G-rich VEGF aptamer as a potential inhibitor of chitin trafficking signal in emerging opportunistic yeast infection

Highlights

• S. cerevisiae as an opportunistic friend for humans can cause critical infection, especially in immune-compromised patients.

• GVEGF as a potential inhibitor against the CHS5-CHS6 complex Formation.

• The capacity of initiation complex formation of the exomer class IV chitin synthase by CHS5–CHS6, but not by CHS5-Bach1.

• The electrostatic surface of CHS6 at C5BD plays a key role in shaping the protein-aptamer interaction.

• Interestingly, protein structures of CHS6 and Bach1 are very similar, but they have only 25% identity at the amino acid sequence.

Abstract

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5–CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5–CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.

Introduction

In recent decades, fungal infections have dramatically increased, particularly in developed countries. S. cerevisiae is currently being developed as opportunistic for fungal pathogens when the immune system is impaired. S. cerevisiae can generate a variety of diseases from systemic infection to death in the severe condition (Pérez-Torrado and Querol, 2015). S. cerevisiae strains present a particular transcription model after infection of the human blood. Another critical point is that the genomes in yeast and mammals that encode very similar proteins. High similarity in protein sequence and function between mammalian and these fungal cells make developing antifungal drug more challenging with less site effect (Baldauf and Palmer, 1993). S. cerevisiae have a specific protective system to resist human defenses which increases the oxidative stress response by activating the Yap1p transcription factor regulation to prevent the lethal effects of reactive oxygen species (ROS) produced by the macrophage (Pérez-Torrado and Querol, 2015). Chitin synthase is one such hopeful curative target because it is absent in plants and vertebrates. Chitin synthase so far has failed to inhibit in clinically, however newly they are significant for fungal infections researchers (Ruiz-Herrera and San-Blas, 2003; Odds et al., 2003). Targeting chitin synthesis and drug efficacy in S. cerevisiae is limited due to the cell wall compensatory responses. Chitin synthesis is controlled by several regulatory mechanisms, some of which are not critical to the synthesis of chitin (Rogg et al., 2012). CHS3, a class IV synthase, produces 90% of the chitin of S. cerevisiae and essential for cell growth (Bulawa, 1992; Banks et al., 2005). Chitin is a glycan ingredient, of β-1, 4-linked N-acetyl-D-glucosamine that exists in most fungal cell walls (Gooday, 1990; Hackman and Goldberg, 1974). In S. cerevisiae, transport of CHS3 to the plasma membrane in the bud neck region is essential in the regulation of the chitin synthesis. CHS5 and CHS6 synthases are necessary for trafficking of the CHS3 from the early endosome and trans‐Golgi network and early endosome to the plasma membrane. CHS6 belongs to a family ChAPs (CHS5-Arf1-binding proteins: Bch1, Bch2, Bud7, CHS6) (Trautwein et al., 2006). The ChAPs family proteins form the exomer complex with CHS5 and are involved in trafficking of specific cargo proteins, from the Golgi to the plasma membrane (Santos and Snyder, 1997). Carrier of the CHS3 to the cell surface can be blocked by remove of the CHS5 or CHS6 genes or concurrent elimination of the BUD7 and BCH1 genes (Trautwein et al., 2006; Sanchatjate and Schekman, 2006; Wang et al., 2006; Starr et al., 2012). Nucleic acid aptamers and the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process were discovered over two decades ago (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Aptamers often nominated ‘chemical antibodies,’ are functionally comparable to traditional antibodies, but offer several advantages, including their relatively small physical size, flexible structure, quick chemical production, versatile chemical modification, high stability and lack of immunogenicity (Zhou and Rossi, 2017). The clinical potential of aptamers highlighted by the FDA approval of several therapeutic aptamers including an aptamer-based drug for macular degeneration (Ng et al., 2006; Kourlas and Schiller, 2006) also by clinical trials for assessment of the safety and efficiency of systemically administered aptamers (Sundaram et al., 2013; Dolinsky et al., 2013). The aptamer is a small nucleic acid with high selectivity and affinity for target receptors and has recently been drawn the attention in applying as an inhibitor. In silico analysis for designing and screening of aptamers has many advantages including the short time for evaluation and understanding of physicochemical properties of the interaction of the aptamer with a receptor (Ahirwar et al., 2016). Locked nucleic acid-modified DNA aptamer targeting the binding region of VEGF on VEGF-receptor and blocking its interaction is shown in various breast cancers (Edwards et al., 2015). Locked residues contribute to the thermal stabilization of the adopted structure and formation of structurally preorganized intermediates that promote folding into a single G-quadruplex (Marušič et al., 2013). Aptamer modifications of the locked nucleic acids turn it to improve binding affinity and higher nuclease resistance (Veedu and Wengel, 2009). Sugar parts of locked nucleic acids with their O2′-C4′-methylene bridge are locked in an N-type conformation, mimicking nucleic acids conformation (Petersen et al., 2002).

In this study, we used high-throughput virtual screening of the aptamer from RCSB data bank to approach an aptamer with highest potential binding interaction to the specific binding site

CHS6; therefore this can block the formation of the exomer complex which subsequently inhibits CHS3 transport to the cell surface as a necessary step for chitin trafficking. Our study investigated an aptamer as a promising target chitin synthase for antifungal drug.