## 13 Human cON PDEs as Potential Drug Targets  
Human antimicrobial defense mechanisms, including the production of type I interferons (IFNs), are triggered by microbial DNA from bacteria, viruses, and potentially  
Figure 11: (**A**) Csm1 protein bound to cA\({}_{4}\) (PDB: 607H). The residues interacting with the ligand are labeled [133]. (**B**) The proposed mechanism of cA\({}_{4}\) hydrolysis by [PERSON]. [PERSON] et al. [117] The 2’-OH is the nucleophilic group responsible for ring opening. The interactions highlighted in blue were not indicated in the crystal structure but were both within 3.7 Å when measured. The proposed hydrolysis mechanism of cA\({}_{6}\) is identical to cA\({}_{4}\) except for 2 additional pockets for all 6 nucleotide bases to bind. Figure 12: (**A**) A high-level overview of CRISPR signal cascade in bacteria. (**B**) An overview of CBASS signaling in bacteria which is used to combat phage propagation through abortive infection. parasites through the cytosolic sensor known as STING. STING signaling is activated by cONs produced by intracellular bacteria or synthesized by the enzyme cGAS in response to cytosolic self-DNA or microbial DNA [32]. ENPP1, the phosphodieserase that hydrolyzes cONs such as 2\({}^{\prime}\),3\({}^{\prime}\)-cGAMP to AMP and GMP, plays a critical role in regulating this pathway. However, ENPP1 is often upregulated in tumor microenvironments, contributing to immunosuppression (see Figure 2B) [134]. Targeting ENPP1 to prevent cON degradation has emerged as a promising strategy to enhance immune activation against both pathogens and tumors. There are currently three ENPP1 inhibitors which have begun undergoing clinical trials (RBS2418, Figure 13[135; 136], and SR-8541A [137], and TXN10128 [138]) for patients with advanced or metastatic solid tumors. The structures of SR-8541A and TNX10128 have not been disclosed. A phase 2 trial is also currently recruiting for SR-8541A for combination therapy with Botensilimab [139]. At the time of writing, there are no outcomes posted on ClinicalTrials.gov for any of the clinical trials for these ENPP1 inhibitors. A plethora of other ENPP1 inhibitors have been discovered in recent years with IC\({}_{50}\) values ranging from low micro-molar to pico-molar. While other reviews attempt to categorize the known ENPP1 inhibitors to probe for structural similarities [134; 140; 141; 142; 143], we will instead focus on more clinically advanced candidates such as RBS2418, or compounds that inhibited ENPP1 with single-digit nanomolar or picomolar inhibitory concentrations (K\({}_{i}\)) as well as additional compounds with higher K\({}_{i}\) values, but representative of structurally different compound classes. Compound **15** was also chosen as a representative ENPP1 inhibitor as the binding mode to the enzyme has been solved (Figure 14). Recently, [PERSON] and co-workers reported interesting VH domains that allosterically inhibit ENPP1 [144]. The active site of ENPP1 anchors both the nucleotide bases as well as one of the two phosphate groups utilizing several residues (Figure 15) [145]. First, Phe, Lys, and Tyr side chains form interactions with the adenosine base and His362 forms an interaction with the guanosine base. This orients the G-2\({}^{\prime}\)phosphate group towards two Zn\({}^{2+}\) ions which are anchored in the active site by several conserved Asp and His residues. The oxygen atom on Thr238 will ultimately become the nucleophilic atom responsible for breaking the G-2\({}^{\prime}\)-phosphoester bond. Then, surrounding water will release the linear substrate from Thr238. After the first hydrolysis, the linear dinucleotide (5\({}^{\prime}\)-pApG-3\({}^{\prime}\)) can then rebind in the opposite orientation with the guanosine base occupying the site which was previously occupied by the adenine base. Hydrolysis occurs again utilizing the same mechanism, ultimately resulting in 5\({}^{\prime}\)-AMP and 5\({}^{\prime}\)-GMP.