Piperazinyl quinolines as chemosensitizers to increase fluconazole susceptibility of Candida albicans clinical isolates

The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.National Institutes of Health (U.S.) (1 R03 MH086456-01

Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(−9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(−9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA

Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles

Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways.National Institutes of Health (U.S.) (NIH-MLPCN program (1 U54 HG005032-1))National Institutes of Health (U.S.) (NIH (1 R03 MH086456-01)

Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles

Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have led to the identification of an indole derivative capable of sensitizing strains demonstrating resistance to fluconazole. This tetracycle (2, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in S. cerevisiae models, and may be a useful probe to uncover alternative resistance pathways

Relative IC₅₀ for two “known” and two “novel” compounds in both BL2-NFκB-Luc and Ramos-NFκB-Luc cell lines.

<p>Relative IC₅₀ is the concentration required to bring the dose-response curve to the halfway point between the top and bottom plateaus of the curve.</p

CD40 knockdown and CD40-luciferase assay in BL2 cells.

<p>(A) Schematic of the canonical CD40 – NF-|B signaling pathway in B cells. (B) RNAi perturbation of <i>CD40</i> in two distinct clones derived from BL2 cells decreases CD40 protein levels by 55% (left) and 40% (middle) compared to the BL2 parent line (black, right); (C) More CD40 on the surface of BL2 cells increases RelA (p65) phosphorylation following activation with tCD40L, as measured by Western blot, with maximum activation at 15 minutes. Results are shown for the same two shRNA lines and parental BL2 cell line as in (B). This is a representative example of multiple experiments. (D) Titration of tCD40L leads to increased luciferase activity. Each experiment was performed in triplicate. The red circle represents ∼80% maximum luciferase activity (64 ng/ml tCD40L). Luciferase activity at baseline (i.e., no tCD40L activation) was subtracted from each measurement to plot results. (E) Titration of IKK inhibitor VII leads to inhibition of luciferase activity following tCD40L activation. Each experiment was performed in duplicate. (F) The luciferase assay is robust, with Z'-factor>0.80 and >60-fold inhibition of luciferase activity without killing cells across different plates.</p

Genetic data on risk of RA and CD40 protein levels.

<p>(A) The regional association plot from analysis of Immunochip (iChip) data in 7,222 CCP+ cases and 15,870 controls. Gene location is shown along the bottom of the graph, with observed –log(P) value along the left Y-axis and recombination rate along the right Y-axis. Each SNP is plotted is a circle, with color scheme (red to white) in reference to the extent of linkage disequilibrium with the index SNP, rs4810485 (labeled as a diamond). (B) The regional association plot from analysis of iChip data and CD40 protein levels in 90 healthy control individuals. (C) A box-whisker's plot of SNP (rs4810485) and CD40 protein levels in B cells from healthy control individuals, where T = non-risk allele and G = risk allele. (D) A box-whisker's plot of SNP (rs4810485) and <i>CD40</i> mRNA levels in PBMC's from two separate collections (total of 1,441 healthy control individuals); T = non-risk allele and G = risk allele.</p

Small molecule screen of CD40-mediated NF-kB signaling in BL2 cells.

<p>(A) Results from duplicate experiments screening 1,982 compounds. Red circles are our positive control (IKK inhibitor VII); grey circles are our neutral controls (DMSO only); and blue circles are test compounds. The red dashed line indicates >2SD from the mean of the neutral controls, which defines our “hit” compounds (n = 81 compounds). (B) Dose-response curves for two compounds known to inhibit inflammation [CID = 5282230 (tranilast)] or NF-|B signaling [CID = 5282360 (4-hydroxy-estradiol)] in the BL2-NF|B-Luc cell lines. (C) Dose-response curves for two compounds not previously implicated in inflammation, NF-κB signaling, CD40 signaling, or other biological pathways related to rheumatoid arthritis: CID = 306804, [4-(1-acetyl-4-oxo-2H-3,1-benzoxazin-2-yl)phenyl] acetate; and CID = 7309015, 8-[(Z)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]-7-hydroxy-4-methylchromen-2-one. Red line = cells activated with tCD40L; black line = cells activated with either CD40 or LPS (in BL2-TLR4-NFκB-Luc cells); green line = cell toxicity, as measured by CellTiter-Glo.</p