Fluorescence Polarization Assay Research Articles

A highly selective and sensitive fluorescence probe for a specific binding site on insect ryanodine receptors.

There is a significant need to study the binding of active compounds to the specific sites on insect ryanodine receptors (RyRs) that are the targets of two novel classes of diamide insecticides to which insects are becoming increasingly resistant. Here, we describe a rapid assay to study the action of potential compounds on the flubendiamide (Flu) binding site of insect RyRs that uses a fluorescence polarization assay with the fluorescence probe Flu-R-L that we synthesized. The IC50 of Flu for inhibiting probe binding on insect RyR was 18.82 ng/mL. The binding of 86 novel phthalic diamide derivatives on insect RyRs was studied using this newly established assay, and the compounds that exhibited high-affinity binding in the assay also possessed in vivo insecticidal activity against Plutella xylostella. Thus, Flu-R-L is a highly selective and sensitive fluorescence probe for studying the binding affinity of novel compounds to the Flu binding site of insect RyRs. The assay based on Flu-R-L is a rapid, accurate, and sensitive method for the screening of potentially bioactive molecules that bind specifically to insect RyRs.

Development and optimization of a cascade of screening assays for inhibitors of TRF2.

TRF2 is a telomere associated protein which plays an important role in telomere maintenance. Knockdown of TRF2 can cause chromosomal end to end fusions and induce DNA damage responses. TRF2 exerts its functions partially by recruiting a number of accessory proteins through its TRF homology domain (TRFH), therefore identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions of TRF2 with its associated proteins is important to elucidate the molecular mechanism of these protein-protein interactions. Development of robust and sensitive screening and evaluation assays is critical to the identification of TRF2 inhibitors, in this paper we reported the development and optimization of a cascade of screening and binding affinity evaluation assays, including a competitive FP (Fluorescence Polarization) assay utilized in our previous research, and two novel label-free DSF (Differential Scanning Fluorescence) and BLI (Biolayer Interferometry) assays. A previously identified TRF2 inhibitor TRF2-27 was used as an internal reference compound and evaluated in all of these assays. According to the results, DSF assay is not suitable for TRF2 screening because of the low ΔTm, while the optimized labeled-free BLI assay was demonstrated to be an accurate and reproducible assay for TRF2 inhibitor screening and characterization.

Identification of catalytic and non-catalytic activity inhibitors against PRC2-EZH2 complex through multiple high-throughput screening campaigns.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) along with embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12), which implements transcriptional repression mainly by depositing trimethylation marks at lysine 27 of histone H3 (H3K27me3). Its catalytic activity is closely correlated with the stability of PRC2, and somatic activating mutation of EZH2 Y641F within the catalytic SET domain drives tumor aggressiveness, drug resistance, and poor prognosis. Here, we report two high-throughput screening (HTS) campaigns targeting EZH2 Y641F and EZH2-EED interaction, respectively. For the EZH2 Y641F mutant, the HTS campaign involved a library of 250,000 compounds using a homogenous time-resolved fluorescence (HTRF) assay and identified 162 hits, while 60,160 compounds were screened against EZH2-EED interaction with a fluorescence polarization (FP) assay resulting in 97 hits. Among the 162 EZH2 Y641F inhibitors, 38 also suppressed EZH2-EED interaction and 80 showed inhibitory effects on the wide-type (WT) EZH2. Meanwhile, 10 of the 97 EZH2-EED interaction inhibitors were active against WT EZH2. These hit compounds provide useful tools for the development of novel PRC2-EZH2 inhibitors targeting its catalytic and non-catalytic activities.

