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. 2018 Mar 15;25(3):279-290.e7.
doi: 10.1016/j.chembiol.2017.12.007. Epub 2018 Jan 4.

Target Identification and Mechanism of Action of Picolinamide and Benzamide Chemotypes with Antifungal Properties

Affiliations

Target Identification and Mechanism of Action of Picolinamide and Benzamide Chemotypes with Antifungal Properties

Verena Pries et al. Cell Chem Biol. .

Abstract

Invasive fungal infections are accompanied by high mortality rates that range up to 90%. At present, only three different compound classes are available for use in the clinic, and these often suffer from low bioavailability, toxicity, and drug resistance. These issues emphasize an urgent need for novel antifungal agents. Herein, we report the identification of chemically versatile benzamide and picolinamide scaffolds with antifungal properties. Chemogenomic profiling and biochemical assays with purified protein identified Sec14p, the major phosphatidylinositol/phosphatidylcholine transfer protein in Saccharomyces cerevisiae, as the sole essential target for these compounds. A functional variomics screen identified resistance-conferring residues that localized to the lipid-binding pocket of Sec14p. Determination of the X-ray co-crystal structure of a Sec14p-compound complex confirmed binding in this cavity and rationalized both the resistance-conferring residues and the observed structure-activity relationships. Taken together, these findings open new avenues for rational compound optimization and development of novel antifungal agents.

Keywords: Sec14p; antifungal; benzamide; chemogenomics; co-crystal; functional variomics; lipid-transfer protein; picolinamide; target identification.

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Conflict of interest statement

Competing financial interests

The authors with the affiliation Novartis Institutes for BioMedical research are employees of Novartis Pharma AG and may own stock in the company. All other authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Chemogenomic profiling and hypersensitivity validation. A) Calculated profiles of the chemogenomic profiling experiment. HIP outlines hits directly affected by the test compound. HOP identifies synthetic interactions with the target. Essential genes are depicted by grey boxes, non-essential genes by black dots. B) Single strain validation of hits from the chemogenomic profiling experiments as recorded in duplicates. C, D) Confirmation of hypersensitivity of the Sec14/sec14 HIP strain against compound 2 and 3 as recorded in duplicates. E) The wildtype and two “bypass Sec14p” strains were spotted on rich medium (YPD) supplemented with DMSO or 20 μM of compound 2 and 3 and incubated at 30 °C for 48 hours. F) Transformants of the temperature-sensitive sec14-1ts yeast strain harboring centromeric (CEN) plasmids carrying either SFH1 or the designated SFH1 activation alleles (SFH1*) were spotted on minimal media supplemented with 120 μM of compound 2, 30 μM of compound 3 or DMSO as indicated and incubated at permissive (left) or restrictive (right) temperature. Transformants harboring YCplac33 (empty vector), or SEC14 expressed from a centromeric (CEN) or a multi-copy plasmid () served as controls.
Figure 2
Figure 2
Synthesis of a collection of benz- and picolinamides for structure-activity relationship studies.
Figure 3
Figure 3
Analysis of lipid transfer inhibition with recombinant Sec14p. A) Fifteen compounds were tested for inhibition of Sec14p-mediated [3H]-PtdIns transfer at a fixed concentration of 20 μM SMI (see Materials and Methods). Values represent mean ± s.e.m. of triplicate assay determinations from two independent experiments. Total radiolabel inputs per assay ranged from 8356–9604 c.p.m., backgrounds from 436–489 c.p.m. and the transfer efficiency from 14–16% of total input [3H]-PtdIns. Activities were normalized against the mock condition set at 100%. B) The five small molecules that inhibited Sec14p [3H]-PtdIns transfer by > 30% in the end-point assays in (a) were tested for inhibition of the structurally unrelated mammalian PITPα. [3H]-PtdIns input for these assays ranged from 10703–11202 c.p.m., assay background from 403–415 c.p.m., and [3H]-PtdIns transfer efficiencies ranged from 10%-11% of total input radiolabel. Values represent mean + s.e.m. of triplicate assay determinations from two independent experiments. C) IC50 values for Sec14p-active compounds. Small molecule inhibitors of Sec14p were titrated into PtdIns-transfer assays to determine IC50 values. [3H]-PtdIns input into these assays varied from 9649 - 11034 c.p.m, backgrounds ranged from 243–648 c.p.m., and transfer efficiencies as functions of total [3H]-PtdIns input ranged from 12–18%. D) IC50 curves for compounds 2 and 3 tested against Sec14p of S. cerevisisae, C. albicans and C.glabrata. IC50 values represent a 95% confidence interval from two independent experiments, with triplicate determinations for each data point
Figure 4
Figure 4
Structural and functional analysis of Sec14p-compound interactions. A) SNPs and mutation frequency as identified by functional variomics for both tested compounds. B) Validation of identified mutations by integrative transformation of indicated SEC14 allele into cells. C) Validation of identified mutations by in vitro testing on recombinant protein. D) Overview of the structure of Sec14p (as cartoon, in marine) in complex with compound 2 (in sticks representation, in grey). The section displayed in detail in panel e is outlined by dotted lines. e) Detailed view (rotated 45° along the x-axis for clarity) of the binding pocket of Sec14p bound to compound 2 as identified by co-crystallization at 2.6 Å resolution. Interacting residues and relevant secondary structure elements of Sec14p are labeled; side chains are colored in marine and shown as sticks. H- and halogen-bonding are visualized as solid lines in blue and green, respectively while hydrophobic interactions are shown as grey dashed lines. p-p stackings are depicted as green dashed lines (light green for parallel stacking; smudge green for perpendicular stacking) with aromatic ring centers as grey spheres. Functional groups of compound 2 are indicated.
Figure 5
Figure 5
Sec14p sequence comparison and investigations into the VV motif. A) Protein sequence alignment of Saccharomyces cerevisiae Sec14p and Sfh1p and Sec14p of the pathogenic fungi Candida albicans, Candida dubliniensis, Candida glabrata, Aspergillus nidulans and Cryptococcus neoformans. Resistance-conferring amino acids identified by the functional variomics screen are marked with black boxes, sites predicted by co-crystal structure to be involved in compound interactions with grey boxes. B) A wildtype strain and isogenic derivatives expressing physiological levels of C. albicans and C. glabrata Sec14 PITPs were spotted on rich medium (YPD) agar medium supplemented with the indicated compounds and incubated at 30 °C for 48 hours. Transplacement of the VV-motif into Sec14pCA (the M154V, C155V double mutant) did not render this PITP sensitive to compound 3 (lower panel).

Comment in

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