About

Anthony O’Donoghue, Ph.D., is an Associate Professor at the Skaggs School of Pharmacy and Pharmaceutical Sciences. His research focuses on the detection and characterization of proteolytic enzymes associated with disease, studying proteases found in infectious organisms, cancerous tissues, immune cells, or human biofluids. His work involves in-depth characterization of target proteases, development of potent and selective inhibitors, and fluorogenic substrates to monitor catalytic activity. He employs a platform technology called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to uncover global proteolytic activity in complex biological samples, applying this to blood, pancreatic cyst fluid, gastric juice of cancer cells, and microbiobes. His workflow includes proteomic and peptidomics analysis to identify proteases and endogenous substrates, enabling the development of inhibitors, protease-activated imaging agents, and protease-activated drugs for disease localization and treatment. Dr. O’Donoghue has contributed to the development of mass spectrometry-based substrate profiling assays, identification of tissue-degrading enzymes from bat fungus, and the development of anti-parasitic compounds targeting parasite proteasomes. His academic background includes a B.S. and Ph.D. in Biochemistry from the National University of Ireland, Galway, and postdoctoral and specialist roles at the University of California, San Francisco. He has held leadership positions such as Director of the Center for Protease Inhibitor Design and President of the International Proteolysis Society, and has been recognized with awards including the Bioanalysis Young Investigator Award and the UCSF Center for Bio-Entrepreneurship Team Award.

Research topics

• Biochemistry
• Biology
• Virology
• Molecular biology
• Chemistry

Selected publications

• mspms: an R package and GUI for multiplex substrate profiling by mass spectrometry

  BMC Bioinformatics · 2026-01-24

  articleOpen accessSenior author

  BACKGROUND: Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) is a powerful method for determining the substrate specificity of proteolytic enzymes, which is essential for developing protease inhibitors, diagnostics, and protease-activated therapeutics. However, the complex datasets generated by MSP-MS pose significant analytical challenges and have limited accessibility for non-specialist users. RESULTS: We developed mspms, a Bioconductor R package with an accompanying graphical interface, to streamline the analysis of MSP-MS data. Mspms standardizes workflows for data preparation, processing, statistical analysis, and visualization. The tool is designed for accessibility, serving advanced users through the R package and broader audiences through a web-based interface. We validated mspms using data from four well-characterized cathepsins (A-D), demonstrating that it reliably captures expected substrate specificities. CONCLUSIONS: mspms is the first publicly available, comprehensive platform for MSP-MS data analysis downstream of peptide identification and quantification. It integrates preprocessing, normalization, statistical testing, and visualization into a single, transparent, and user-friendly framework, making it a valuable resource for the protease research community. The package is distributed via Bioconductor, and a graphical interface is available online for interactive use.

  PublisherDOI

• Additional file 2 of mspms: an R package and GUI for multiplex substrate profiling by mass spectrometry

  Figshare · 2026-01-01

  articleOpen accessSenior author

  Supplementary Material 2

  PublisherDOI

• Additional file 2 of mspms: an R package and GUI for multiplex substrate profiling by mass spectrometry

  Figshare · 2026-01-01

  articleOpen accessSenior author

  Supplementary Material 2

  PublisherDOI

• An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

  Journal of Biological Chemistry · 2025-02-07 · 2 citations

  articleOpen access

  Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the endoplasmic reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality, we expressed and purified recombinant RHBDL4 and employed it in a Förster resonance energy transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the nonprime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

  PublisherOA PDFDOI

• Structural Insights into Salinosporamide a Mediated Inhibition of the Human 20S Proteasome

