We have developed a general method to discover covalent inhibitors of catalytic cysteines using fragment-based drug discovery. NMR rate studies were used to compare several electrophiles in order to identify one which is moderately reactive with cysteine and which demonstrates similar reactivity regardless of the structure of the fragment it is attached to. Electrophiles were also tested for their ability to exclusively label the catalytic cysteine of an enzyme with multiple surface cysteines. (E)-methyl 4-aminobut-2-enoate was identified as an electrophile which meets these criteria, although other electrophiles are still being investigated.  This amine handle of this electrophile was then coupled to a library of fragments containing a carboxylic acid group. This library was selected on the basis of meeting the �rule of three� criteria as well as maximizing structural diversity. Pseudo first-order NMR kinetics studies with N-acetyl cysteine methyl ester have confirmed that all members of this library have rate constants within a factor of 2 of each other.The electrophilic fragments can then be screened in batches of ten against the enzyme of interest, and any binders can be identified by mass spectrometry. Whole protein ESI-MS is used to identify any hits based on the mass shift of the protein peak.  Selectivity for the catalytic cysteine is confirmed by tryptic digestion and MALDI-TOF MS of the tryptic peptides. Our choice of electrophile ensures that any hits will be selected based on their ability to bind to the active site of the enzyme and not simply due to their greater reactivity. We have chosen the HECT E3 ubiquitin ligase Nedd4-1 as a model enzyme to validate this methodology, and screening against this target is currently underway.  However, we anticipate that this technology will be applicable to all enzymes with catalytic cysteines, including cysteine proteases.

Fragment-based approach to covalent inhibitors of catalytic cysteines

246th National Meeting (2013)

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