Comparison of four serological tests for the diagnosis of swine brucellosis

The purpose of this article is to present results of a research in which the results of four serological tests were compared for the diagnosis of swine brucellosis in pigs from Brazilian herds in which the outbreak occurred in relation to other brucellosis-free. Rose Bengal (RBT), complement fixation (CFT), agglutination plus 2-mercaptoethanol test (TAT+ME), and commercial fluorescence polarization assay (FPA) were used to test 333 sera (271 sows and 62 finishing pigs) from a Brucella suis infected swine herd and 1,100 swine sera from brucellosis free pigs taken at a slaughterhouse. Considering infected all the 271 sows from the outbreak, and interpreting results of the FPA according to the manufacturer directions, sensitivities observed were 95.94% for FPA, 94.83% for RBT, 93.73% for CFT, and 92.25% for TAT+ME. Considering infected all the pigs from the infected herd with a positive result in at least one test, sensitivities observed were 98.92% for RBT, 97.13% for CFT, 96.06% for FPA, and 94.98% for TAT+ME. Specificities of the tests were CFT and TAT+ME, 100%; FPA, 99.55%; and RBT 99.27%. Results indicated a good performance of all the tests, and the agreement among them was almost perfect.

Grifolin, neogrifolin and confluentin from the terricolous polypore Albatrellus flettii suppress KRAS expression in human colon cancer cells.

In our search for bioactive mushrooms native to British Columbia, we determined that the ethanol extracts from fruiting bodies of the terrestrial polypore Albatrellus flettii had potent anti-cell viability activity. Using bioassay-guided fractionation, mass spectrometry and nuclear magnetic resonance, we successfully isolated three known compounds (grifolin, neogrifolin and confluentin). These compounds represent the major anti-cell viability components from the ethanol extracts of A. flettii. We also identified a novel biological activity for these compounds, specifically in down-regulating KRAS expression in two human colon cancer cell lines. Relatively little is known about the anti-cell viability activity and mechanism of action of confluentin. For the first time, we show the ability of confluentin to induce apoptosis and arrest the cell cycle at the G2/M phase in SW480 human colon cancer cells. The oncogenic insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) has been previously shown to regulate KRAS mRNA expression in colon cancer cells, possibly through its ability to bind to the KRAS transcript. Using a fluorescence polarization assay, we show that confluentin dose-dependently inhibits the physical interaction between KRAS RNA and full-length IMP1. The inhibition also occurs with truncated IMP1 containing the KH1 to KH4 domain (KH1to4 IMP1), but not with the di-domain KH3 and KH4 (KH3&4 IMP1). In addition, unlike the control antibiotic neomycin, grifolin, neogrifolin and confluentin do not bind to KRAS RNA. These results suggest that confluentin inhibits IMP1-KRAS RNA interaction by binding to the KH1&2 di-domains of IMP1. Since the molecular interaction between IMP1 and its target RNAs is a pre-requisite for the oncogenic function of IMP1, confluentin should be further explored as a potential inhibitor of IMP1 in vivo.

Mesenchymal stem cell exosome characterisation and high-throughput quantification by fluorescence polarisation spectroscopy

Background & Aim Mesenchymal Stem Cells (MSC) are a heterogenous subpopulation of adherent cells that are isolated from adult connective tissue, multipotent and capable of self-renewal. They are an attractive tool in cell therapy for multiple diseases and recent studies have demonstrated a therapeutic effect using MSC-derived exosomes instead of the cells themselves. Exosomes play an important role in cell-to-cell communication and have diverse biological roles; however, the production of EVs as a medicinal product presents serious challenges in terms of isolation, quantification and release. Current methods for quantification and testing rely on either the expression of specific membrane-associated proteins, such as CD81 and CD63, or on the measurement of physical properties such as size or density. The aim of this work is to develop a novel and high-throughput EV quantification platform for MSCs-derived exosomes using fluorescent polarisation (FP). FP is an accessible spectroscopic method that relies on the interaction between a small labeled probe and a target protein, taking advantage of differences in rotational energy of the free vs. bound probe. The change in polarisation between the unbound and bound probe can be used the devise a quantitative test of the concentration of EVs. Methods, Results & Conclusion EV-binding probes should exhibit strong and specific binding to the target and be highly stable in solution. Based on these characteristics, different probes have been identified in the current study. As prominent exosomes surface markers, the CD81 and CD63 tetraspanins have been targeted and candidate probes have been chosen based on their interaction with these markers. An enzyme-linked immunosorbent assay (ELISA) system has been established to assess the ability of the candidate probes to detect surface markers and capture exosomes. Following probe validation, candidate probes will be labeled with a suitable fluorophore and their utility in an FP assay format assessed in comparison with gold standard techniques for exosome quantification. As part of this study, MSCs-derived exosomes have been harvested from various culture conditions using ultracentrifugation and characterised through standard methods including western blot, Dynamic Light Scattering and Transmission Electron Microscopy. This assay will provide a rapid and high-throughput solution for exosome quantification directly in cell culture conditioned media, in a cost effective and timely manner.

