The FDA approvals of afatinib and ibrutinib in 2013 led to a heightened interest in cysteine-reactive covalent inhibitors. However, there are few methods to discover new cysteine-reactive inhibitors for enzymes for which reversible binding scaffolds are not known. To this end, we rationally designed a chemical system to attach a cysteine-reactive electrophile to drug-like fragments without significant alterations in the thiol reactivity of the attached electrophile, ensuring that specific binding and not increased reactivity will produce candidate inhibitors. We applied this method, which we call irreversible tethering, to discover inhibitors of the HECT E3 ubiquitin ligase Nedd4-1, an enzyme with no validated inhibitors. Nedd4-1 has a catalytic cysteine and a non-catalytic surface cysteine, and is implicated in viral budding, cancers, and neurodegenerative diseases. We screened our electrophilic fragment library and discovered two fragments which reacted with Nedd4-1 as determined by mass spectrometry. Surprisingly, we found that these inhibitors did not react with the more reactive catalytic cysteine of Nedd4-1, but the other surface cysteine near the non-covalent ubiquitin binding site. This site binds to ubiquitinated substrates in order to extend the length of the ubiquitin chain. The X-ray crystal structure of the most potent fragment in complex with Nedd4-1 was solved, demonstrating that it forms a stable covalent bond with the ubiquitin-binding site cysteine of Nedd4-1 and has specific interactions with residues around this cysteine. This structure has been used to further optimize the fragment into a more potent inhibitor. Due to their proximity to the non-covalent ubiquitin binding site, our inhibitor reduces the binding affinity of Nedd4-1 for ubiquitin. In vitro enzymatic assays have shown that these molecules inhibit Nedd4-1 polyubiquitination processivity and switch it to a distributive mechanism. Click chemistry and in-gel fluorescence with an alkyne tagged analog of this inhibitor have shown that the inhibitor reacts with Nedd4-1 in TC71 cells with good selectivity. However, we have not yet been able to show that the optimized inhibitor impairs Nedd4-1 function in cells, perhaps due to insufficient potency or a limitation in its mechanism of action. Further optimization of the inhibitor could result in a compound to study the diseases in which Nedd4-1 polyubiquitination is implicated.

Last modified

• 02/19/2018

Creator

• Stefan Kathman

DOI

• https://doi.org/10.21985/N2WD5M

Subject

• Chemistry

Keyword

• Electrophile
• Covalent
• Fragment
• Ubiquitin
• Nedd4-1

Date created

• 2016-01-01

Resource type

• Dissertation

Rights statement

• In Copyright