Virtual Screening Yields Inhibitors of Novel Antifungal Drug Target, Benzoate 4‑Monooxygenase

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)­benzoic acid (compound <b>2</b>) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process

Virtual Screening Yields Inhibitors of Novel Antifungal Drug Target, Benzoate 4‑Monooxygenase

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)­benzoic acid (compound <b>2</b>) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process

Schematic presentation of the structure of TRIM28.

<p>The conserved RBCC motif is present at the N-terminal domain; the variable C-domain includes the PHD and BROMO domains. Image adapted from Hatakeyama <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688-Hatakeyama1" target="_blank">[45]</a>. Author's approval was obtained for the use of this image; original image is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688.s002" target="_blank">Figure S2</a>.</p

Schematic presentation of the whole workflow.

<p>1. Material preparation – expanding a GBM cell line, laminin grown and enriched in stem-like cells, 2. Llama immunization with whole GBM cells, 3. Construction of a nanobody library, 4. Phage display cycle – enrichment of antigen specific nanobodies on various biological samples (whole protein extract from GBM tissues, membrane protein-enriched fractions from GBM tissues and GBM stem-like cell lines), 5. ELISA - selection of GBM specific nanobodies, 6. Nanobody production – large scale production and purification of specific nanobodies, 7. Nanobody:antigen pairs – immobilizing the nanobodies and binding to the corresponding antigens, 8. Antigen identification by mass spectrometry, 9. Antigen validation by Western blot. See Materials and methods for additional experimental details.</p

Sequences of the nanobodies that were chosen for the large-scale production.

<p>The amino acid sequence of the CDR3 regions of the nanobodies is given in alphabetic order.</p

Antigen validation.

<p>Western blot membrane after probing with a monoclonal anti-GAPDH antibody (36 kDa band) used as an internal control for amount of protein loaded in each lane, a monoclonal anti-β-actin antibody (48 kDa band) and monoclonal anti-TRIM28 antibody (100 kDa band). Samples: GSCm – membrane protein-enriched extract isolated from GBM stem-like cell lines; GBMm – membrane protein-enriched extract isolated from GBM tissues; NBTm – membrane protein-enriched extract isolated from human brain samples; GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples; GBMw – whole protein extract from GBM tissues; NBTw – whole protein extract from human brain samples.</p

Relative band intensities of antigens validated with Western blot.

<p>The relative band intentisy of the antigens, TRIM28 and β-actin, was calculated as the ratio between arbitrary units of the band of the antigen and the arbitrary units of the band of the internal control, GAPDH [AU(antigen)/AU(GAPDH)]. Samples: GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples.</p