  Molecules · 2025-03-20 · 4 citations

  articleOpen access

  The 20S proteasome, a critical component of the ubiquitin–proteasome system, plays a central role in regulating protein degradation in eukaryotic cells. Marizomib (MZB), also known as salinosporamide A, is a natural γ-lactam-β-lactone compound derived from Salinispora tropica and is a potent 20S proteasome covalent inhibitor with demonstrated anticancer properties. Its broad-spectrum inhibition of all three proteasome subunits and its ability to cross the blood–brain barrier has made it a promising therapeutic candidate for glioblastoma. In addition to this, MZB also demonstrates significant inhibition against the 20S proteasome of Trichomonas vaginalis (Tv20S), a protozoan parasite, suggesting its potential for parasitic treatments. Here, we present the cryo-EM structure of the human 20S proteasome in complex with MZB at 2.55 Å resolution. This structure reveals the binding mode of MZB to all six catalytic subunits within the two β-rings of the 20S proteasome, providing a detailed molecular understanding of its irreversible inhibitory mechanism. These findings enhance the therapeutic potential of MZB for both cancer and parasitic diseases at the molecular level and highlight marine-derived natural products in targeting the proteasome for therapeutic applications.

  PublisherOA PDFDOI

• Aza-peptide aldehydes and ketones: synthesis and evaluation as human 20S proteasome inhibitors

  Future Medicinal Chemistry · 2025-10-16 · 1 citations

  articleOpen access

  AIMS: Aza-peptide aldehydes and ketones were developed as a new class of peptidyl analogues to inhibit the human constitutive (c)20S proteasome as alternative therapeutics to treat multiple myeloma (MM). MATERIAL AND METHODS: Eleven new aza-peptide aldehydes and ketones were designed based on their preference to bind at the ß5 catalytic subunit of c20S proteasome with benzyloxycarbonyl(Cbz)-Leu-Leu-Leu (MG132-like) and morpholinyl(Mp)-Homophenylalanyl(HPh)-Leu-Phe-Leu (Carfilzomib-like) sequences, synthesized, structurally characterized and evaluated for their inhibitory potency in competitive kinetic assays in vitro. Additionally, cell viability assays and molecular modeling experiments were designed and performed in support. RESULTS: values only for the cancer cells after 48 h. CONCLUSIONS: Overall, aza-peptide aldehydes and ketones are a new class of selective human c20S proteasome inhibitors with the potential for further development as alternative therapeutics for multiple myeloma.

  PublisherOA PDFDOI

• Discovery of benzyl carbamate inhibitors of coronavirus M ^pro enzymes from a legacy collection of cysteine protease inhibitors

  Journal of Enzyme Inhibition and Medicinal Chemistry · 2025-11-17 · 1 citations

  articleOpen accessCorresponding

  , and this behaviour was supported by covalent and noncovalent computational simulations. This study highlights the importance of revisiting legacy assets to identify starting points for new antiviral drugs.

  PublisherDOI

• Chalcogen derivatives for the treatment of African trypanosomiasis: biological evaluation of thio and seleno- semicarbazones and their azole derivatives

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-23

  preprintOpen access

  ABSTRACT Human African Trypanosomiasis (HAT) is caused by Trypanosoma brucei . Drug therapy remains challenging due to drug resistance and/or toxicity. New drugs are needed. Using thiosemicarbazones as a starting point, we employed a S to Se isosteric replacement strategy to design 44 analogs which were evaluated against T. brucei in vitro . Compounds were divided into eleven groups of four derivatives corresponding to thio-, selenosemicarbazones, and their cyclic counterparts, thio- and selenazoles. We selected three groups which contained a total of six derivatives that inhibited parasite growth by >70%. Then, we investigated the mechanism of action of these compounds, performing quantitative assays to measure their inhibition of the T. brucei cathepsin L-like protease ( Tbr CATL) and DPPH antioxidant activities. The lead compound ( Se O3 ) showed antioxidant capacity and the best activity against T. brucei (EC 50 = 0.47 µM). Nevertheless, its toxicity should be improved. We also predicted the interactions of these compounds with Tbr CATL utilizing molecular dynamics. We demonstrate that the Se derivatives are more active than their S analogues, and that the selenazole ring decreases Se -associated toxicity. Also, thio- and selenosemicarbazones are more potent against Tbr CATL than the cyclic derivatives. We conclude that Tbr CATL inhibition should be combined with antioxidant activity to obtain active compounds against T. brucei .

