Structure-function analysis of a pH-responsive molecular switch required for fungal pathogenicity

Most fungi are extremely useful, for example, the production of antibiotics, bread, wine, and beer all involve fungi. However, like bacteria and viruses, fungi can cause life-threatening diseases. Unlike bacteria and viruses, fungal cells share many similarities with those of humans. For this reason it has been very difficult to identify drugs which will kill fungi, without causing harm to the human patient.

Infectious diseases caused by fungi are a worldwide problem and recent estimates suggest that fungal infections cause at least as many deaths as malaria and tuberculosis. There are currently only three classes of drugs with which to treat fungal infections, and there are limitations associated with all of them. This research project will characterize a group of fungal proteins, already proven to be required for survival in the mammalian host, with a view to finding drugs which inhibit them. We will focus upon a specific class of signaling protein which is outstandingly amenable to drug discovery, known as G protein coupled receptors (GPCRs). Fungal cells use GPCR-like proteins to gather information about their surroundings, it is therefore likely that chemistries to inhibit these proteins already exist but have been overlooked in conventional drug screening approaches.

Roughly half of currently licensed drugs target GPCRs. GPCR-like proteins are located at the surface of the cell, and have a characteristic structure composed of seven membrane-spanning segments. GPCRs are sensory molecules which detect signals outside of the cell, and convey this information via intracellular signaling molecules, to elicit a cellular response. The success of GPCRs as drug targets is due to their location at the surface of the cell, the existence of important pockets or folds in the proteins which can be blocked by drugs, and the availability of multiple technologies and tools to monitor GPCR-mediated signaling.

Most fungi use a GPCR-like protein (called PalH or Rim21) to sense the pH of their surroundings. In pathogenic fungi the loss of PalH, or the transcription factor (PacC) it communicates with, prevents fungal survival in the mammalian host. Recently, in the major mould pathogen of humans, Aspergillus fumigatus, we found that loss of pH signalling impacts multiple processes associated with fungal infection including cell wall biosynthesis and penetration of the lung lining. Additionally we found that mutants defective in pH signaling became highly susceptible to killing by echinocandin antifungal drugs, which are usually unable to kill Aspergillus species. When we tested this observation in a mammalian infection model, we found that pH non-signaling mutants were cleared much more efficiently in echinocandin-treated mice. Thus alone, and in combination with existing antifungal drugs, inhibition of fungal pH signaling could be a valuable treatment option.

During this research we will find out how PalH sensors detect extracellular pH by systematically mutating the sensor. Mutations which prevent pH signaling will inform us upon which segments of the protein are required for signaling. Additionally we will try to make enough of the PalH protein in the laboratory to allow formation of crystals and analysis of the protein structure. In practice the resolution of membrane protein structure has been very challenging but recent methodological advances have improved success rates and access to a 3D model of protein structure will unlock many possibilities for computational analysis of the protein and possible inhibitors. Finally, informed by the structural and mutational analyses, we will develop assays using fluorescent proteins, which will be useful for high throughput screening of PalH inhibitors. The tools and insight generated by this research will open new avenues for antifungal drug discovery. In fungal genomes many hundreds of GPCR-like proteins are encoded but to date the number characterized is less than 1 per cent.

Invasive fungal infections (IFIs) pose a severe and fatal threat to patients undergoing organ or stem cell transplantation, anti-cancer therapy, or suffering from AIDs. On a global scale IFIs cause equivalent mortality to TB and malaria, but our ability to cure IFIs remains critically restricted by the limited repertoire and suboptimal efficacy of licensed antifungal drugs.

Fungi use a GPCR-like protein (called PalH or Rim21), absent in humans, to sense external pH. In pathogenic fungi the loss of PalH, or the transcription factor (PacC) it communicates with, prevents fungal survival in the mammalian host. Recently, a major mould pathogen of humans, Aspergillus fumigatus, we found that loss of pH signalling impacts multiple processes associated with fungal infection including penetration of the lung lining.

