The transition from a pharmacophore-guided approach to a receptor-guided approach in the design of potent protein kinase C ligands.

The pharmacophore-guided approach used in the first phase of the design of novel protein kinase C (PKC) ligands was based on the study of the geometry of bioequivalent pharmacophores present in diacylglycerol (DAG) and in the more potent phorbol ester tumor promoters. A number of potent DAG lactones were generated by this approach, in which the glycerol backbone was constrained into various heterocyclic rings to reduce the entropic penalty associated with DAG binding. Based on the information provided by X-ray and NMR structures of the cysteine-rich, C1 phorbol ester/DAG binding domain, the DAG lactones were further modified to optimize their interaction with a group of highly conserved hydrophobic amino acids along the rim of the C1 domain. This receptor-guided approach culminated with the synthesis of a series of "super DAG" molecules that can bind to PKC with low nanomolar affinities. These compounds provide insight into the basis for PKC ligand specificity. Moreover, some of the compounds reviewed herein show potential utility as antitumor agents.

Monocarboxylic-based phosphotyrosyl mimetics in the design of GRB2 SH2 domain inhibitors.

Three monocarboxylic-containing analogues, O-carboxymethyltyrosine (cmT, 5), 4-(carboxymethyl)phenylalanine (cmF, 6), and 4-(carboxydifluoromethyl)phenylalanine (F2cmF, 7) were utilized as phosphotyrosyl (pTyr) replacements in a high affinity B-bend mimicking platform, where they exhibited IC50 values of 2.5 microM, 65 microM and 28 microM, respectively, in a Grb2 SH2 domain Biacore binding assay. When a terminal N(alpha)-oxalyl axillary was utilized to enhance ligand interactions with a critical SH2 domain Arg67 residue (alphaA-helix), binding potencies increased from 4- to 10-fold, resulting in submicromolar affinity for cmF (IC50 = 0.6 microM) and low micromolar affinity for F2cmF (IC50 = 2 microM). Cell lysate binding studies also showed inhibition of cognate Grb2 binding to the p185erbB-2 phosphoprotein in the same rank order of potency as observed in the Biacore assay. These results indicate the potential value of cmF and F2cmF residues as pTyr mimetics for the study of Grb2 SH2 domains and suggest new strategies for improvements in inhibitor design.

Potent inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands.

Development of Grb2 Src homology 2 (SH2) domain binding inhibitors has important implications for treatment of a variety of diseases, including several cancers. In cellular studies, inhibitors of Grb2 SH2 domain binding have to date been large, highly charged peptides which relied on special transport devices for cell membrane penetration. Work presented in the current study examines a variety of pTyr mimetics in the context of a high-affinity Grb2 binding platform. Among the analogues studied are new non-phosphorus-containing pTyr mimetics 23a and 23b which, when incorporated into tripeptide structures 18f and 20f, are able to inhibit Grb2 SH2 domain binding with affinities among the best yet reported for non-phosphorus-containing SH2 domain inhibitors (IC50 values of 6.7 and 1.3 microM, respectively). The present study has also demonstrated the usefulness of the Nalpha-oxalyl group as an auxiliary which enhances the binding potency of both phosphorus- and non-phosphorus-containing pTyr mimetics. When combined with the (phosphonomethyl)phenylalanine (Pmp) residue to give analogues such as L-20d, potent inhibition of Grb2 SH2 domain binding can be achieved both in extracellular assays using isolated Grb2 SH2 domain protein and in intracellular systems measuring the association of endogenous Grb2 with its cognate p185erbB-2 ligand. These latter effects can be achieved at micromolar to submicromolar concentrations without prodrug derivatization. The oxalyl-containing pTyr mimetics presented in this study should be of general usefulness for the development of other Grb2 SH2 domain antagonists, independent of the beta-bend-mimicking platform utilized for their display.

Identification of HIV-1 integrase inhibitors based on a four-point pharmacophore.

The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the 'open' part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 microM, among which 10 had IC50 values of less than 10 microM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.

Salicylhydrazine-containing inhibitors of HIV-1 integrase: implication for a selective chelation in the integrase active site.

