Identification and Validation of Carbonic Anhydrase II as the First Target of the Anti-Inflammatory Drug Actarit

<b>Background and purpose:</b> Identifying the macromolecular targets of drug molecules is a fundamental aspect of drug discovery and pharmacology. Several drugs remain without known targets (orphan) despite large-scale in silico and in vitro target prediction efforts. Ligand-centric chemical-similarity-based methods for in silico target prediction have been found to be particularly powerful, but the question remains of whether they are able to discover targets for target-orphan drugs. <b>Experimental Approach:</b> We used one of these in silico methods to carry out a target prediction analysis for two orphan drugs: actarit and malotilate. The top target predicted for each drug was carbonic anhydrase II (CAII). Each drug was therefore quantitatively evaluated for CAII inhibition to validate these two prospective predictions. <b>Key Results:</b> Actarit showed in vitro concentration-dependent inhibition of CAII activity with submicromolar potency (IC₅₀ = 422 nM) whilst no consistent inhibition was observed for malotilate. Among the other 25 targets predicted for actarit, RORγ (RAR-related orphan receptor-gamma) is promising in that it is strongly related to actarit’s indication, rheumatoid arthritis (RA). <b>Conclusion and Implications:</b> This study is a proof-of-concept of the utility of MolTarPred for the fast and cost-effective identification of targets of orphan drugs. Furthermore, the mechanism of action of actarit as an anti-RA agent can now be re-examined from a CAII-inhibitor perspective, given existing relationships between this target and RA. Moreover, the confirmed CAII-actarit association supports investigating the repositioning of actarit on other CAII-linked indications (e.g., hypertension, epilepsy, migraine, anemia and bone, eye and cardiac disorders).