Ricin is a potent cytotoxin which has been used widely in the construction of therapeutic agents such as immunotoxins. Recently it has been used by governments and underground groups as a poison. There is interest in identifying and designing effective inhibitors of the ricin A chain (RTA). In this study computer-assisted searches indicated that pterins might bind in the RTA active site which normally recognizes a speci®c adenine base on rRNA. Kinetic assays showed that pteroic acid could inhibit RTA activity with an apparent K i of 0.6 mM. A 2.3 A Ê crystal structure of the complex revealed the mode of binding. The pterin ring displaces Tyr80 and binds in the adenine pocket making speci®c hydrogen bonds to active site residues. The benzoate moiety of pteroic acid binds on the opposite side of Tyr80 making van der Waals contact with the Tyr ring and forming a hydrogen bond with Asn78. Neopterin, a propane triol derivative of pterin, also binds to RTA as revealed by the Xray structure of its complex with RTA. Neither pterin-6-carboxylic acid nor folic acid bind to the crystal or act as inhibitors. The models observed suggest alterations to the pterin moiety which may produce more potent and speci®c RTA inhibitors.

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.

Fifteen novel non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of the NCI DIS 3D database. The pharmacophore query used in the search was derived directly from the X-ray determined structures of protease/inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, NSC 32180, a “dimer” of 4-hydroxycoumarin, and NSC 117027, a “tetramer” of 2-hydroxy quinone, had ID50 values of 0.32 and 0.75 μM for HIV-1 protease inhibition, respectively, and two other inhibitors had ID50 values close to 1 μM. Among the potent inhibitors, NSC 158393 not only demonstrated activity against HIV-1 protease (ID50 1.7 μM) but also exhibited promising antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5 μM). Validation of the pharmacophore used in the search was accomplished by conformational analysis. The binding modes of the most potent inhibitor found in our studies, NSC 32180, were predicted employing docking and molecular dynamics techniques.