A systems biology analysis of thioredoxin reductase inhibition to support discovery of a novel antifungal treatment

Description

Fungal diseases affect approximately 1.2 billion individuals worldwide with at least 1.5 million deaths each year. The emergence of drug resistant strains has made clinical treatment failures more frequent.

Fungal thioredoxin reductase 1 (TRR1) is essential for viability in all three major human fungal pathogens – Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, and is structurally distinct from the human thioredoxin reductase enzyme. This offers an exciting opportunity to develop a small-molecule drug with broad spectrum single agent activity against clinically resistant fungi.
Because pharmacological inhibition may not completely suppress Trr1 activity all the time, elucidating the effects of titrated TRR1 inhibition is required to determine the minimum requirement for a novel treatment and to identify and circumvent potential tolerance/resistance mechanisms in major human fungal pathogens.

Plan of Investigation:
Quantification of the dose-effect relationship of TRR1 inhibition. Using engineered fungal strains that express TRR1 from conditional promoters, we will modulate TRR1 expression levels, assay the impact on thioredoxin oxidation status and correlate these metrics with fungal fitness/viability. This will inform the degree of Trr1 inhibition required to attenuate virulence and establish a toolkit with which to predict and optimise efficacious therapeutic regimens.

Mouse model. Using established models of invasive fungal disease, the impact of in vivo titration of TRR1 expression will be assessed by quantitation of tissue fungal burden using qRT-PCR to determine the dynamics of fungal clearance following TRR1 knock-down in established fungal infection. Infected organs will be excised, homogenised and cultured for enumeration of viable fungal colonies, and histopathological analysis will assess the extent of fungal colonisation and inflammatory pathology in tissues. Peripheral blood will be sampled at relevant intervals for serological detection of fungal infection, thereby establishing a means for onward use of non-invasive clinical observation markers.

Systems Biology modelling. A mathematical model will be developed to quantitatively describe thioredoxin oxidation, serological markers and cell survival in vitro. This model will be expanded for in vivo mouse model data, and ultimately forecast possible clinical fungal load responses.

Disease modelling for invasive fungal infection. We will survey the literature to construct a model that connects fungal load and patient survival for invasive fungal infection, which will be used in conjunction with the systems biology model to explore possible clinical outcomes.

The student will undergo a highly interdisciplinary training to acquire and integrate in vitro, in vivo and in silico techniques relevant to drug discovery and development.

The student will gain tremendous skills for drug discovery and development by the training provided in this PhD project. As quantitative modelling is concerned, the student will receive training with S1 to develop and validate systems biology models to encompass data generated at different dimensional and temporal scales. This student will learn to develop nonlinear mixed effects modelling to recapitulate pharmacokinetics data. The project will also equip the student with essential clinical statistics experience by performing survival analysis. These skills are in short supply in the UK and globally for drug discovery and development.

The student will also receive training with S2 in the molecular manipulation of fungal pathogens to modulate drug target levels (conditional target expression and quantification at the mRNA and protein level), and in the biochemical and cell biology approaches to assess direct and indirect readouts of target engagement.
The student will receive training via S3 on in vivo approaches to infection modelling, including a range of analytical techniques for quantitation of pathogen dynamics during interaction with whole animal hosts, and mode of action and efficacy of antifungal therapies.

This project will deliver training in a suite of valuable wet lab skills to assist in defining PK/PD relationships which are broadly applicable to different therapeutic areas. Specific to this project is the training in redox biology and the importance of protein oxidation in mediating fungal virulence.

These skills will prepare the student for further developing a successful career in drug discovery and development, either in the academia or the industry. Rotations in the co-supervisors’ research groups
Rotation in S2’s laboratory will provide training to the student in molecular, biochemical, and cell biology approaches to study the impact of modulating Trr1 expression levels (as a proxy for target engagement) on fungal redox signalling and fitness. Not only will this provide the quantitative data necessary for the modelling, it will also provide wet lab training to the student which will nicely complement the mathematical modelling theme of the PhD. Specifically, the student will gain experience in engineering a range of human fungal pathogens to conditionally express TRR1, and the molecular (qRT-PCR) and biochemical (western blotting) tools to quantify the levels of the target (Trr1). Trr1 functions to reduce thioredoxin (which becomes oxidised upon reducing oxidised protein targets such as the transcription factor Cap1) and the student will gain experience in detection of thioredoxin oxidation and secondary readouts (i.e. regulation of Cap1-dependent gene expression).

The student will also directly assess the impact of Trr1 inhibition on fungal growth and survival both in vitro and in vivo. Via S3 the student will learn best practice in analysis of in vivo pathogen dynamics in the murine host including application of analytical methodologies that quantify pathogen burden, antimicrobial efficacy and immunopathology.