Firefly luciferase in chemical biology: A compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter

Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report

Fragment-based discovery of a regulatory site in thioredoxin glutathione reductase acting as "doorstop" for NADPH entry

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectivity inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases

High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase

The secretory and transmembrane isoforms of Prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small molecule modulators of PAP, we used a 1,536-well based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate (DiFMUP) as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog [8-(4-chlorophenylthio) cGMP; pCPT-cGMP] inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells

Author Correction: A consensus-based transparency checklist.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Incorporating Baseline Outcome Data in Individual Participant Data Meta-Analysis of Non-randomized Studies.

Background In non-randomized studies (NRSs) where a continuous outcome variable (e.g., depressive symptoms) is assessed at baseline and follow-up, it is common to observe imbalance of the baseline values between the treatment/exposure group and control group. This may bias the study and consequently a meta-analysis (MA) estimate. These estimates may differ across statistical methods used to deal with this issue. Analysis of individual participant data (IPD) allows standardization of methods across studies. We aimed to identify methods used in published IPD-MAs of NRSs for continuous outcomes, and to compare different methods to account for baseline values of outcome variables in IPD-MA of NRSs using two empirical examples from the Thyroid Studies Collaboration (TSC). Methods For the first aim we systematically searched in MEDLINE, EMBASE, and Cochrane from inception to February 2021 to identify published IPD-MAs of NRSs that adjusted for baseline outcome measures in the analysis of continuous outcomes. For the second aim, we applied analysis of covariance (ANCOVA), change score, propensity score and the naïve approach (ignores the baseline outcome data) in IPD-MA from NRSs on the association between subclinical hyperthyroidism and depressive symptoms and renal function. We estimated the study and meta-analytic mean difference (MD) and relative standard error (SE). We used both fixed- and random-effects MA. Results Ten of 18 (56%) of the included studies used the change score method, seven (39%) studies used ANCOVA and one the propensity score (5%). The study estimates were similar across the methods in studies in which groups were balanced at baseline with regard to outcome variables but differed in studies with baseline imbalance. In our empirical examples, ANCOVA and change score showed study results on the same direction, not the propensity score. In our applications, ANCOVA provided more precise estimates, both at study and meta-analytical level, in comparison to other methods. Heterogeneity was higher when change score was used as outcome, moderate for ANCOVA and null with the propensity score. Conclusion ANCOVA provided the most precise estimates at both study and meta-analytic level and thus seems preferable in the meta-analysis of IPD from non-randomized studies. For the studies that were well-balanced between groups, change score, and ANCOVA performed similarly

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Item does not contain fulltextMicroscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science

Deletions in VANGL1 are a risk factor for antibody-mediated kidney disease

We identify an intronic deletion in VANGL1 that predisposes to renal injury in high risk populations through a kidney-intrinsic process. Half of all SLE patients develop nephritis, yet the predisposing mechanisms to kidney damage remain poorly understood. There is limited evidence of genetic contribution to specific organ involvement in SLE.(1,2) We identify a large deletion in intron 7 of Van Gogh Like 1 (VANGL1), which associates with nephritis in SLE patients. The same deletion occurs at increased frequency in an indigenous population (Tiwi Islanders) with 10-fold higher rates of kidney disease compared with non-indigenous populations. Vangl1 hemizygosity in mice results in spontaneous IgA and IgG deposition within the glomerular mesangium in the absence of autoimmune nephritis. Serum transfer into B cell-deficient Vangl1(+/-) mice results in mesangial IgG deposition indicating that Ig deposits occur in a kidney-intrinsic fashion in the absence of Vangl1. These results suggest that Vangl1 acts in the kidney to prevent Ig deposits and its deficiency may trigger nephritis in individuals with SLE

Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization

What do parents think about parental participation in school-based interventions on energy balance-related behaviours? a qualitative study in 4 countries

<p>Abstract</p> <p>Background</p> <p>Overweight and obesity in youth has increased dramatically. Therefore, overweight prevention initiatives should start early in life and target modifiable energy balance-related behaviours. Parental participation is often advocated as important for school-based interventions, however, getting parents involved in school-based interventions appears to be challenging based on earlier intervention experiences. The purpose of this study was to get insight into the determinants of and perspectives on parental participation in school-interventions on energy balance-related behaviours (physical activity, healthy eating, sedentary behaviours) in parents of ten- to twelve-year olds in order to develop an effective parental module for school-based interventions concerning energy balance-related behaviours.</p> <p>Methods</p> <p>Four countries (Belgium, Hungary, Norway and Spain) conducted the focus group research based on a standardised protocol and a semi-structured questioning route. A variation in parental socio-economic status (SES) and parental school involvement was taken into account when recruiting the parents. The audio taped interviews were transcribed, and a qualitative content analysis of the transcripts was conducted in each country.</p> <p>Results</p> <p>Seventeen focus group interviews were conducted with a total of 92 parents (12 men, 80 women). Physical activity was considered to be a joint responsibility of school and parents, nutrition as parent's responsibility but supported by the school, and prevention of sedentary behaviours as parent's sole responsibility. Parents proposed interactive and practical activities together with their child as the best way to involve them such as cooking, food tasting, nutrition workshops, walking or cycling tours, sport initiations together with their child. Activities should be cheap, on a convenient moment, focused on their children and not on themselves, not tutoring, not theoretical, and school-or home-based.</p> <p>Conclusions</p> <p>Parents want to be involved in activities related to energy balance-related behaviours if this implies 'doing things together' with their child at school or at home.</p