About

Michael C. Fitzgerald is a Professor of Chemistry and a Professor of Biochemistry at Duke University. He serves as the Chair of Chemistry and is a member of the Duke Cancer Institute. His academic and research roles are based at the 3222 French Science Center, Durham, North Carolina. As a faculty member, he is involved in research and teaching within the Department of Biochemistry and the broader scientific community at Duke. His professional activities include leadership in the department and participation in initiatives related to biochemistry and cancer research.

Research topics

• Biology
• Biochemistry
• Cell biology
• Chemistry
• Computational biology
• Immunology
• Genetics

Selected publications

• MESH1-mediated coenzyme A degradation drives ferroptosis sensitivity and muscle pathology

  Journal of Clinical Investigation · 2026-04-25

  articleOpen access

  Coenzyme A (CoA) facilitates fatty acid synthesis, energy production, gene regulation, and antioxidant function. While CoA biosynthesis is well-characterized, the mechanisms governing CoA degradation remain poorly understood. Here, we identify the Metazoan Homolog of SpoT, MESH1, as a CoA phosphatase that dephosphorylates CoA at the 3' position of the ribose ring to form dephospho-CoA (dp-CoA). Recent studies have shown that CoA, similar to glutathione (GSH), is a cysteine-derived metabolite that protects cells against ferroptosis. Ferroptosis induced by blocking cystine import depletes CoA biosynthesis, while CoA restoration rescues cells from ferroptosis. We found that MESH1 knockdown preserved CoA levels by preventing its degradation, contributing to ferroptosis protection, indicating the bifunctional role of MESH1 in regulating CoA and previously reported NADPH. Mechanistically, MESH1 knockdown elevates CoA levels, maintaining functional mitochondrial thioredoxin system, thereby preventing mitochondrial lipid peroxidation. In Drosophila, we found that dMesh1 overexpression leads to ferroptosis-mediated muscle atrophy, which can be rescued by increasing CoA and NADPH levels. Taken together, these findings establish MESH1 as a key phosphatase that governs ferroptosis sensitivity by coordinating CoA and NADPH homeostasis, unveiling a novel link between CoA degradation, mitochondrial integrity, and muscle health.

  PublisherOA PDFDOI

• Data-Independent Acquisition (DIA) Strategy for Measuring Protein Stability Using Stability of Proteins from Rates of Oxidation (SPROX)

  Journal of the American Society for Mass Spectrometry · 2026-03-11

  articleOpen accessSenior authorCorresponding

  The stability of proteins from the rates of oxidation (SPROX) technique is a mass spectrometry-based approach for making protein folding stability measurements on the proteomic scale. The development and application of SPROX, to date, have primarily relied on the use of quantitative bottom-up proteomics and data-dependent acquisition (DDA) strategies using isobaric mass tags. Use of isobaric mass tags is attractive, as it enables the mass spectrometry readout in SPROX to be highly multiplexed. However, the use of such isobaric mass tags is restricted to DDA strategies, which can be limited in their proteomic coverage compared with data-independent acquisition (DIA) strategies. Reported here is a new “one-pot” SPROX workflow that employs a DIA readout and a label-free quantification strategy. Analysis of the proteins in an E. coli cell lysate using the DIA-SPROX strategy allowed for the calculation of transition midpoints with reasonable accuracy. The proteins from a S. cerevisiae cell lysate were also assessed for ligand-induced changes in their transition midpoints upon the introduction of cyclosporine A (CsA) to identify the protein targets of this well-studied ligand. The DIA-SPROX strategy developed here successfully identified known protein targets of CsA with a low false positive rate using a combination of two different software, Spectronaut and DIA-NN, for DIA data processing. We also find that the proteomic coverage obtained using DIA-SPROX is comparable to the coverage obtained in conventional DDA-SPROX experiments. Significantly, this comparable coverage can be achieved without a fractionation strategy (e.g., methionine-containing peptide enrichment) in DIA-SPROX.

  PublisherDOI

• Extrinsic and intrinsic factors affect copper‐induced protein precipitation across eukaryotic and prokaryotic proteomes

  Protein Science · 2025-05-15 · 1 citations

  articleOpen accessSenior authorCorresponding

  The susceptibility of a protein to aggregation upon exposure to copper ions (Cu) has been recognized as a contributor to Cu-induced cellular dysfunction and toxicity. Different cell types succumb to Cu to varying degrees, indicating innate differences between species in the mechanisms used to tolerate exposure to Cu in excess of their biological needs. Investigated here are properties associated with metal-induced protein precipitation (MiPP) compared across cell lysates generated from three cell lines from three different species: Escherichia coli, Candida albicans, and the human prostate cancer cell line 22Rv1. The human cell line was the most sensitive to Cu-induced protein precipitation, while C. albicans was the most tolerant. This trend aligns with the relative susceptibilities of these cells to Cu-induced cytotoxicity. The unique susceptibilities of these proteomes to precipitation by Cu were examined to identify factors that influence a protein's relative sensitivity to this effect. Identified were intrinsic factors such as frequency and solvent accessibility of known metal-binding amino acids, as well as external factors related to the molecular composition of their native cell lysates. Overall, our findings help to elucidate the biomolecular basis underpinning the unique capacity of adventitious Cu to have differential effects on eukaryotic and prokaryotic organisms and the level of Cu needed to induce protein precipitation.

