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Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD\u2019s inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated<jats:italic>in silico<\/jats:italic>by molecular docking to human NE, expressed in<jats:italic>Escherichia coli<\/jats:italic>and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity<jats:italic>in vitro<\/jats:italic>and RL-KD presented the best performance. We further tested RL-KD<jats:italic>in vivo<\/jats:italic>in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity<jats:italic>in vitro<\/jats:italic>. 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