  PublisherOA PDFDOI

• Discovery and characterization of protease activities in acidified bovine milk

  Food Chemistry · 2025-08-05

  articleOpen accessSenior authorCorresponding

  Protease activities present in acidified bovine milk were investigated using a peptide digestion assay coupled to mass spectrometry analysis. Results from this assay showed considerable overlap of cleavages at pH 3.5 and 5.5, suggesting shared protease activities at these pH values; however, overall activity at pH 5.5 was significantly lower. Two main proteases were identified at pH 3.5: cathepsin D (endoprotease), and a previously unreported tripeptide aminopeptidase. Fluorimetric assays for measurement of activity of both proteases across the pH range 3.0–6.7 were developed. Cathepsin D activity was highest pH 3.0, and declined progressively with increasing pH; in contrast, the tripeptide aminopeptidase activity was restricted to pH 3.0–4.0. Both proteases were detected in a set of 26 individual milk samples, indicating that they might be part of a proteolytic system in milk. This study provides insights about the proteases present in milk and highlights the role of pH in their regulation. • Distinct proteases are active in bovine milk at different pH conditions • Cathepsin D is the main endoprotease active at pH 3.5 • A previously undiscovered tripeptide aminopeptidase is also active at pH 3.5 • Cathepsin D and tripeptide aminopeptidase may work in tandem to generate tripeptides • Novel fluorogenic assays were developed to quantify milk protease activity

  PublisherDOI

• Comprehensive proteolytic profiling of Aedes aegypti mosquito midgut extracts: Unraveling the blood meal protein digestion system

  PLoS neglected tropical diseases · 2025-02-06 · 8 citations

  articleOpen access1st authorCorresponding

  To sustain the gonotrophic cycle, the Aedes aegypti mosquito must acquire a blood meal from a human or other vertebrate host. However, in the process of blood feeding, the mosquito may facilitate the transmission of several bloodborne viral pathogens (e.g., dengue, Zika, and chikungunya). The blood meal is essential as it contains proteins that are digested into polypeptides and amino acid nutrients that are eventually used for egg production. These proteins are digested by several midgut proteolytic enzymes. As such, the female mosquito's reliance on blood may serve as a potential target for vector and viral transmission control. However, this strategy may prove to be challenging since midgut proteolytic activity is a complex process dependent on several exo- and endo-proteases. Therefore, to understand the complexity of Ae. aegypti blood meal digestion, we used Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to generate global proteolytic profiles of sugar- and blood-fed midgut tissue extracts, along with substrate profiles of recombinantly expressed midgut proteases. Our results reveal a shift from high exoproteolytic activity in sugar-fed mosquitoes to an expressive increase in endoproteolytic activity in blood-fed mosquitoes. This approach allowed for the identification of 146 cleaved peptide bonds (by the combined 6 h and 24 h blood-fed samples) in the MSP-MS substrate library, and of these 146, 99 (68%) were cleaved by the five recombinant proteases evaluated. These reveal the individual contribution of each recombinant midgut protease to the overall blood meal digestion process of the Ae. aegypti mosquito. Further, our molecular docking simulations support the substrate specificity of each recombinant protease. Therefore, the present study provides key information of midgut proteases and the blood meal digestion process in mosquitoes, which may be exploited for the development of potential inhibitor targets for vector and viral transmission control strategies.

  PublisherDOI

Frequent coauthors

• Pavla Fajtová

  153 shared

• William H. Gerwick

  Scripps Institution of Oceanography

  123 shared

• Jehad Almaliti

  University of California, San Diego

  84 shared

• Conor R. Caffrey

  83 shared

• Elany Barbosa da Silva

  University of California, San Diego

  77 shared

• Mateus Sá Magalhães Serafim

  69 shared

• Zhenze Jiang

  Center for Discovery

  67 shared

• Charles S. Craik

  University of California, San Francisco

  65 shared

Awards & honors

• North America representative to International Proteolysis So…
• Bioanalysis Young Investigator Award (2013)
• UCSF Center for Bio-Entrepreneurship Team Award (2009)
• UC Education Abroad Program Scholar (2002)
• Irish Research Council for Science, Engineering & Technology…