Biochemical characterization of SARS-CoV-2 nucleocapsid protein

The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients’ sera, proving the importance of this antigen in host immunity and diagnostics.

Analytic Sensitivity of an ELISA Test on Pooled Sera Samples for Detection of Bovine Brucellosis in Eradication Stages in Uruguay.

Bovine brucellosis has been under eradication in Uruguay since 1998. The eradication program includes, among other interventions, individual sera sampling of beef animals at slaughter, and annual serum testing of all dairy cows—accounting for two million samples annually. At a herd prevalence of 0.8%, a pooled-sera sample approach could reduce the economic burden of the surveillance system by reducing the testing and operational costs. Our objective was to evaluate the analytic sensitivity of an indirect ELISA test for Brucella abortus in serum pools. Sixty-two Brucella abortus-positive bovine sera samples (based upon rose bengal and fluorescent polarization assay) were used as the positive control samples. Rose bengal-negative sera from negative farms were used to dilute the positive samples to the desired concentrations. Positive samples were diluted by using 1 ml of positive sera and 1 ml of negative sera (1/2 dilution) up to 1/1,024. Data were analyzed using generalized linear mixed models with a binary outcome (positive or negative), dilution number as a fixed effect, and a random effect for sample ID. Analytic sensitivity was 99.0% [95% confidence interval (CI): 96.3–99.7], 98.3% (95% CI: 93.1–99.6), 97.3% (95% CI: 87.4–99.4) for dilutions 1/2, 1/4, and 1/8, respectively. The analytical sensitivity, however, decreased when diluted to greater proportions. Given the current herd prevalence in Uruguay, it seems plausible that the use of a pooled sample approach could be adopted by policymakers to reduce the cost of the surveillance program and increase the number of samples being tested.

Kinase inhibitor roscovitine as a PB2 cap-binding inhibitor against influenza a virus replication

In this study, we examined the impact of roscovitine, a cyclin-dependent kinase inhibitor (CDKI) that has entered phase I and II clinical trials, on influenza A viruses (IAVs) and its antiviral mechanism. The results illustrated that roscovitine inhibited multiple subtypes of influenza strains dose-dependently, including A/WSN/1933(H1N1), A/Aichi/2/68 (H3N2) and A/FM1/47 (H1N1) with IC50 value of 3.35 ± 0.39, 7.01 ± 1.84 and 5.99 ± 1.89 μM, respectively. Moreover, roscovitine suppressed the gene transcription and genome replication steps in the viral life cycle. Further mechanistic studies indicated that roscovitine reduced viral polymerase activity and bound specifically to the viral PB2cap protein by fluorescence polarization assay (FP) and surface plasmon resonance (SPR). Therefore, we believed roscovitine, as a PB2cap inhibitor, was a prospective antiviral agent to be developed as therapeutic treatment against influenza A virus infection.

Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists

SummaryOne of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylalanine analogs, which displayed nanomolar Ki values. Their antiproliferative potential as well as their proapoptotic action, manifested by an increase in caspase-3 activity, was examined using various cell lines.

