Laura J. Byrnes, Won Young Choi, Paul Balbo, Mary Ellen Banker, Jeanne Chang, Shi Chen, Xuemin Cheng, Yang Cong, Jeff Culp, Hongxia Di, Matt Griffor, Justin Hall, Xiaoyun Meng, Barry Morgan, James J. Mousseau, Jennifer Nicki, Thomas O'Connell, Simeon Ramsey, Alex Shaginian, Suman Shanker, John Trujillo, Jinqiao Wan, Fabien Vincent, Stephen W. Wright, Felix Vajdos
ACS Chemical Biology (2024).
https://doi.org/10.1021/acschembio.4c00397
Abstract
Peptidyl arginine deiminases (PADs) are enzymes implicated in various inflammatory and autoimmune diseases. Developing isoform-specific inhibitors has remained a challenge. This study reports the discovery of a potent, noncovalent, and selective PAD2 inhibitor, PF-3166, identified through DNA-encoded library (DEL) screening. Biochemical and biophysical assays revealed that PF-3166 inhibits PAD2 via a novel Ca²⁺-competitive allosteric mechanism. X-ray crystallography confirmed that PF-3166 induces conformational changes that eject Ca²⁺ from the high-affinity CA1 site and prevent Ca²⁺ binding at the CA2 site, both essential for enzyme activation. PF-3166 demonstrated selective cellular activity in human neutrophils, offering a valuable tool for elucidating PAD2 function in disease contexts.
Summary
A large-scale DEL screen targeting PAD2 yielded three compound series. Among them, PF-3166 stood out as a potent and selective PAD2 inhibitor (IC₅₀ = 150–600 nM), while the other two (PF-2635 and PF-8522) bound PAD2 but lacked inhibitory activity. Structural studies revealed that all three compounds bind at the PAD2 dimer interface. However, only PF-3166 induced significant conformational changes that disrupted Ca²⁺ binding at both CA1 and CA2 sites, leading to enzyme inhibition. This novel allosteric mechanism was further validated in cellular assays, where PF-3166 selectively inhibited PAD2-dependent citrullination in human neutrophils.
Highlights
1. First report of a noncovalent, selective PAD2 inhibitor (PF-3166) from DEL screening. 2. PF-3166 inhibits PAD2 via a novel allosteric mechanism involving Ca²⁺ displacement from CA1 and CA2 sites. 3. X-ray structures reveal compound-induced conformational changes that prevent Ca²⁺ binding and enzyme activation. 4. PF-3166 is selective over PAD3 and PAD4 and shows cellular activity in human neutrophils. 5. Represents a new chemical tool for studying PAD2 biology and therapeutic potential.
Conclusion
PF-3166 is a first-in-class, cell-active, and selective PAD2 inhibitor that acts through a novel allosteric mechanism by disrupting Ca²⁺ binding at two critical sites (CA1 and CA2), essential for PAD2 activation. Its discovery underscores the power of DEL screening in uncovering unique inhibitory mechanisms. Despite liabilities such as high molecular weight and low permeability, PF-3166 provides a strong foundation for further optimization and a valuable tool for dissecting PAD2’s role in inflammation, autoimmunity, and cancer.