In previous studies we identified N,N'-bis(salicylhydrazine) (1) as a lead compound against purified recombinant HIV-1 integrase. We have now expanded upon these earlier observations and tested 45 novel hydrazides. Among the compounds tested, 11 derivatives exhibited 50% inhibitory concentrations (IC50) of less than 3 microM. A common feature for activity among these inhibitors is the hydroxyl group of the salicyl moiety. Although the active inhibitors must contain this hydroxyl group, other structural modifications can also influence potency. Removal of this hydroxyl group or replacement with an amino, bromo, fluoro, carboxylic acid, or ethyl ether totally abolished potency against integrase. Several asymmetric structures exhibited similar potency to the symmetric lead inhibitor 1. The superimposition of the lowest-energy conformations upon one another revealed three sites whose properties appear important for ligand binding. Site A is composed of the 2-hydroxyphenyl, the alpha-keto, and the hydrazine moieties in a planar conformation. We propose that this site could interact with HIV-1 integrase by chelation of the metal in the integrase active site as inhibition of HIV-1 integrase catalytic activity and DNA binding were strictly Mn2+-dependent. The hydrophobic sites B and C are probably responsible for complementarity of molecular shape between ligand and receptor. Our data indicate that only those compounds which possessed sites A, B, and C in a linear orientation were potent inhibitors of HIV-1 integrase. Although all the active inhibitors possessed considerable cytotoxicity and no apparent antiviral activity in CEM cells, the study presents useful information regarding ligand interaction with HIV-1 integrase protein.

Identification of a nucleotide binding site in HIV-1 integrase.

HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3'-azido-3'-deoxythymidine (AZT) analog 3',5-diazido-2', 3'-dideoxyuridine 5'-monophosphate (5N3-AZTMP) and proteolytic mapping identified the amino acid 153-167 region of integrase as the site of photocrosslinking. Docking of 5N3-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.

Recognition and interaction of small rings with the ricin A-chain binding site.

Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings.

Pharmacophores in drug design and discovery.

Compounds containing a specific pharmacophore--the minimum structural features necessary for enzyme binding--can be retrieved from a database such as the National Cancer Institute repository by means of three-dimensional (3D) searching, which allows the retrieval of all compounds possessing a specified set of atoms with a given 3D geometry. The ways in which pharmacophores can be found and characterized and the details of the 3D searching methods are described. Elaboration of compounds found in such searches and their subsequent development as lead drugs is also discussed.

Mathematics as a basis for chemistry.

Mathematicians are increasingly publishing papers in which mathematics is applied to chemical problems. Examination of some of these papers reveals that while they contain genuine contributions to chemistry they tend to avoid the more interesting and difficult problems. The "forward" problem of estimation of physical properties from a compound's structure, for example, has seen many publications, a proportion of which have been successful. The "reverse" problem, prediction of the structures which possess given properties, is only rarely examined and never by mathematicians. This is unfortunate because these problems are mathematically complex and mathematicians could make significant contributions by bringing their skills to bear on such questions.

Structure-based identification of a ricin inhibitor.

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 specific adenine base on rRNA. Kinetic assays showed that pteroic acid could inhibit RTA activity with an apparent Ki of 0.6 mM. A 2.3 A crystal structure of the complex revealed the mode of binding. The pterin ring displaces Tyr80 and binds in the adenine pocket making specific 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 X-ray 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 specific RTA inhibitors.

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

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.

Discovery of HIV-1 integrase inhibitors by pharmacophore searching.

Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152, compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206,876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 microM and are promising lead compounds for further HIV-1 integrase inhibitor development.

Hydrazide-containing inhibitors of HIV-1 integrase.

Inhibitors of HIV integrase are currently being sought as potential new therapeutics for the treatment of AIDS. A large number of inhibitors discovered to date contain the o-bis-hydroxy catechol structure. In an effort to discover structural leads for the development of new HIV integrase inhibitors which do not rely on this potentially cytotoxic catechol substructure, NSC 310217 was identified using a three-point pharmacophore search based on its assigned structure N-(2-hydroxybenzoyl)-N-(2-hydroxy-3-phenoxypropyl)hydrazine (1). When a sample of NSC 310217 was obtained from the NCI repository, it was shown to exhibit potent inhibition of HIV-1 integrase (3'-processing IC50 = 0.6 microgram/mL). In work reported herein, we demonstrate that NSC 310217, rather than containing 1, which has no inhibitory potency against HIV-1 integrase, is comprised of roughly a 1:1 mixture of N-(2-hydroxybenzoyl)-N'-(2-hydroxy-3-phenoxypropyl)hydrazine (6) and N,N'-bis-salicylhydrazine 7, with all inhibitory potency residing with compound 7(IC50 = 0.7 microM for strand transfer). In subsequent structure-activity studies on 7, it is shown that removing a single amide carbonyl (compound 14, IC50 = 5.2 microM) or replacing one aromatic ring system with a naphthyl ring (compound 19, IC50 = 1.1 microM) can be accomplished with little loss of inhibitory potency. Additionally, replacing a single hydroxyl with a sulfhydryl (compound 23, IC50 = 5.8 microM) results in only moderate loss of potency. All other modifications examined, including the replacement of a single hydroxyl with an amino group (compound 22), resulted in complete loss of potency. Being potent, structurally simple, and non-catechol-containing, compounds such as 7 and 14 may provide useful leads for the development of a new class of HIV integrase inhibitor.