Optimization of Click Chemistry for Bioconjugation

Application of the expanded genetic code has created specialized proteins that enable new methods to investigate biological systems. Biomaterials and antibody‐drug conjugations and often employ “click chemistry” utilizing copper(I)‐catalyzed azide‐alkyne cycloaddition. We have functionalized green fluorescent protein (GFP) by incorporating a non‐standard amino acid (nsAA), p‐Azido‐phenylalanine. However, the literature on the rate and yield of this reaction remains incomplete. To enhance bioconjugation, we have explored and optimized reaction conditions and stoichiometry. We employed two corroborating assays to test single‐ and double‐click reaction events: size exclusion chromatography and a fluorescence polarization assay. We evaluated the efficiency of a single click reaction between a small molecule fluorophore and our functionalized GFP. Completed click reactions were run through a purification column and fluorescence readings of eluted samples were used to evaluate yield. Analysis of this single click event was also conducted through a fluorescence polarization assay. This allows the determination of the reaction rate and yield in real time. Additionally, yield of double click reactions between two functionalized proteins and a polyethylene glycol (PEG) molecule with two alkyne handles has been evaluated using SDS‐PAGE. Through these efforts, we report reaction yields that surpass previously reported values. These advances support the improved employment of click chemistry for broad applications in biofunctionalization such as the development of biomaterials and antibody‐drug conjugatesSupport or Funding InformationFunding and support provided by the Cal Poly Chemistry and Biochemistry Department, the Center for Applications in Biotechnology, and the National Science Foundation.

Connectivity Enhanced Structure Activity Relationship (ceSAR): A Novel Approach to Increase Accuracy and Speed of Virtual Drug Discovery

Experimental drug discovery and repurposing is a costly and time‐consuming process. Here, a novel approach, dubbed connectivity enhanced structure activity relationship (ceSAR) is introduced. ceSAR combines traditional virtual screening methodologies with cheminformatics and Library of Integrated Network‐Based Cellular Signatures (LINCS) gene expression signatures of genetic and chemical perturbations. This methodology combines structural similarity of small molecules with transcriptional connectivity and virtual screening‐predicted specific interacting molecules for the enhancement of structure activity relationship (SAR) and lead compound discovery. The transcriptional connectivity aspect of ceSAR is implemented as an R Shiny package, titled Sig2Lead, that employs connectivity between chemical and genetic signatures included in the LINCS library to identify small molecule inhibitors of user‐targeted pathways. Chemical libraries are reduced with ceSAR by first measuring concordance of transcriptomic signatures between genetic and chemical perturbation, identifying putative pathway inhibitors, followed by traditional docking approaches to re‐score the remaining library to identify compounds likely to interact with the target of interest. This combination identifies compounds that are both pathway inhibitors and have shape complementarity to the target of interest, making the most likely scenario that the molecules are directly interacting with the target protein of interest to modulate the pathway. Through integration of these orthogonal approaches, both an increased accuracy and speed can be observed, with the speed increase being several orders of magnitude.Support or Funding InformationThis work was supported in part by the National Institutes of Health grants U54 HL127624ceSAR combines transcriptional connectivity and virtual screening to identify novel inhibitors from a screened chemical library. The library is first reduced using transcriptional connectivity to a knockdown of the target gene to restrict the search to putative pathway inhibitors. These pathway inhibitors are then reduced further using traditional virtual screening approaches to find compounds with shape complementarity to the protein of interest.Figure 1Comparison of results of an early drug screen using a combination of docking with AutoDock4.2.6 and Sig2Lead shows a dramatic reduction in compounds without losing the best‐performing compounds. If used in this case, the ceSAR method would have excluded 51 of the 147 tested compounds, of which 45 had no observable IC50 via a Fluorescence Polarization assay, thus driving library enrichment. This reduction would have saved time and reagents through preventing testing of as many spurious compounds.Figure 2

Structure-based design, synthesis, and evaluation of Bcl-2/Mcl-1 dual inhibitors.

