GSK484

GSK484 is a PADI4-selective in vitro chemical probe. PAD4 is a calcium-dependent enzyme which catalyzes the reaction in which protein arginine residues are converted into citrulline, with the release of ammonia. This pathway is anticipated to be relevant to multiple immune and inflammatory processes, oncology, and the regulation of stem cell maintenance. GSK484 reversibly binds to the low-calcium form of PAD4 (IC50 = 50 nM, mouse neutrophils) and has been shown to be selective over PAD1-3 in cellular assays and with assays employing recombinant enzymes. The probe has been studied in cells but has not been evaluated with in vivo models. It is not clear that the probe will function in vivo due to the need for low concentrations of calcium to render the target in optimal form for binding. This may be difficult to achieve in a physiological model. 

There is solid experimental evidence supporting GSK484 as a PAD4-specific inhibitor in biochemical systems, including nanomolar potencies in fluorescence polarization, ligand-competition, and NH₃-release assays, X-ray crystallography, and convincing SAR. Users should note GSK484 is significantly more active in the absence of calcium-bound PAD4. Mass spectrometry and dialysis experiments support a reversible, competitive binding mechanism in the absence of calcium and a more complicated mode of action at higher calcium concentrations. 50-target biochemical activity profiling demonstrates reasonable selectivity compared to unrelated proteins in vitro. There is also solid experimental evidence supporting GSK484 as a relatively PAD4-specific inhibitor in certain cell-based systems, including Western blotting, cell imaging, and chemoproteomic mass spectrometry. Note there is an approximately 10-20-fold decrease in potency in most cell-based experiments, which could be due to a variety of factors including calcium status, intracellular concentration, and nonspecific protein binding, and parameters of the biochemical assays, among others. GSK484 is relatively nontoxic to certain cell lines at 10 uM. Users should also note that while the original manuscript presents biochemical and cellular selectivity data for PAD1-4 but not PAD6. There is an inactive analog, GSK106. I would highly recommend including this control compound in any experiments utilizing GSK484.

I recommend this probe for use in cell-based systems, with the caution to carefully consider calcium concentrations when designing and interpreting experiments. Depending on the experimental question, I would also consider monitoring the contributions of the related PDA1-3 and PDA6, since there is always potential for other PDA engagement in cell-based systems. The original report does not provide data regarding use in model organisms, so it is unclear how GSK484 may engage PDA4 in complex organisms given its calcium-dependent activity. I do not endorse this compound for use in animal models, though additional data demonstrating useful (e.g., potent, selective, consistent with proposed mechanism of action) target engagement in vivo may enable a future recommendation.