Justin Hall

Peptidylarginine deiminases (PAD1-4) are calcium dependent enzymes responsible for protein citrullination, a post-translational modification converting arginine residues to citrulline. Elevated levels of citrullinated proteins have been associated with rheumatoid arthritis, neurodegenerative diseases, and cancers. Though highly selective PAD4 inhibitors have been described, inhibitors to the broader family currently are limited to covalent substrate analogs. Herein, we describe an allosteric binding pocket common to PAD1-4 suitable for the identification of potent, non-covalent enzyme inhibitors. A ligand-based virtual screen is utilized to identify a PAD4 inhibitor for which surface plasmon resonance confirms target binding but non-competitively with a known PAD4 ligand. We further show through co-crystal structure analysis that the ligand binds PAD4 at an allosteric pocket resulting in stabilization of a catalytically inactive, calcium-deficient enzyme conformation. A ligand designed based on this site potently inhibits all four PAD isozymes and prevents protein citrullination in neutrophils with a broader protein repertoire than observed with a PAD4-selective inhibitor.

Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large‐scale industrial organizations. The link between ligand affinity (K _D) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K _D to ΔTm correlate, we define equations relating K _D and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K _D and ΔTm for irreversible denaturation.

Cyclic GMP‐AMP synthase (cGAS) is activated by ds‐DNA binding to produce the secondary messenger 2′,3′‐cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds‐DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2′,3′‐cGAMP formation, and interestingly, describe a catalytic mechanism where 2′,3′‐cGAMP may be a minor product of cGAS compared with linear nucleotides.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (K_D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF-patient-derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT-001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high-affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high-affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high-affinity small molecule binding is to be achieved.