The simplest model for PalH-mediated pH sensing would be one in which the PalH pH sensor exists in multiple conformational states, each having a different affinity for, and/or ability to activate, the PalF arrrestin partner. Plausible pH-dependent triggers of altered conformational equilibrium might include binding of a ligand, altered protonation state of amino acid side chains, modulation of interactions between PalH amino terminus and extracellular loops (ECLs), PalH oligomerisation, altered lipid-protein interactions, or alteration of physical characteristics of the cell such as cell volume, or cell wall composition. To distinguish between these multiple possibilities, we will probe the relative contributions of particular amino acid side chains, and carboxy- and amino-terminal domains to functionality of the PalH receptor. We will then redirect mutant constructs into recombinant protein expression vectors, yeast membrane two hybrid (YTMH) vectors, and A. fumigatus FRET and bimolecular fluorescence complementation strains to interrogate the effects of mutations on protein structure, arrestin engagement and receptor oligomerisation.

The global market for antifungal therapeutics was estimated to be in the region of $9.4 billion US in 2010 (GBI research) and is anticipated to exceed $12 billion by 2016 with expenditure in the EU accounting for around a third of this (BCC research). The highest grossing agents in 2010 were the systemic drugs, voriconazole ($825 million), caspofungin ($611 million) and itraconazole ($341 million) (Evaluate Pharma). The significance of Aspergillus and candida sp. to health has lead one pharmaceutical company (Merck) to express a public interest in acquiring an Aspergillus/Candida (or mould specific) anti-infective agent.

The health burden of diseases caused by fungi is considerable. Where severe invasive infections are concerned, deep-organ fungal infection occurs as a negative corollary to otherwise successful, but nonetheless expensive, treatments for serious illness. According to a working group of the Health Protection Agency Advisory Committee for Fungal Infection and Superficial Parasites, in 2002, almost 10,000 patients in England were estimated to have suffered a deep-organ fungal infection; almost half of them died from their fungal disease. New classes of antifungal drugs are urgently required and the antifungal drug development pipeline is currently sparse.

The annual healthcare costs go way beyond just the cost of therapeutics. The cost of invasive infections caused by Aspergillus sp. in the US alone was estimated at $633 million in 1996 and includes the cost of failed chemotherapy, bone marrow or organ transplantation procedures (Dasbach EJ, Davies GM, Teutsch SM. Burden of aspergillosis-related hospitalizations in the United States. Clin Infect Dis 2000;31:1524-8.).

The pipeline for new antifungal compounds is sparse. Very few antifungal compounds are currently in development. Three new triazoles (albaconazole, isavuconazole and ravuconazole) are in clinical trials however, as with the current triazoles they all target the lanosterol 14-demethylase and are likely to be dogged by similar resistance issues. The glucan synthase inhibitor in development by Merck has been stalled in Phase I since 2009, as have the novel compounds corifungin (Acea biotech), T-2307 (Toyama) and the HDAc inhibitor of Methylgene. A number of other compounds show pre-clinical promise, particularly E1210 an inhibitor of GPI-anchor biosynthesis (EISAI) however this too has stalled. All of this indicates potential problems in development.

Molecular switches: Currently synthetic molecular switches are of interest in the field of nanotechnology for application in molecular computers. The pH-responsive molecular switch under study in this proposal could be of future value for the development of novel medical devices, drug delivery strategies, and medical monitoring devices. We will work to train academic and commercial partners in the application and use of these tools, protecting where necessary any intellectual property.

Live cell imaging assays for fungal signal transduction: Researchers and commercial enterprises seeking live cell assays for inhibition of intracellular signalling pathways will benefit from this research. The FRET and Bimolecular Fluorescence reporter strains are a novel development for Apsergillus species as the platform we have developed allows a single-step transformation to introduce interacting partners of interest. These tools have commercial worth as they can facilitate high throughput screening for chemicals which inhibit fungal signalling pathways. We will work to train academic and commercial partners in the application and use of these tools, protecting where necessary any intellectual property.