In this report, multiple DNA-conjugate libraries, comprising a total of over 500 billion small molecular ligand candidates, have been selected to identify novel NLRP3 (Nucleotide-binding oligomerization domain, Leucine-rich Repeat and Pyrin domain containing 3) nanomolar inhibitors.
NLRP3 activation results in a protective pro-inflammatory response by the release of the pro-inflammatory cytokines IL-1β and IL-18, subsequently leading to pyroptotic cell death. However, alteration of NLRP3 function has been associated with a number of organ-specific autoimmune diseases, including a variety of acute and chronic CNS and systemic diseases.
Therefore, for the identification of orally active CNS-penetrant NLRP3 inhibitors for the treatment of neurosensory disease, a DECL selection campaign was performed on a HIS-tagged NLRP3 protein construct with retained ATPase activity, and employing, in separate screening trials, previously reported blocking agents to better understand the binding modalities and mechanisms of action of the newly selected ligands.
Out of the multiple DNA-conjugate libraries tested, the most interesting and relevant binding results were displayed by indazole compounds selected from a 3-building block split-&-pool library comprising 98.8 million candidates.
Compound 1 was able not only to inhibit the NLRP3 inflammasome pathway with an IC50 as good as 25nM but also showed, in SPR studies, a specific nanomolar binding (KD range 104–123 nM) to the NACHT domain of NLRP3, distinct from those of previously reported ligands.
SAR optimization of the initial lead provided an equally potent analog, Compound 2 (IC50 33 nM), with improved metabolic stability suggesting high potential for further improvement of Compound 1‘s drug-like features.
