BACKGROUND: Owing to the economic relevance of resistance evolution against herbicides, new chemical entities addressing unprecedented molecular targets are urgently needed to develop future sustainable weed control solutions. As part of our discovery research, the new class of aminoisothiazolamides was investigated. RESULTS: Aminoisothiazolamide 3-amino-4-chloro-N-(cyclohexylmethyl)isothiazole-5-carboxamide 1a and several of its derivatives displayed potent herbicidal and fungicidal in vivo activity in initial glasshouse tests. Lysyl-tRNA synthetase 1 (KRS1) was identified as the putative target for 1a and was validated as a key contributor to the biochemical mode-of-action of aminoisothiazolamides. Thermal stability shift analysis with KRS1 from Arabidopsis thaliana (AtKRS1) revealed that 1a specifically increased the thermostability of this enzyme, proving the KRS1 enzyme as the aminoisothiazolamide target. It turned out that the inhibition of AtKRS1 and HsKRS was strongly correlated, as was the inhibition of AtKRS1 and the herbicidal activity of the aminoisothiazolamides. Hence, in vivo acute toxicity tests were initiated at a very early project stage complementing the enzyme tests. CONCLUSION: The observed toxicological effects paired with the anticipated likelihood to overcome this problem, owing to the highly conserved active sites in different species, finally resulted in the conclusion to stop the further exploration of the otherwise promising class of herbicidal aminoisothiazolamides. Thus, we opted to discard several further herbicidal lead structures before the start of in-depth investigations when sequence analyses suggested similar levels of conservation between the respective binding pockets in plants and mammalians. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.