  PublisherOA PDFDOI

• Discovery of Host-Directed Small Molecules with Broad Anti-Leishmanial Efficacy

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-04 · 1 citations

  preprintOpen access

  ABSTRACT Leishmaniasis, a neglected tropical disease affecting nearly 10% of the global population, suffers from limited therapeutic options and rising drug resistance. To address this, we developed 343 analogs of AR-12, a compound that has previously illustrated host-directed anti-leishmanial effects. Primary screening using a luminescence-based assay revealed 66 analogs with greater selectivity than the parent compound, AR-12. Sixteen promising candidates, selected for high potency (IC₅₀ < 1 µM) or high selectivity (>15), underwent secondary screening via Giemsa staining. Four lead compounds (53, 134, 197, and 354) demonstrated therapeutic indices greater than 40. Tertiary assays confirmed their broad in vitro efficacy against both Leishmania donovani and L. mexicana . Notably, 197 exhibited potent host-directed activity and proteomic analysis identified lysozyme as a mechanistic target, implicating it in the host-mediated clearance of intracellular parasites. These findings highlight the dual host– and pathogen-directed mechanisms of these compounds and support their potential as the basis for new therapeutic strategies. Further optimization and clinical exploration of these leads are warranted to meet the urgent need for effective leishmaniasis treatments. AUTHOR SUMMARY Leishmaniasis is a parasitic infectious disease with limited therapeutic options and rising drug resistance. Drugs that act directly against Leishmania can further drive drug resistance. Host-directed therapies work to enable the host responses to enhance pathogen clearance and reduce disease progression. Host-directed therapies both limit the emergence of new drug resistance and combat drug resistant infections. In this work we screened a library of 343 AR-12 analogs for host-directed anti-leishmanial activity. This work identified 4 hit compounds. We then did proteomic analysis to understand the mechanism of action of the primary lead compound, identifying the protein lysozyme as having a role in host-directed activity against Leishmania infection.

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• Copper Activates a Redox Switch to Reversibly Inhibit Glyceraldehyde-3-Phosphate Dehydrogenase

  Biochemistry · 2025-10-23

  articleOpen access

  Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the most conserved proteins across all kingdoms of life, has a multitude of moonlighting functions beyond its enzymatic role in glycolysis. Metal binding to GAPDH has previously been reported to inhibit enzymatic activity in several prokaryotic and eukaryotic systems, although the mechanism of inhibition has not been elucidated. In this study, we examined the effects of zinc, silver, and copper ions on Escherichia coli GAPDH (ecGAPDH) and explore the mechanism of inhibition via enzymatic activity assays under aerobic and anaerobic conditions, electron paramagnetic spectroscopy, and mass spectrometry. This study shows that Zn2+ does not affect ecGAPDH activity, while Cu2+ causes redox inactivation that oxidizes the protein upon reduction to Cu+. Cu+ binds tightly to the protein (log Ka = 15.2 ± 0.2, pH 7.4), with diminished affinity in the presence of G3P substrate. Although the anaerobic binding of Cu+ or Ag+ moderately diminishes catalytic turnover, these ions sensitize the protein to rapid and complete oxidative inactivation in the presence of oxygen. Oxidative modification of the active site cysteine, including glutathionylation, is reversible. This oxidative process, which occurs upon exposure to Cu and Ag, bestows GAPDH the ability to act as an all-purpose redox switch responsive to toxic metals as well as reactive oxygen species. This work provides insight into shared mechanisms by which cells use redox inactivation of sentinel enzymes like GAPDH to redirect metabolic processes for cellular protection.

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• Leveraging Vulnerabilities in Copper Trafficking for Synergistic Antifungal Activity

  ACS Chemical Biology · 2025-10-17

  articleOpen access

  Candida albicans is an opportunistic fungal pathogen that causes millions of infections per year, for which more efficacious treatments are needed. Observations that azole antifungals incite C. albicans to adjust a variety of metal-dependent processes led us to hypothesize that vulnerabilities in metallohomeostasis incurred by drug stress could be leveraged by compounds that interrupt metal trafficking. Here, we show that tetrathiomolybdate (TTM), a copper (Cu) chelator that interferes with Cu trafficking and use, inhibits growth of C. albicans on its own and synergizes with select azoles to enhance antifungal activity. Proteomic and biochemical experiments revealed that TTM causes differential expression and stabilization of proteins involved in fermentation and oxidative stress responses in C. albicans. The synergy between TTM and azoles was found to arise from increased expression and stability of the nitric oxide dioxygenase Yhb1, a response driven by the decreased stability and activity incurred by TTM of CuZn superoxide dismutase 1. Addition of imidazole-based antifungals highjacks this stress response by inhibiting Yhb1. This study highlights the centrality of Cu homeostasis as a regulatory hub connecting energy production, oxidative stress management, and overall cellular fitness in ways that can be pharmacologically manipulated to enhance efficacy of existing antifungal agents.

