Date of Award

5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Bioanalysis

Research Advisor

Glen E. Palmer, PhD

Committee

Jarrod R. Fortwendel, PhD; Kirk E. Hevener, PhD; Brian Peters, PhD; Todd B. Reynolds, PhD

Keywords

Antifungal development;Candida albicans;Chemical biology;Coenzyme A biosynthesis;Pantothenate kinase;Pantothenate synthetase

Abstract

An estimated 1.5 million people die each year from invasive fungal infections (IFIs) involving dissemination to the deeper organs via the bloodstream, with estimated healthcare costs of over $7 billion in the U.S. alone. Collectively, several Candida species account for more than 75% of disseminated fungal infections in the U.S., with attributable mortality rates ranging 35-75%. Unfortunately, the prospect of curing these infections is limited by the modest efficacy of the available antifungal drugs: the azoles, echinocandins, and amphotericin B. Approximately one-third of patients with disseminated Candida infections are non-responsive to treatment with the azoles, and favorable response rates are just 52-73% for the echinocandins and just 62% for amphotericin B. As such, there is an urgent need for development of new antifungal drugs with improved clinical efficacy.

Coenzyme A (CoA) is a universal and essential cofactor for several key metabolic pathways including fatty acid oxidation and synthesis, the tricarboxylic acid cycle, and sterol biosynthesis. Nearly all organisms must synthesize their own CoA from pantothenic acid (vitamin B5-PA) through a five-step sequence of reactions requiring ATP and cysteine. Several bacterial species can take up exogenous PA through the PanF transporter, or produce it de novo from beta-alanine and pantoate, in a reaction catalyzed by pantothenate synthetase (PS). Similarly, the model yeast, Saccharomyces cerevisiae, can acquire exogenous PA through the pantothenate symporter (Fen2p) or synthesize it de novo using PS, encoded by the PAN6 gene. In contrast, mammalian cells lack the enzymes required for endogenous PA production, instead depending upon a sodium-driven multivitamin transporter to import from exogenous sources. Thus, this has raised interest in exploiting the fundamental difference in physiology to develop pathogen-selective antimicrobial agents targeting enzymes involved in PA production. Pantothenate kinase (PanK), encoded by the CAB1 gene in S. cerevisiae, catalyzes the first step in the conversion of PA to CoA. PanK has been confirmed to be essential for the viability of S. cerevisiae as well as several bacterial species. However, the CoA biosynthetic pathway is not well characterized in one of the most medically important human fungal pathogens, C. albicans. Therefore, we sought to investigate the importance of pantothenate uptake, synthesis, and conversion to CoA for C. albicans survival and virulence.

A Candida albicans fen2∆/∆ mutant was viable in vitro and virulent in a mouse model of disseminated infection. In contrast, the growth of C. albicans strains with doxycycline repressible expression of PAN6 (PTETO-PAN6) or CAB1 (PTETO-CAB1), was arrested in the presence of doxycycline, even in medium supplemented with pantothenate. Furthermore, overexpression of C. albicans FEN2 was not sufficient to restore growth of PTETO-PAN6 even in the presence of PA. This suggests that C. albicans is unable to uptake sufficient exogenous PA to support growth. Moreover, neither strain was virulent in doxycycline treated mice. Collectively, these results establish that Cab1p and Pan6p are essential for C. albicans survival and virulence, and therefore, are valid targets for antifungal development.

To facilitate the discovery of Pan6p or Cab1p inhibitors, we validated high-throughput compatible screening assays. Target-based whole-cell and biochemical screens identified small molecules that specifically target C. albicans Cab1p or Pan6p. This includes a compound that targets CaCab1p and has broad spectrum antifungal activity as well as in vivo efficacy. In addition, we solved the first eukaryotic pantothenate synthetase crystal structure, C. albicans PS, to support identification and optimization of compounds that specifically interact with the fungal protein in an effort to expand development of novel antifungal therapies with improved patient outcomes.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0002-4409-004X

DOI

10.21007/etd.cghs.2024.0657

Additional Files
2024-007-Regan-DOA.pdf (103 kB)
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