We have previously shown that a small peptide bearing the hydrolytically stable phosphotyrosyl (pTyr) mimetic, (difluorophosphonomethyl) phenylalanine (F2Pmp), is an extremely potent inhibitor of PTP1B, with an IC50 value of 100 nM [Burke, T. R., Kole, H. K., & Roller, P. P. (1994) Biochem. Biophys. Res. Commun. 204, 129-134]. We further demonstrated that removal of the peptide portion and incorporation of the difluorophosphonomethyl moiety onto a naphthalene ring system, but not a phenyl ring system, resulted in good inhibitory potency [Kole, H. K., Smyth, M. S., Russ, P. L., & Burke, T. R., Jr. (1995) Biochem, J. 311, 1025-1031]. In order to understand the structural basis for this inhibition, and to aid in the design of further analogs, we solved the X-ray structure of [1, 1-difluoro-1-(2-naphthalenyl)-methyl]phosphonic acid (6) complexed within the catalytic site of PTP1B, solved to 2.3 A resolution. In addition to showing the manner in which the phosphonate group is held within the catalytic site, the X-ray structure also revealed extensive hydrophobic interactions with the naphthalene ring system, beyond that possible with an analog bearing a single phenyl ring. It is further evident that, of the two fluorine atoms, the pro-R alpha-fluorine interacts with the enzyme to a significantly greater degree than the pro-S alpha-fluorine, forming a hydrogen bond to Phe 182. On the basis of a computer-assisted molecular modeling analysis, it was determined that addition of a hydroxyl to the naphthyl 4-position, giving [1, 1-difluoro-1-[2-(4-hydroxynaphthalenyl)] methyl]phosphonic acid (8), could potentially replace a water molecule situated in the PTP1B-6 complex, thereby allowing new hydrogen-bonding interactions with Lys 120 and Tyr 46. Compound 8 was therefore prepared and found to exhibit a doubling of affinity (Ki = 94 microM) relative to parent unsubstituted 6 (Ki = 179 microM), supporting, in principle, the development of high-affinity ligands based on molecular modeling analysis of the enzyme-bound parent.

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys29) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys29 was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys29 and His199, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu245 and the P2 Arg residue was clearly demonstrated, and it was found that the benzyl group of the P1 substrate residue occupies a large hydrophobic pocket thought to represent the S'1 subsite. This may have important implications for structure-based design of cathepsin B inhibitors.