Our sequence and structure analysis revealed several additional residues that are not conserved across the phosphatases and may impact covalent inhibition. Arg 47 and Asp 48 in PTP1B lie on the pTyr loop, alongside Tyr 46, which forms the conserved hydrophobic floor of the active site (Figure ). These two residues diverge across the phosphatases tested and are likely to impact selectivity for the multi-ring molecules in our library (Figure ). For example, we observed a distinctive improvement in potency against HePTP for compound 71 (Figure ). This molecule has a hydroxy group that is well-positioned to engage the unique Thr 127 on the HePTP pTyr loop (Asp 48 in PTP1B). A previous investigation exploited an electrostatic interaction with Asp 48 to design selective non-covalent inhibitors of PTP1B, as this charged residue is not present in all classical tyrosine phosphatases. We also identified two residues with modest conservation (Leu 110 and Asn 111) that buttress the catalytic cysteine (Cys 215 in PTP1B) (Figure ). A recent investigation showed that the dynamics of Leu 110 in PTP1B play a role in enzyme catalysis. SHP1 and SHP2 both have a unique Thr residue in place of Leu 110, and this Thr residue hydrogen-bonds to the backbone carbonyl preceding the catalytic cysteine in those enzymes. Thus, although Leu 110 and Asn 111 do not make direct contact with substrates or covalent ligands, we hypothesize that changes at these positions will impact the orientation, dynamics, and reactivity of the catalytic cysteine. Perhaps the most surprising difference between phosphatases observed in our screens was the divergence in ligand engagement by SHP1 and SHP2. The catalytic domains of these enzymes have 59% sequence identity, which increases to 88% identity within 10 Å of the active site, and both proteins share a common domain architecture and regulatory mechanism. Despite this high degree of structural homology, SHP1 is more readily inhibited by aryl chloroacetamides than SHP2, and it has a unique preference for isoxazole-containing molecules (Figure ). Based on docking models and sequence alignments, there are no ligand-proximal residues that differentiate SHP1 and SHP2. The only potentially significant active site difference we identified between the two enzymes is the first residue of the WPD loop (Figure ,B, Thr 177 in PTP1B, Leu 415 in SHP1, and Arg 421 in SHP2). A previous study showed that the T177A mutation in PTP1B stabilizes a closed state of the WPD loop but only marginally impacts catalytic activity. Mutations to the bulkier Leu or Arg found in SHP1 and SHP2 have not been examined, but we anticipate that they will have a substantial impact on loop dynamics and covalent inhibition.