Interrogation of the Active
Sites of Protein Arginine
Deiminases (PAD1, -2, and -4) Using Designer Probes
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Abstract
Protein arginine deiminases (PADs) are involved in a
number of
cellular pathways, and they catalyze the transformation of peptidyl
arginine residue into a citrulline as part of post-translational modifications.
To understand ligand preferences, a group of probe molecules were
investigated against PAD1, PAD2, and PAD4. These probe molecules carried
a well-known covalent modifier of the catalytic cysteine residue,
2-chloroacetamidine moiety, which was tethered to an α-amino
acid via a carbon linker. The chain length for the linker varied from
0 to 4. Time-dependent assays indicated that 2-chloroacetamidine (2CA)
with no linker inhibited all PAD enzymes with a similar trend in the
second-order rate constants, although with poor affinity. Among the
other three probe molecules, compound <b>3</b> with a three-carbon
linker exhibited the best second-order rate constants for optimal
ligand reactivity with the binding site. These analyses provide insights
into the relative patterns of covalent inactivation of PAD isozymes
and the design of novel inhibitors targeting PAD enzymes as potential
therapeutic targets