No abstract

Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21, and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.

The structures of a large number of HIV-1 integrase inhibitors have in common two aryl units separated by a central linker. Frequently at least one of these aryl moieties must contain 1,2-dihydroxy substituents in order to exhibit high inhibitory potency. The ability of o-dihydroxy-containing species to undergo in situ oxidation to reactive quinones presents a potential limitation to the utility of such compounds. The recent report of tetrameric 4-hydroxycoumarin-derived inhibitor 5 provided a lead example of an inhibitor which does not contain the catechol moiety. Compound 5 represents a large, highly complex yet symmetrical molecule. It was the purpose of the present study to determine the critical components of 5 and if possible to simplify its structure while maintaining potency. In the present study, dissection of tetrameric 5 (IC50 = 1.5 microM) into its constituent parts showed that the minimum active pharmacophore consisted of a coumarin dimer containing an aryl substituent on the central linker methylene. However, in the simplest case in which the central linker aryl unit consisted of a phenyl ring (compound 8, IC50 = 43 microM), a significant reduction in potency resulted by removing two of the original four coumarin units. Replacement of this central phenyl ring by more extended aromatic systems having higher lipophilicity improved potency, as did the addition of 7-hydroxy substituents to the coumarin rings. Combining these latter two modifications resulted in compounds such as 3,3'-(2-naphthalenomethylene)bis[4,7-dihydroxycoumarin] (34, IC50 = 4.2 microM) which exhibited nearly the full potency of the parent tetramer 5 yet were structurally much simpler.

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.

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.

The human immunodeficiency virus type one integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE), tyrphostins, and curcumin confer inhibitory activity against HIV-1 integrase. We have investigated the actions of several recently described protease inhibitors, possessing novel structural features, on HIV-1 integrase. NSC 158393, which contains four 4-hydroxycoumarin residues, was found to exhibit antiviral, antiprotease, and antiintegrase activity. Both the DNA binding and catalytic activities (3'-processing and strand transfer) of integrase were inhibited at micromolar concentrations. Disintegration catalyzed by an integrase mutant containing only the central catalytic domain was also inhibited, indicating that the binding site for these compounds resides in the central 50-212 amino acids of HIV-1 integrase. Binding at or near the integrase catalytic site was also suggested by a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. NSC 158393 inhibited HIV-2, feline, and simian immunodeficiency virus integrases while eukaryotic topoisomerase I was inhibited at higher concentrations, suggesting selective inhibition of retroviral integrases. Molecular modeling studies revealed that the two hydroxyls and two carbonyl moieties in NSC 158393 may represent essential elements of the pharmacophore. Antiviral efficacy was observed with NSC 158393 derivatives that inhibited both HIV protease and integrase, and the most potent integrase inhibitors also inhibited HIV protease. Hydroxycoumarins may provide lead compounds for development of novel antiviral agents based upon the concurrent inhibition of two viral targets, HIV-1 integrase and protease.

A searcheable database of three-dimensional structures has been developed from the chemistry database of the NCI Drug Information System (DIS), a file of about 450,000 primarily organic compounds which have been tested by NCI for anticancer activity. The DIS database is very similar in size and content to the proprietary databases used in the pharmaceutical industry; its development began in the 1950s; and this history led to a number of problems in the generation of 3D structures.