this series showing an IC50 of 2.5 nM in the TR-FRET assay with a Ki value in the subnanomolar range. Our docking study indicated that the C2-phthalimidopropyl group in compound 24a provided an extra hydrogen bonding interaction with the key residue Arg415 that may be responsible for the observed boost in binding affinity. In addition, compounds 12b, 15, and 24a were shown to activate the Nrf2 signaling pathway in NCM460D cells resulting in elevated mRNA levels of GSTM3, HMOX1 and NQO1 by 2.4–11.7 fold at 100 μM as compared to the vehicle control.

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.

Quality control of fluorescence polarization assay over gel-based assay.

Desmoplakin (DSP) is an integral part of the desmosome, the intercellular junction that links the intermediate filaments of adjacent cells. Specific mutations in DSP (R451G, S507F, S442F, and S299R) result in calpain hypersensitivity. Our long-term objective is to reduce this hypersensitivity by occluding the protease binding site of DSP with small molecule drugs or other molecules. To do this type of screening efficiently, we need a reliable, robust, and inexpensive DSP screening method. However, existing DSP expression constructs are low yield. Also, existing methods of observing degradation rates are done via a protein and time-intensive gel-based assay. Last, calpain is expensive. Here we describe a new fluorescence polarization assay to detect DSP degradation using both a shorter fluorescein isothiocyanate (FITC) labeled DSP construct and using trypsin as the protease. We show that this shorter construct is susceptible to degradation however the mutants degrade at a faster rate. We also show that trypsin was a reasonable substitute for calpain. Having now developed these new reagents and methods, we are well-positioned to screen for ways to rescue DSP mutants.

Sero-prevalence of brucellosis in ruminants and awareness of stakeholders on the disease in wet markets in Maswa district, Tanzania

Brucellosis is a zoonotic disease with economic drawbacks worldwide. Livestock wet-markets are known premises for human-animal interaction, livestock products consumption and live animal trades in village setups. However, the contribution of wet-market procedures and the knowledge of stakeholders towards zoonotic diseases are not well documented. The present study assessed the sero-prevalence of brucellosis in on-sale ruminants, knowledge of stakeholders on the disease and practices favoring disease transmission so as to generate evidence-based disease transmission risk at wet-markets. A cross sectional study involved collection of 496 blood samples from cattle, goats and sheep in wet-markets in Maswa district, Tanzania. Sera were harvested and tested for brucellosis using fluorescent polarization assay. Structured questionnaire about the epidemiology and control of brucellosis was administered to 400 wet-market stakeholders. Data were analyzed by computing descriptive statistics. The sero-prevalences of 11.2% (95% CI: 7.6 – 15.8) in cattle, 3.92% (95% CI: 1.1 – 9.7) in sheep and 1.39% (95% CI: 0.2 – 4.9) in goats were recorded. Un-screened ruminants from different herds were mixed during auction. Sold animals moved to different destinations while un-sold ones returned home. Interview results revealed that 91% of respondents had low knowledge about brucellosis. The mean knowledge score on index-summated scale was 18.2%. The results imply that wet markets are a risk epicenter for transmission of brucellosis and stakeholders are not informed about it. It is recommended that wet-markets be brucellosis surveillance points and strategies for brucellosis control should consider an awareness and training package to the community

Cascade-amplified fluorescence polarization assay for miRNA based on aggregation strategy of Y-shaped DNA

MicroRNAs (miRNAs) have emerged as one of the promising biomarkers. Their aberrant levels are intimately connected to various diseases. However, Sensitive assay of low-abundance miRNAs remain a grand challenge. Herein, a novel sensitive cascade-amplified fluorescence polarization (FP) sensing platform for miRNA detection was developed based on aggregation strategy of Y-shaped DNA via streptavidin (SA). The miRNA-155 was employed as the model target analyte. In the presence of miRNA-155, the duplex-specific nuclease (DSN) cycling amplification and catalytic hairpin assembly (CHA) cycling amplification were triggered successively. Owing to the target-triggering dual cyclic amplification, numerous Y-shaped DNA nanostructure could be easily generated according to the assembly order of hairpin probes. Then, SA was added to the system, inducing the aggregation of Y-shaped DNA nanostructure to form a large-size of DNA/SA complexes. Taking advantages of cascade-amplified strategy, a significant enhancement of the FP signal was achieved and a sensitive assay for miRNA detection was realized with the detection limit of 1 × 10-13 M. Beyond that, the proposed method could distinguish single-base mismatch and achieved satisfactory result for the expression level assay of miRNA-155 in different cell lines, holding great potential in the applications in miRNA-related clinical diagnosis and biochemical researches.

Discovery of Novel 3-Phenylpiperidine Derivatives Targeting the β-Catenin/B-Cell Lymphoma 9 Interaction as a Single Agent and in Combination with the Anti-PD-1 Antibody for the Treatment of Colorectal Cancer.

Direct disruption of the β-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential strategy for colorectal cancer (CRC) treatment through inhibiting oncogenic Wnt activity. Herein, a series of 3-phenylpiperidine derivatives were synthesized and evaluated as β-catenin/BCL9 PPI inhibitors. Among them, compound 41 showed the best IC50 (0.72 μM) in a competitive fluorescence polarization assay and a KD value of 0.26 μM for the β-catenin protein. This compound selectively inhibited the growth of CRC cells, suppressed Wnt signaling transactivation, and downregulated oncogenic Wnt target gene expression. In vivo, 41 showed potent anti-CRC activity and promoted the infiltration and function of cytotoxic T lymphocytes while decreasing the infiltration of regulatory T-cells (Tregs). Furthermore, the combination of 41 and the anti-PD-1 antibody (Ab) efficiently enhanced anti-CRC efficacy, first verifying the in vivo efficacy of the small-molecule β-catenin/BCL9 PPI inhibitor and anti-PD-1 Ab in combination.

Chemical synthesis of a reported p47phox/p22phox inhibitor and characterization of its instability and irreproducible activity.

The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology.

Rationally designed stapled peptides allosterically inhibit PTBP1-RNA-binding.

The diverse role of the splicing factor PTBP1 in human cells has been widely studied and was found to be a driver for several diseases. PTBP1 binds RNA through its RNA-recognition motifs which lack obvious pockets for inhibition. A unique transient helix has been described to be part of its first RNA-recognition motif and to be important for RNA binding. In this study, we further confirmed the role of this helix and envisioned its dynamic nature as a unique opportunity to develop stapled peptide inhibitors of PTBP1. The peptides were found to be able to inhibit RNA binding via fluorescence polarization assays and directly occupy the helix binding site as observed by protein crystallography. These cell-permeable inhibitors were validated in cellulo to alter the regulation of alternative splicing events regulated by PTBP1. Our study demonstrates transient secondary structures of a protein can be mimicked by stapled peptides to inhibit allosteric mechanisms.

Dissecting Selectivity Determinants of Small-Molecule Inhibitors of SH2 Domains Via Fluorescence Polarization Assays.

Fluorescence polarization (FP) assays can be used to identify small-molecule inhibitors that bind to SH2 domain-containing proteins. We have developed FP assays by which to identify inhibitors of the SH2 domains of the two closely-related transcription factors STAT5a and STAT5b. Point mutation of selected amino acids in the putative binding site of the protein is a valuable tool by which to gain insight into the molecular mechanism of binding. In this chapter, we describe the cloning and application of point mutant proteins in order to transfer the binding preference of selected SH2 domain-binding STAT5b inhibitors to STAT5a, with results that highlight the importance of considering a role for residues outside the SH2 domain in contributing to the binding interactions of SH2 domain inhibitors.