PF-022 (1) is a novel polycyclic benzothiophene kinase inhibitor selective for mitogen-activated protein kinase-activated protein kinase 2 (MK2). Compound 1 emerged as an inhibitor bearing submicromolar potency against MK2 (IC50 5 nM) and demonstrated projected human pharmacokinetics sufficient for oral dosing. However, following a single, oral administration of 1 to beagle dogs, animals experienced an acute liver injury characterized by increases in biomarkers associated with hepatotoxicity; particularly noteworthy was the reversible elevation in bile salts and total bilirubin. Accompanying this observation was an ADME appraisal which included hepatic bioactivation of 1 in multiple species and the in vitro inhibition of P-glycoprotein (P-gp; IC50 21 μM). Simply attenuating the bioactivation via structural modification proved ineffective in improving the in vivo tolerability of this polycyclic scaffold. Hence, disruption of hepatobiliary transporters by the compound series was hypothesized as the likely mechanism contributing to the acute hepatotoxicity. Indeed, closer in vitro examination employing transporter gene overexpressing MDCK cell lines and membrane vesicles revealed potent compound-dependent inhibition of human multi-drug resistance-associated protein 2 (MRP2/ABCC2; IC50 38 μM) and bile salt export pump (BSEP/ABCB11; IC50 10 μM), two crucial hepatobiliary transport proteins accountable for bilirubin and bile salt homeostasis, respectively. Subsequent introduction of pKa-altering modifications to a second generation compound PF029 proved successful in reducing its affinity for these key efflux transporters (MRP2 IC50 >>80 μM; BSEP IC50 > 70 μM; P-gp > 90 μM), consequently mitigating this overt organ toxicity in dogs.