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• Covalent inhibition of Plasmodium falciparum Ubc13 impairs global protein synthesis

  iScience · 2025-04-28

  articleOpen access

  ubiquitinome studies. Nascent protein synthesis was reduced following NSC697923 exposure, supporting a role for PfUbc13 and K63-Ub in mediating protein translation. These findings expand our knowledge of PfUbc13-dependent processes in these pathogenic parasites and highlight this enzyme as a potential antimalarial drug target.

  PublisherDOI

• Small molecules reveal differential shifts in stability and protein binding for G-quadruplex RNA

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-12 · 2 citations

  preprintOpen access

  The potential of therapeutically targeting RNA with small molecules continues to grow yet progress is hindered by difficulties in determining specific mechanisms of action, including impacts on RNA-protein binding. RNA G-quadruplexes (rGQs) are a particularly promising target due to their range of biological functions, structural stability, and hydrophobic surfaces, which promote small molecule and protein interactions alike. Challenges arise due to 1) the low structural diversity among rGQs, thereby limiting binding selectivity, and 2) a lack of knowledge regarding how small molecules can manipulate rGQ-protein binding on a global scale. We first leveraged a small molecule library privileged for RNA tertiary structures that displayed differential binding to rGQs based on loop length, consistent with computational predictions for DNA GQs. We next utilized an RT-qPCR-based assay to measure stability against enzymatic readthrough, expected to be a common mechanism in rGQ function. We discovered small molecules with significant, bidirectional impacts on rGQ stability, even within the same scaffold. Using Stability of Proteins from Rates of Oxidation (SPROX), a stability-based proteomics method, we then elucidated proteome level impacts of both stabilizing and destabilizing rGQ-targeting molecules on rGQ-protein interactions. This technique revealed small molecule-induced impacts on a unique subset of rGQ-binding proteins, along with proteins that exhibited differential changes based on the identity of the small molecule. The domain and peptide-level insights resulting from SPROX allow for the generation of specific hypotheses for both rGQ function and small molecule modulation thereof. Taken altogether, this methodology helps bridge the gap between small molecule-RNA targeting and RNA-protein interactions, providing insight into how small molecules can influence protein binding partners through modulation of target RNA structures.

  PublisherOA PDFDOI

• Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome

  Cell chemical biology · 2024-03-15 · 17 citations

  articleOpen access
  PublisherOA PDFDOI

• Stability-Based Proteomics for Investigation of Structured RNA–Protein Interactions

  Analytical Chemistry · 2024-02-11 · 7 citations

  articleOpen accessSenior authorCorresponding

  RNA–protein interactions are essential to RNA function throughout biology. Identifying the protein interactions associated with a specific RNA, however, is currently hindered by the need for RNA labeling or costly tiling-based approaches. Conventional strategies, which commonly rely on affinity pull-down approaches, are also skewed to the detection of high affinity interactions and frequently miss weaker interactions that may be biologically important. Reported here is the first adaptation of stability-based mass spectrometry methods for the global analysis of RNA–protein interactions. The stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) methods are used to identify the protein targets of three RNA ligands, the MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU), in LNCaP nuclear lysate. The 315 protein hits with RNA-induced conformational and stability changes detected by TPP and/or SPROX were enriched in previously annotated RNA-binding proteins and included new proteins for hypothesis generation. Also demonstrated are the orthogonality of the SPROX and TPP approaches and the utility of the domain-specific information available with SPROX. This work establishes a novel platform for the global discovery and interrogation of RNA–protein interactions that is generalizable to numerous biological contexts and RNA targets.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM061680

  NIH · $1.4M · 2008

• NIH Grant R21CA127064

  NIH · $386k · 2010

• Anti-Malarial Drug Target Discovery using Energetics-Based Proteomics Methods

  NIH · $420k · 2018–2020

• Elucidating the Molecular Basis of Cellular Metal Stress by using Mass Spectrometry-Based Proteomic Methods

  NIH · $2.1M · 2022–2026

• NIH Grant S10RR020971

  NIH · $992k · 2007

Frequent coauthors

• Michael J. Campa

  Duke University

  27 shared

• Edward F. Patz

  27 shared

• Michael Z. Wang

  24 shared

• Yan Tong

  22 shared

• Patrick D. DeArmond

  Nationwide Children's Hospital

  22 shared

• Jagat Adhikari

  Washington University in St. Louis

  20 shared

• Kendall D. Powell

  19 shared

• Graham M. West

  Pfizer (United States)

  19 shared

Labs

• The Fitzgerald LabPI