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

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.

Coumarin-based inhibitors of HIV integrase.

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.

Potent inhibitors of human immunodeficiency virus type 1 integrase: identification of a novel four-point pharmacophore and tetracyclines as novel inhibitors.

A four-point pharmacophore was constructed from energy-minimized structures of chicoric acid and dicaffeoylquinic acid. The search of 206,876 structures in the National Cancer Institute 3D database yielded 179 compounds that contain this pharmacophore. Thirty-nine of these compounds were tested in an in vitro assay specific for human immunodeficiency virus type 1 integrase (IN). Each retrieved structure was fit to the pharmacophore, and the conformation that afforded the best fit was identified. Twenty of the 39 compounds tested exhibited IC50 values of < 20 microM. Among the most potent inhibitors, tetracyclines emerged as a new class of inhibitors. Although the parent tetracycline exhibited marginal potency against purified IN, all substituted tetracyclines tested showed 5-100-fold increased potency. Disintegration assays with truncated IN mutants indicated that tetracyclines inhibit the IN catalytic core domain. To investigate whether chelation of divalent metals is implicated in differential potency of tetracyclines, enzyme assays were performed in the presence of both Mn2+ or Mg2+; no significance difference in potency was observed. Rolitetracycline inhibited IN/DNA complex formation in the presence of EDTA, which suggests that inhibition was metal independent. Rolitetracycline reversed DNA binding of IN after the complex was allowed to form before the addition of drug. Selectivity of tetracyclines was also examined in an assay specific for topoisomerase I, and none of the tetracyclines tested induced topoisomerase I-mediated cleavable complex or inhibited camptothecin-induced cleavable complex. Remarkable potency against the IN in the absence of divalent metals and the core enzyme coupled with water solubility makes tetracyclines potential candidates for X-ray crystal structure determination with IN.

Molecular modeling in the discovery of drug leads.

The National Cancer Institute of the U.S.A. maintains a repository of about 500,000 chemicals which it has tested at some time for anticancer activity. As new chemotherapeutic targets present themselves, methods have been developed by which this large database can be re-examined without resorting to expensive high-volume biological screening. Electronic screening, the method described in this paper, begins with the identification of a target enzyme. The pharmacophore used by the enzyme in binding to substrates is identified, and then all compounds in the database that contain the pharmacophore are found. These compounds are then further filtered, for example, by physical properties such as solubility, and the relatively small number of compounds that survive are submitted for biological testing. This use of a primary electronic screen in the search for ligands of protein kinase C is described. The screen is very fast, and the method is quite generally applicable to different enzymes.

Antiretroviral agents as inhibitors of both human immunodeficiency virus type 1 integrase and protease.

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.

Molecular modeling and site-directed mutagenesis studies of a phorbol ester-binding site in protein kinase C.

The protein kinase C (PKC) binding site used by PKC activators such as phorbol esters and diacylglycerols (DAGs) has been characterized by means of molecular modeling and site-directed mutagenesis studies. Based upon a NMR-determined solution structure of the second cysteinerich domain of PKC alpha, molecular modeling was used to study the structures of the complexes formed between the PKC receptor and a number of PKC ligands, phorbol esters, and DAGs. Site-directed mutagenesis studies identified a number of residues important to the binding of phorbol esters to PKC. Analysis of the molecular modeling and mutagenesis results allows the development of a binding model for PKC ligands for which the precise binding nature is defined. The calculated hydrogen bond energies between the protein and various ligands in this binding model are consistent with their measured binding affinities. The binding site for phorbol esters and DAGs is located in a highly conserved, hydrophobic loop region formed by residues 6-12 and 20-27. For the binding elements in phorbol esters, the oxygen at C20 contributes most to the overall binding energy, and that at C3 plays a significant role. The oxygen atom at C12 is not directly involved in the interaction between phorbol esters and PKC. Our results also suggest that the oxygens at C9 and C13 are involved in PKC binding, while the oxygen at C4 is of minimal significance. These results are consistent with known structure-activity relationships in the phorbol ester family of compounds. Comparisons with the X-ray structure showed that although the X-ray data support the results for oxygens at C3, C12, and C20 of phorbol esters, they suggest different roles for oxygens at C4, C9, and C13. Several factors which may contribute to these discrepancies are discussed.

Discovery of novel, non-peptide HIV-1 protease inhibitors by pharmacophore searching.

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 microM for HIV-1 protease inhibition, respectively, and two other inhibitors had ID50 values close to 1 microM. Among the potent inhibitors, NSC 158393 not only demonstrated activity against HIV-1 protease (ID50 1.7 microM) but also exhibited promising antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5 microM). 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.