High-Throughput Screen for Identifying Small Molecules That Target Fungal Zinc Homeostasis

Resistance to traditional antifungal drugs has increased significantly over the past three decades, making identification of novel antifungal agents and new targets an emerging priority. Based on the extraordinary zinc requirement of several fungal pathogens and their well-established sensitivity to zinc deprivation, we developed an efficient cell-based screen to identify new antifungal drugs that target the zinc homeostasis machinery. The screen is based on the zinc-regulated transcription factor Zap1 of Saccharomyces cerevisiae, which regulates transcription of genes like the high-affinity zinc transporter ZRT1. We generated a genetically modified strain of S. cerevisae that reports intracellular zinc deficiency by placing the coding sequence of green fluorescent protein (GFP) under the control of the Zap1-regulated ZRT1 promoter. After showing that the GFP fluorescence signal correlates with low intracellular zinc concentrations in this strain, a protocol was developed for screening small-molecule libraries for compounds that induce Zap1-dependent GFP expression. Comparison of control compounds and known modulators of metal metabolism from the library reveals a robust screen (Z′ = 0.74) and validates this approach to the discovery of new classes of antifungal compounds that interfere with the intracellular zinc homeostasis. Given that growth of many pathogenic organisms is significantly impaired by zinc limitation; these results identify new types of antifungal drugs that target critical nutrient acquisition pathways

Antifungals: Need to Search for a New Molecular Target

In the 1990s, drug resistance has become an important problem in a variety of infectious diseases including human immunodeficiency virus infection, tuberculosis, and other bacterial infections which have profound effects on human health. At the same time, there have been dramatic increase in the incidence of fungal infections, which are probably the result of alterations in immune status associated with the acquired immuno deficiency syndrome epidemic, cancer chemotherapy, and organ and bone marrow transplantation. The rise in the incidence of fungal infections has exacerbated the need for the next generation of antifungal agents, since many of the currently available drugs have undesirable side effects, are ineffective against new or reemerging fungi, or lead to the rapid development of the resistance. This review will focus on the pathogenic yeast Candida albicans, since a large body of work on the factors and mechanism associated with antifungal drug resistance in this organism is reported sufficiently. It will certainly elaborate the probable molecular targets for drug design, discovered to date

Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192.

Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1

Comparison of the distance between catalytic calcium and the ligand in the protein ligand complex.

<p>The position of the ligand substrate in the active site was monitored by plotting the distance between catalytic calcium and the centre of the mass of substrate. (A-D) depict the distance between the catalytic calcium of the rh-PON1_(wt) (<b>—</b>), rh-PON1_(H115W,R192) (<b>—</b>),rh-PON1_{(H115W,R192K)} (<b>—</b>), and rh-PON1_{(H115W,R192I)} (<b>—</b>) proteins and the bound ligands over the course of the MD simulations. The ligands used were panel (A)–Pxn; panel (B)—Pha, panel (C)—<i>δ</i>-val, and panel (D)—TBBL.</p

OP-hydrolyzing activity of the rh-PON1 enzymes.

<p>(A) compares the Pxn-hydrolyzing activity of the recombinant enzymes. The Pxn-hydrolyzing activity was determined using direct assay. Equal amounts of the rh-PON1 enzymes were incubated with 1 mM paraoxon in the activity buffer (50 mM Tris-HCl, pH 8.0 and containing 1 mM CaCl₂) and the hydrolysis of Pxn was recorded at 405 nm. (B) and (C) depict the CPO- and DFP- hydrolyzing activity of the enzymes, determined by using an indirect AChE-inhibition assay, as described in the Experimental procedure. The concentration of CPO and DFP used were 75 μM and 200 μM (final concentration), respectively. The hydrolytic activity of rh-PON1_(wt) was taken 100% and the percentage activities of the rh-PON1 mutants were calculated. Enzymatic assays were performed in duplicate. Various mutants were named with single letter code representing the particular amino acid at position 192. <b>Legends:</b> wt, rh-PON1_(wt); K, rh-PON1_{(H115W,R192K)}; R, rh-PON1_(H115W,R192); Q, rh-PON1_{(H115W,R192Q);} N, rh-PON1_{(H115W,R192N)}; D, rh-PON1_{(H115W,R192D)}; E, rh-PON1_{(H115W,R192E);} S, rh-PON1_{(H115W,R192S);} T, rh-PON1_{(H115W,R192T)}; W, rh-PON1_{(H115W,R192W)}; Y, rh-PON1_{(H115W,R192Y);} F, rh-PON1_{(H115W,R192F);} L, rh-PON1_{(H115W,R192L)}; I, rh-PON1_{(H115W,R192I)}; V, rh-PON1_{(H115W,R192V);} P, rh-PON1_{(H115W,R192P);} G, rh-PON1_{(H115W,R192G)}; A, rh-PON1_{(H115W,R192A)}.</p

Kinetic parameters for hydrolysis of Pxn by rh-PON1 mutants.

<p>a,b,c- Data derived from Bajaj et al. (2014) Biochimie, 22: 210–214.</p

Molecular surface representation of the active site of rh-PON1_{(H115W,R192I)} protein containing <i>δ</i>-val (A) and TBBL (B) ligands.

<p>The molecular surface of the active site of the protein is shown in grey colour and the active site residues W115, D183, H184, I178, D269 and the catalytic calcium are indicated by red, blue, green, yellow, cyan and magenta colours, respectively. <i>δ</i>-val and TBBL are shown in stick model and colour by atom type (red—oxygen; yellow—sulphur; orange—carbon). Note that in the rh-PON1_{(H115W,R192I)} containing TBBL <b>(B)</b>, the oxygen atom of TBBL is oriented towards the carboxyl oxygen of D183 (blue).</p

H-bonding network in the active site of the TBBL-bound rh-PON1 protein complexes.

<p>The amino acid residues of the rh-PON1 proteins are shown in stick format and colour by atom type (<b>red</b>–oxygen; <b>blue</b>–nitrogen). The yellow broken lines show H-bonding interaction between the amino acid residues. Catalytic calcium is represented by yellow spheres. <b>Panels (A-D)</b> depict rh-PON1_(wt), rh-PON1_(H115W,R192), rh-PON1_{(H115W,R192K)}, and rh-PON1_{(H115W,R192I)} proteins. Differential H-bonding network around position 192 was observed in these proteins.</p

Lactone-hydrolyzing activity of the rh-PON1 enzymes.

<p>(A) and (B) compare the TBBL- and <i>δ</i>-val-hydrolyzing activity of the rh-PON1 enzymes, respectively. The TBBL-hydrolyzing activity was determined using Ellman-based colorimetric assay. Equal amounts of the rh-PON1 enzymes were separately incubated with 0.5 mM TBBL in the activity buffer containing 0.3 mM DTNB and the hydrolysis of TBBL was monitored at 412 nm. The <i>δ</i>-val-hydrolyzing activity of the rh-PON1 enzymes was determined by pH-indicator assay. The enzymes were incubated with 1 mM (in 50 mM bicine buffer pH 8.3, 1 mM CaCl₂) and the hydrolysis of <i>δ</i>-val was monitored at 577 nm using <i>m</i>-cresol purple as the indicator. The hydrolytic activity of rh-PON1_(wt) was taken 100% and the percentage activities of all the rh-PON1 mutants were calculated. Enzymatic assays were performed in duplicate. Various mutants were named with single letter code representing the particular amino acid at position 192. <b>Legends:</b> same as in the legends of Fig 3.</p