Related Experiment Video
Updated: Jul 16, 2026
Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source
Published on: May 29, 2021
Structure-Guided Discovery and Biochemical Validation of Novel Small-Molecule Inhibitors Predicted to Target the
1Medizinische Klinik und Poliklinik I, LMU University Hospital, 80336 Munich, Germany.
Abstract:
Crimean-Congo hemorrhagic fever virus (CCHFV) remains a major public health threat due to its high mortality rates and the absence of approved antiviral therapies. The viral ovarian tumor (OTU) protease is a critical virulence factor that suppresses host innate immunity through its deubiquitinase activity, making it an attractive therapeutic target. In this study, we employed a structure-guided approach to identify and validate novel small-molecule inhibitors targeting the non-catalytic Y89-W99 pocket of the OTU protease. Recombinant OTU protease was successfully expressed, purified, and refolded, yielding a soluble and enzymatically active protein. Cellular assays confirmed that the enzyme retains robust deubiquitinase activity, significantly reducing global ubiquitin conjugates in mammalian cells. In silico analysis of a putative DUB inhibitor library identified several candidate inhibitors with favorable binding interactions within the Y89-W99 pocket. Biochemical validation using a fluorometric Ub-AMC assay revealed that multiple small molecules strongly inhibit OTU activity, including OTUi-10 (~93% inhibition), OTUi-13 (~87%), OTUi-1 (~85%), OTUi-4 and OTUi-11 (~81%), and OTUi-9 (~76%). Additional moderate inhibitors included OTUi-12 (~67%), OTUi-19 and OTUi-21 (~66%), and OTUi-5 (~57%). In silico drug-likeness and toxicity profiling filtered the library to four fully compliant candidates, OTUi-4, OTUi-10, OTUi-11, and OTUi-12, all free of predicted toxicity alerts. These findings suggest that the Y89-W99 pocket may be a pharmacologically relevant site worthy of further investigation and identify OTUi-10, OTUi-4, and OTUi-11 as promising preliminary hit compounds. The results also provide initial insights that may guide future optimization and mechanistic studies of OTU protease inhibitors targeting CCHFV.
Insights
New small molecules show promise in inhibiting Crimean-Congo hemorrhagic fever virus (CCHFV) ovarian tumor (OTU) protease, a key factor in viral virulence. This research identifies potential new antiviral therapies by targeting the OTU protease
Area of Science:
- Biochemistry
- Virology
- Drug Discovery
Background:
- Crimean-Congo hemorrhagic fever virus (CCHFV) poses a significant public health risk due to high mortality and lack of effective treatments.
- The viral ovarian tumor (OTU) protease is crucial for CCHFV virulence, suppressing host immunity via deubiquitinase activity, making it a prime therapeutic target.
Purpose of the Study:
- To identify and validate novel small-molecule inhibitors targeting the non-catalytic Y89-W99 pocket of the CCHFV OTU protease using a structure-guided approach.
Main Methods:
- Recombinant OTU protease was expressed, purified, and validated for enzymatic activity.
- In silico analysis of a deubiquitinase (DUB) inhibitor library was performed to identify potential binders.
- Biochemical assays (Ub-AMC) were used to evaluate the inhibitory activity of candidate molecules against OTU protease.
Main Results:
- Several small molecules demonstrated potent inhibition of OTU protease activity, with OTUi-10 showing ~93% inhibition.
- In silico drug-likeness and toxicity profiling identified four compliant candidates (OTUi-4, OTUi-10, OTUi-11, OTUi-12).
- OTUi-10, OTUi-4, and OTUi-11 were highlighted as promising preliminary hit compounds with no predicted toxicity.
Conclusions:
- The Y89-W99 pocket of the CCHFV OTU protease is a viable pharmacological target for antiviral drug development.
- The identified hit compounds, particularly OTUi-10, OTUi-4, and OTUi-11, warrant further investigation for CCHFV therapeutic strategies.