Based on our previously reported Bcl-2/Mcl-1 dual inhibitor 4-thiomorpholinyl-2-cyano-3-amidinophenalenone (A1) that simultaneously occupies the p2 and p4 hydrophobic pockets of Bcl-2 and Mcl-1, we optimized molecules with different bond angles of the groups extending to the p4 pocket and bulky hydrophobic groups to explore p2. Research on structure-activity relationship resulted in a new derivative B4 that is capable of occupying both the p2 and p4 more deeply and completely than A1, with Ki values determined by fluorescence polarization assay (FPAs) improving to 0.31 μM for Bcl-2 and 0.16 μM for Mcl-1. Furthermore, B4 exhibited selective lethality on cancer cells over normal cells. It showed stronger apoptosis induction than (-)-gossypol on a Bcl-2/Mcl-1-dependent cancer cell line and killed an Mcl-1-dependent cell line which is resistant to ABT-199 treatment.

Fluorescence polarization assay to detect the presence of traces of ciprofloxacin

Detection of ciprofloxacin residues in milk by sensitive and rapid methods is of great interest due to its use in the treatment of dairy livestock health. Current analytical approaches to antibiotics detection, are laboratory-based methods and they are time-consuming and require trained personnel. To cope this problem, we propose an assay, based on fluorescence polarization principle, able to detect the presence of ciprofloxacin in diluted milk sample without any pre-treatment. The proposed method is based on the use of ciprofloxacin-protein conjugate labeled with near infrared fluorescence dye, which upon binding to specific antibody causes an increase of the fluorescence polarization emission signal. The developed assay allows for the detection of ciprofloxacin at a concentration of 1ppb, which represents an amount lower than the maximum residual limit (MRL) of ciprofloxacin in milk, as set by the European Union regulation (100 ppb).

Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.

BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to nonhomologous cysteine residues within the C-terminal BRD4 bromodomain (BRD4-BD2), we performed a midthroughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify BRD4. Subsequent mass spectrometry, NMR, and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to BRD4 among human bromodomains. This site is orthogonal to the BRD4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays nor an acetylated histone peptide in AlphaScreen assays. Finally, we tethered our top-performing covalent fragment to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace BRD4 from chromatin.

Interaction of an IκBα Peptide with 14-3-3.

Inflammatory responses mediated by the transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) play key roles in immunity, autoimmune diseases, and cancer. NF-κB is directly regulated through protein–protein interactions, including those with IκB and 14-3-3 proteins. These two important regulatory proteins have been reported to interact with each other, although little is known about this interaction. We analyzed the inhibitor of nuclear factor kappa B α (IκBα)/14-3-3σ interaction via a peptide/protein-based approach. Structural data were acquired via X-ray crystallography, while binding affinities were measured with fluorescence polarization assays and time-resolved tryptophan fluorescence. A high-resolution crystal structure (1.13 Å) of the uncommon 14-3-3 interaction motif of IκBα (IκBαpS63) in a complex with 14-3-3σ was evaluated. This motif harbors a tryptophan that makes this crystal structure the first one with such a residue visible in the electron density at that position. We used this tryptophan to determine the binding affinity of the unlabeled IκBα peptide to 14-3-3 via tryptophan fluorescence decay measurements.

The seroprevalence of brucellosis and molecular characterization of Brucella species circulating in the beef cattle herds in Albania.

BackgroundBrucellosis is a ubiquitous zoonotic disease globally. It is endemic among bovines, sheep, and goats in Albania. The national control and eradication programs for brucellosis has been applied on sheep and goat farms as well as large dairy cattle farms, i.e., those with more than ten milking cows. The current study aims at estimating the herd and average individual animal prevalence of brucellosis in the national beef cattle herds, the missing information that was essential to propose the most appropriate control measures for this sub-population. Rose Bengal Test (RBT), Fluorescence Polarization Assay (FPA), and Enzyme-Linked Immunosorbent Assay (ELISA) were used as serological tests and classical bacteriology for isolation. Results were also used to investigate the difference in sensitivity between the assays used.MethodologyIn total, 655 animals from 38 beef cattle herds from six southern districts of Albania were sampled. Sera were tested using RBT, FPA, and ELISA. Fifteen positive cows and a bull from eight high-prevalence positive herds were slaughtered, and particular tissue samples were collected for bacteriology.ResultsThe overall herd seroprevalence in the tested beef cattle population was 55%, while the overall average within-herd prevalence (including only positive herds) was 38.3%, 42.7%, and 45.6% determined by the RBT, ELISA, and FPA, respectively. FPA was used for the first time in the diagnosis of bovine brucellosis in Albania, and its sensitivity was higher than RBT and ELISA. Three B. abortus strains were identified, two from the supra-mammary lymph node of two cows and one from the epididymis of a seropositive bull.ConclusionBrucellosis was highly prevalent in beef cattle in the southern part of Albania, and B. abortus was isolated from this subpopulation. To the best of our knowledge, this is the first statistically based survey of bovine brucellosis in beef herds in Albania. Using the FPA in parallel with other serological tests improved overall diagnostic sensitivity. Test and slaughter policy is not a rational approach for the control of brucellosis in beef cattle in Albania, and vaccination is only applicable, including strict control of the movement of animals.

