When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket. Saturated IZD-containing peptides are more potent inhibitors than unsaturated analogs because the IZD heterocycle and phenyl ring directly attached to it bind in a nearly orthogonal orientation with respect to each other, a conformation that is close to the energy minimum of the saturated IZD-phenyl moiety. These results explain why the heterocycle is a potent phosphonate mimetic and an ideal starting point for designing small nonpeptidic inhibitors. Protein-tyrosine phosphatase 1B (PTP1B)2 is considered to be one of the best validated drug targets for the treatment of type II diabetes. The enzyme is localized to the cytoplasmic face of the endoplasmic reticulum where it negatively regulates insulin signaling by dephosphorylating phosphotyrosine (Tyr(P)) residues in the kinase regulatory domain of the insulin receptor (IR) (1, 2). Mice lacking the homolog of PTP1B have lower blood glucose levels and improved insulin responsiveness compared with normal and diabetic mice through enhanced IR signaling in peripheral tissues (3, 4). Similar results were also observed when an antisense oligonucleotide was injected into mice (5). These compelling biological results coupled with the wealth of structural data, which have been generated since the crystal structure of PTP1B was determined in 1994 (6), have contributed to the rapid design of many potent inhibitors (7-13). Unfortunately, poor physicochemical properties have limited their development as drug candidates.Crystal structures of PTP1B in complex with phosphorylated peptides corresponding to the IR kinase activation segment (14) and an autophosphorylated site of the epidermal growth factor receptor (15) reveal that the highly negatively charged substrates bind to multiple positively charged sites (Fig. 1a). The catalytic site, which is located at the base of a shallow pocket called the primary phosphate-binding pocket or A site, is the most polar region of the protein, and it contains the phosphatebinding signature motif (Cys 215 -Arg 221 ) common to all members of the protein-tyrosine phosphatase family (16). A second phosphate-binding pocket (B site), adjacent to the A site, was identified from the crystal structures of the protein in complex with a small aryl phosphonate (17) and a bisphosphorylated peptide (14). The B site, which is larger and shallower than the A site and has lower binding affinity for aryl phosphates, is noncatalytic but may play an important role in determining substrate specificity. A third phosphate-binding site (C site) was identified when the distal Tyr(P) mimetic of a bisphosphonate-containing inhibitor unexpectedly bound in a large flat region of the protein near Lys 41 and Arg 47 (18). Overall, the active site of PTP1B possesses very few desirable drug-design features. The highly charged A site and flat, solvent-exposed B and C sites significantly increase the difficulty of designing pote...

Structure-based design led to the discovery of novel (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine (pTyr) mimetics that when incorporated into dipeptides are exceptionally potent, competitive, and reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). The crystal structure of PTP1B in complex with our most potent inhibitor 12 revealed that the (S)-IZD heterocycle interacts extensively with the phosphate binding loop precisely as designed in silico. Our data provide strong evidence that the (S)-IZD is the most potent pTyr mimetic reported to date.

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp 48 , and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp 48 and the backbone NH of Arg 47 via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe 182 of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular -stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.Type II diabetes, also known as non-insulin-dependent diabetes mellitus, is characterized by a deficiency in insulin signaling despite normal or greatly elevated levels of insulin. Although the cause of insulin resistance is unknown, inhibiting enzymes that negatively regulate the signaling pathway may restore insulin responsiveness. Protein-tyrosine phosphatase 1B (PTP1B), 2 a key negative regulator of insulin signaling, has emerged as an attractive target for the treatment of type II diabetes. PTP1B directly inactivates the insulin receptor (IR) by dephosphorylating tyrosine residues in the regulatory domain (1, 2) and its overexpression inhibits IR signaling (3). The most convincing evidence for its potential use as a therapeutic target, however, is that PTP1B knock-out mice (4) and those injected with an antisense oligonucleotide (5, 6) displayed increased insulin sensitivity.A number of PTP1B inhibitors containing highly charged non-hydrolyzable phosphonate mimetics have been reported (7-13). Poor membrane permeability, however, has limited their development as drug candidates. The lack of inhibitors with suitable physicochemical properties can be attributed to the need to interact with the highly positively charged primary phosphate binding pocket and the surrounding flat, solventexposed region.We have recently reported the structure-based design of a novel isothiazolidino...

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