Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel Na1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on Na1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings

Assessing the activity of ETC inhibitors against atovaquone-resistant <i>T</i>. <i>gondii</i> parasites.

(A-G) Dose-response curves depicting the percent proliferation of WT (black) or atovaquone-resistant (ATVR, red) T. gondii parasites in the presence of increasing concentrations of (A) atovaquone, (B) buparvaquone, (C) auranofin, (D) trifloxystrobin, (E) azoxystrobin, (F) MMV024397, or (G) MMV688853. Values are expressed as a percent of the average fluorescence from a no-drug control at mid-log phase growth in the fluorescence proliferation assay, and represent the mean ± SEM of three (or four for (E)) independent experiments performed in triplicate; error bars that are not visible are smaller than the symbol. Inset bar graphs depict the EC50 ± SEM (nM) of three (or four for (E)) independent experiments. Paired t-tests were performed and p-values are shown.</p

Identification of selective inhibitors of the ETC in <i>P</i>. <i>falciparum</i>.

Dose-response curves depicting the proliferation of WT (black) or yeast dihydroorotate dehydrogenase (yDHODH)-expressing (red) P. falciparum parasites in the presence of increasing concentrations of (A) the known ETC inhibitor atovaquone, (B) chloroquine, a compound that does not inhibit the ETC, (C) buparvaquone, (D) auranofin, (E) trifloxystrobin, (F) azoxystrobin, (G) MMV024397, or (H) MMV688853 after 96 h of culture. Values are expressed as a percentage of the average proliferation of the drug-free control, and represent the mean ± SEM of three independent experiments performed in triplicate; error bars that are not visible are smaller than the symbol.</p

Screening the MMV ‘Pathogen Box’ for inhibitors of O₂ consumption in <i>T</i>. <i>gondii</i>.

The O2 consumption rate (OCR) of extracellular T. gondii parasites was measured in a 96-well plate using a Seahorse XFe96 extracellular flux analyzer. Compounds from the MMV ‘Pathogen Box’ were added to wells at a final concentration of 1 μM, and the change in OCR was monitored in real-time after each addition. Percent inhibition of OCR by each of the 400 compounds was calculated relative to complete inhibition observed after addition of the known OCR inhibitors atovaquone (1 μM) and antimycin A (10 μM), with each compound represented by a dot. A >30% inhibition cut off was applied (dotted line), with seven compounds inhibiting OCR by >30% at 1 μM (coloring of dots corresponds to coloring of labels of the chemical structures shown below). These hits included MMV689480/buparvaquone (burgundy), the endochin-like quinolone (ELQ) MMV671636 (green), MMV688754/trifloxystrobin (pink), MMV688978/auranofin (orange), MMV024397 (red), the aminopyrazole carboxamide MMV688853 (dark blue), and MMV021057/azoxystrobin (light blue). Data are from a single experiment, with the plate layouts and data points summarized in S1 Table.</p

Characterizing Complex III inhibition by the candidate ETC inhibitors in <i>T</i>. <i>gondii</i>.

(A-G) Complex III activity assays showing the change in absorbance of equine heart CytC at 550 nm over time (measured every 15 s) in the presence (+ para, dark shade) or absence (- para, light shade) of parasite extracts, and in the absence of drug (no drug, DMSO vehicle control, black or gray), or in the presence of (A) auranofin (orange, 10 μM), (B) atovaquone (green, 1.25 μM), (C) buparvaquone (burgundy, 5 μM), (D) trifloxystrobin (pink, 2.5 μM), (E) azoxystrobin (light blue, 80 μM), (F) MMV024397 (red, 20 μM) or (G) MMV688853 (dark blue, 20 μM). Decylubiquinol (DUBH2) was added at the indicated times. Data are from a single experiment and are representative of three independent experiments. (H) Complex III activity assays showing the change in absorbance of equine heart CytC at 550 nm over time where change in absorbance in the absence of parasite extracts (i.e. background absorbance) was subtracted from the change in absorbance in the presence of parasite extracts. Data are from a single experiment and are representative of three independent experiments. (TIF)</p

Effects of the identified MMV Pathogen Box compounds on <i>T</i>. <i>gondii</i> proliferation.

Determination of the inhibitory properties of the identified compounds on the proliferation of (A) human foreskin fibroblast (HFF) cells or (B) T. gondii parasites, including wild type (WT) RH, WT ME49, atovaquone-resistant (ATVR) ME49, and ELQ-300-resistant (ELQR) RH and the corresponding ELQ sensitive (ELQS) parental strains. Data are reported as average EC50 (nM) ± SEM from three or more independent experiments. The selectivity index (SI) was calculated by dividing the EC50 against HFF cells by the EC50 against WT RH T. gondii parasites, with SI values >1 indicating increased selectivity towards the parasite. The fold change (FC) was calculated by dividing the EC50 against ATVR or ELQR parasites by the EC50 against WT ME49 or ELQS parasites respectively, with FC values >1 indicating increased resistance and FC values R or ELQR strains. Paired t-tests were performed to compare the EC50 of the compounds in WT vs ATVR or ELQS vs ELQR parasites, and p-values are depicted as ns = not significant (p > 0.05), * p p < 0.01. ND = not determined.</p

MMV688853 dually targets <i>Tg</i>CDPK1 and the ETC in <i>T</i>. <i>gondii</i> parasites.

