Instead, the ability of such compound to bind to ER and alter its function should be investigated. Monitoring the presence of a vast number of different EDCs in soil and water and studying their biochemical effect on ER is considered one of the key current challenges for ensuring healthy ecosystems in both developed and in-development countries. For these reasons, analytical methods that exploit ER as a biorecognition element to detect the presence of EDCs are particularly attractive: if a chemical binds to ER in the assay, then it means that it can potentially interfere with the hormone signaling pathways and thus be toxic. X-ray structures have shown that the human ER binds ligands in a highly hydrophobic pocket that can accommodate EDCs of different sizes and chemical properties. Due to the high sequence identity, it is likely that the general conformation of the ER ligand binding site is conserved; however, local structural differences and a certain degree of conformational flexibility have to be present to account for the different properties and affinities of EDC compounds. These differences may, at least in principle, be exploited for the rational design of modified receptors capable of recognizing classes of EDCs with different affinity and selectivity. In this manuscript, we aimed to modulate the binding properties of the estrogen receptor protein by rationally modifying the amino acid composition of the ligand binding domain. By combining sequence alignment and structural analysis of known ER-ligand complexes with computational analysis, we were able to predict single point variants of the estrogen receptor ligand binding domain with altered binding properties with respect to the wild type ER ligand binding domain. These predictions were experimentally confirmed by producing and characterizing the most relevant OSI-774 EGFR/HER2 inhibitor recombinant ERaLBD variants. In particular, we were able to generate a single point ER mutant with a 6-fold increased binding affinity towards some EDCs, reaching i.e. an IC50 value of 2 nM for 17a-ethinylestradiol ligand. 17a-Ethinylestradiol is an orally bio-active hormone and one of the most commonly used medications, identified as an emerging aquatic pollutant due to its bio-accumulation in surface waters. Due to the increased affinity of one of our ER variants for this and other compounds, utilizing such mutated ER instead of the wt-ERaLBD as biorecognition element in an assay or biosensor would result in increased sensitivity. We used two different docking algorithms with either rigid or flexible docking options.