Discovery and Development of Small-Molecule Inhibitors of Glycogen Synthase.

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

Association of brucellosis to abortions in humans and domestic ruminants in Kagera ecosystem, Tanzania.

Brucellosis is a worldwide zoonotic disease of socio-economic importance. Understanding the association of this disease with pregnancy outcome has the potential of contributing to the reduction of its reproductive burden in humans and animals among pastoral communities in Tanzania. A prospective cohort study was conducted in Kagera Region on pregnant women (n=76) and gravid ruminants (121 cattle, 125 goats and 111 sheep). Exposed and non-exposed groups to brucellosis were followed for 6months (from 15 November 2017 to 15 April 2018). Sera were collected and analysed using Rose Bengal Test (RBT) and Fluorescence polarization assay (FPA) test. Measures of effect, univariable and multivariable logistic regression analyses were computed. Positivity to both RBT and FPA tests was 21% (95% CI: 12.5-32) in pregnant women and 5% (95% CI: 3.1-8) in gravid ruminants. Among aborted cases, four women (out of nine), two cows (out of seven), two goats (out of 26) and zero sheep (out of 11) were positive to brucellosis. The abortion rate in humans and ruminants was 11.8% and 12.3%, respectively. Seropositivity to brucellosis was similar in aborted and non-aborted cases in humans (p=.08) and in ruminants (p=.2). At the population level, brucellosis was associated with abortions (population attributable risk: PAR) at 3.5% in pregnant women and at 0.5% in gravid ruminants in the study area. Infections to brucellosis were increased in exposed pregnant women (OR=19; 95% CI: 1.8-203, p=.01) and in cattle (OR=11; 95% CI: 1.3-88, p=.02). There is an indication that brucellosis could be contributing to abortions in pregnant women and domestic ruminants Kagera Region. Molecular tools could support more the results from serological tests to avoid cross-reaction with other pathogen agents. Control of brucellosis in animals is likely to reduce the threat of abortions in humans.

Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors

Signal transducer and activator of transcription 5B (STAT5B) is constitutively activated in multiple cancers as a result of hyperactivating mutations or dysregulation of upstream effectors. Therapeutic strategies have predominantly targeted the Src homology 2 (SH2) domain to inhibit STAT phosphorylation, a prerequisite for STAT5B transcriptional activation. An alternative approach for STAT5B pharmacologic inhibition involves targeting the DNA-binding domain (DBD). However, this strategy remains relatively unexplored and is further hindered by the lack of a high-throughput in vitro engagement assay. Herein, we present the development and optimization of a STAT5B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the STAT5B DBD though displacement of a fluorescently labelled oligonucleotide. The assay can generate a complete DNA-binding profile in 10 min, with signal stability up to 2 h, and minimal changes under a range of conditions including 10 % (v/v) glycerol, 15 % (v/v) DMSO, 1 mM NaCl, 0.02 % (w/v) BSA, and 1 mM EDTA. This assay is compatible with both unphosphorylated and phosphorylated STAT5B and demonstrates suitability for high-throughput screening with a Z′ factor of 0.68 ± 0.07 and a signal to noise ratio of 6.7 ± 0.84.