(A) Schematic depicting the small glycine gatekeeper residue of WT TgCDPK1 (white) which enables inhibition by 3-MB-PP1 and MMV688853. Mutation of this residue to a larger methionine residue (TgCDPK1G128M, red) blocks inhibitor access to the binding site and thereby confers resistance to these compounds. (B) Percent invasion of parasites expressing WT TgCDPK1 (white) or TgCDPK1G128M (red) into host cells in the absence of drug (DMSO vehicle control), or the presence of MMV688853 (5 μM), 3-MB-PP1 (5 μM) or atovaquone (1 μM), normalized relative to the no-drug control. At least 100 parasites were counted per experiment, with data representing the mean ± SEM of three independent experiments. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p-values shown. (C) Intracellular proliferation assays depicting the percent of vacuoles containing 1–8+ (gray tones) or abnormal (orange) parasites when parasites expressing WT TgCDPK1 (white) or TgCDPK1G128M (red) were cultured in the absence of drug (DMSO vehicle control), or the presence of MMV688853 (5 μM), 3-MB-PP1 (5 μM) or atovaquone (1 μM) for 20 h. Abnormal morphology was defined as vacuoles that contained misshapen parasites (representative images in S6 Fig). At least 100 vacuoles were counted per condition, with data representing the mean ± SEM of three independent experiments. (D) Dose-response curves depicting the O2 consumption rate (OCR) of parasites expressing WT TgCDPK1 (black) or TgCDPK1G128M (red) incubated with increasing concentrations of MMV688853 as a percentage of a no-drug (DMSO vehicle) control. Data represent the mean ± SEM of three independent experiments. Inset bar graph depicts the EC50OCR ± SEM (nM) of three independent experiments. The p-value from a paired t-test is shown. (E) OCR of parasites expressing WT TgCDPK1 (white) or TgCDPK1G128M (red) incubated in the absence of drug (DMSO vehicle control), or in the presence of MMV688853 (5 μM), 3-MB-PP1 (5 μM) or atovaquone (1 μM), expressed as a percentage of the OCR prior to addition of compounds. Data represent the mean ± SEM of three independent experiments. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p-values shown. (F) Dose-response curves depicting the percentage proliferation of parasites expressing WT TgCDPK1 (black) or TgCDPK1G128M (red) in the presence of increasing concentrations of MMV688853 over 6 days. Values are expressed as a percent of the average fluorescence from the no-drug control at mid-log phase growth in the fluorescence proliferation assay, and represent the mean ± SEM of five independent experiments conducted in triplicate; error bars that are not visible are smaller than the symbol. Inset bar graph depicts the EC50 ± SEM (nM) of five independent experiments. The p-value from a paired t-test is shown.</p

ELQ-300-resistant parasites exhibit cross-resistance to MMV688853 in O₂ consumption rate activity assays.

(A-C) Dose-response curves depicting the OCR of intact WT (black) or atovaquone-resistant (ATVR, red) T. gondii parasites in the presence of increasing concentrations of (A) atovaquone, (B) ELQ-300 or (C) MMV688853. (D-F) Dose-response curves depicting the OCR of intact parental ELQ-300 sensitive (ELQS; black) or ELQ-300-resistant (ELQR, blue) T. gondii parasites in the presence of increasing concentrations of (D) atovaquone, (E) ELQ-300 or (F) MMV688853. Values represent the percent OCR relative to the no-drug (100% OCR) and inhibitory atovaquone-treated (D-F) or antimycin A-treated (A-D; 0% OCR) controls, and depict the mean ± SEM of three independent experiments, each conducted in at least duplicate; error bars that are not visible are smaller than the symbol. Inset bar graphs depict the EC50OCR ± SEM (nM) of three independent experiments. Where relevant, paired t-tests were performed and p-values are shown.</p

Assessing the activity of ETC inhibitors against atovaquone-resistant <i>P</i>. <i>falciparum</i> parasites.

(A-H) Dose-response curves depicting the percent proliferation of WT (black) or atovaquone-resistant (ATVR, red) P. falciparum parasites in the presence of increasing concentrations of (A) atovaquone, (B) chloroquine, (C) buparvaquone, (D) auranofin, (E) trifloxystrobin, (F) azoxystrobin, (G) MMV024397, or (H) MMV688853 after 96 h of culture, as measured using a SYBR Safe-based proliferation assay. Values are expressed as a percent of the average fluorescence from the no-drug control, and represent the mean ± SEM of three independent experiments performed in triplicate; error bars that are not visible are smaller than the symbol. Inset bar graphs depict the EC50 ± SEM of three independent experiments. Paired t-tests were performed and p-values are shown.</p