Targeting pathogenic T cells with Ag-specific tolerizing DNA vaccines encoding autoantigens is a powerful and feasible therapeutic strategy for Th1-mediated autoimmune diseases. However, plasmid DNA contains abundant unmethylated CpG motifs, which induce a strong Th1 immune response. We describe here a novel approach to counteract this undesired side effect of plasmid DNA used for vaccination in Th1-mediated autoimmune diseases. In chronic relapsing experimental autoimmune encephalomyelitis (EAE), combining a myelin cocktail plus IL-4-tolerizing DNA vaccine with a suppressive GpG oligodeoxynucleotide (GpG-ODN) induced a shift of the autoreactive T cell response toward a protective Th2 cytokine pattern. Myelin microarrays demonstrate that tolerizing DNA vaccination plus GpG-ODN further decreased anti-myelin autoantibody epitope spreading and shifted the autoreactive B cell response to a protective IgG1 isotype. Moreover, the addition of GpG-ODN to tolerizing DNA vaccination therapy effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models. In conclusion, suppressive GpG-ODN effectively counteracted the undesired CpG-induced inflammatory effect of a tolerizing DNA vaccine in a Th1-mediated autoimmune disease by skewing both the autoaggressive T cell and B cell responses toward a protective Th2 phenotype. These results demonstrate that suppressive GpG-ODN is a simple and highly effective novel therapeutic adjuvant that will boost the efficacy of Ag-specific tolerizing DNA vaccines used for treating Th1-mediated autoimmune diseases.

Analysis of mRNA from multiple sclerosis lesions revealed increased amounts of transcripts for several genes encoding molecules traditionally associated with allergic responses, including prostaglandin D synthase, histamine receptor type 1 (H1R), platelet activating factor receptor, Ig Fc epsilon receptor 1 (Fc epsilon RI), and tryptase. We now demonstrate that, in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), mediated by T helper 1 (Th1) T cells, histamine receptor 1 and 2 (H1R and H2R) are present on inflammatory cells in brain lesions. Th1 cells reactive to myelin proteolipid protein expressed more H1R and less H2R than Th2 cells. Pyrilamine, an H1R antagonist, blocked EAE, and the platelet activating factor receptor antagonist CV6209 reduced the severity of EAE. EAE severity was also decreased in mice with disruption of the genes encoding Ig Fc gamma RIII or both Fc gamma RIII and Fc epsilon RI. Prostaglandin D synthase and tryptase transcripts were elevated in EAE brain. Taken together, these data reveal extensive involvement of elements of the immune response associated with allergy in autoimmune demyelination. The pathogenesis of demyelination must now be viewed as encompassing elements of both Th1 responses and "allergic" responses.

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.

The substrate specificity of cytochrome P450, defined as the ability of a compound to promote NAD(P)H and O-2 utilization in the production of either organic or reduced oxygen metabolites, is largely determined by steric and hydrophobic interactions. P450 specificity may therefore be determined by the ''fit'' of a compound within the active site. A receptor-constrained three-dimensional screening program (DOCK) has been used to select 11 compounds predicted to fit within the P450(cam) active site and 5 compounds predicted to fit within the L244A P450(cam) but not wild-type active site. The 16 compounds were evaluated as P450(cam) substrates by measuring (a) binding to the enzyme, (b) stimulation of NADH and O-2 consumption, (c) enhancement of H2O2 production, and (d) formation of organic metabolites. Seven of the compounds predicted to fit in the active site, and none of the compounds predicted not to fit, were found to be substrates. Compounds predicted to fit very tightly within the active site were poor or non-substrates. The L244A P450(cam) mutant was constructed, expressed, purified, and shown to readily oxidize some of the larger compounds incorrectly predicted to be substrates for the wild-type enzyme. The 5 ligands selected to fit the L244A but not wild-type sites were not detectable substrates, presumably because they fit too tightly into the active site. Retroactive adjustments of the docking program based on an analysis of the docking parameters, particularly variation of the minimum distance allowed between ligand and protein atoms, allow correct predictions for the activity of 15 of the 16 compounds with wild-type P450(cam). The DOCK predictions for the L244A mutant were also improved by changing the minimum contact distances to disfavor the larger compounds. The results indicate that ligands that fill the; active site are marginal or non-substrates. A degree of freedom of motion is required for substrate positioning and catalytic function. if parameters are chosen to allow for this requirement, P450(cam) substrate predictions based on Ligand docking in the active site can be reasonably accurate.

Irreversible inhibition of the HIV-1 protease by agents that specifically alkylate its catalytic aspartate residues is a potentially useful approach for circumventing the evolution of HIV strains that are resistant to protease inhibitors. Five haloperidol- and two FMOC-based epoxides of differing reactivities have been synthesized and tested as irreversible inhibitors of the HIV-1 protease (HIV-1 PR). Of these, two trisubstituted epoxides, a cis-1,2- disubstituted epoxide, a 1,1-disubstituted epoxide, and a monosubstituted epoxide function as irreversible inhibitors, but two trans-1,2-disubstituted epoxides do not. The most effective of the epoxides (6) inactivates HIV-1 PR with K-inact = 65 mu M and V-inact = 0.009 min(-1). 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), a nonspecific inactivating agent for aspartyl proteases, has been used to validate a protocol for establishing the stoichiometry and site of protein alkylation. Mass spectrometric analysis of the inactivated enzyme shows that one molecule of either EPNP or the cyclic 1,2-disubstituted epoxide 6 is covalently bound per HIV-1 PR dimer. Mass spectrometric sequencing of labeled proteolytic peptides shows that both inhibitors are covalently bound to a catalytic aspartate residue. The covalent binding of three alpha,beta-unsaturated ketone derivatives of haloperidol has been similarly examined. Analysis of HIV-1 PR inactivated by these agents establishes that they bind covalently to the two cysteines and the N-terminal amino group but not detectably to the catalytic aspartate residues. The results indicate that aspartate-targeted inactivation of HIV-1 PR depends on (a) matching the reactivity of the alkylating functionality to that of the aspartates, preferably by exploiting the two-aspartate catalytic motif of the protease to activate the alkylating agent, and (b) appropriate positioning of the alkylating functionality within the active site. These requirements are readily met by a monosubstituted, 1,1-disubstituted, or cyclic cis-1,2-disubstituted epoxide but not by trans-1,2-disubstituted epoxides or alpha,beta-unsaturated ketones.

Publisher Summary This chapter describes the substrate docking algorithms and the prediction of substrate specificity. The utility of computer-assisted molecular docking for the prediction of cytochrome P450 substrates has been explored. P450cam is used as the model P450 enzyme because several high resolution crystal structures are available for it, although the approach should be applicable to any other isoform of P450 for which an active site structure is available. The chapter reviews computational approaches to analysis of the two subsequent steps, the degree of uncoupling, and the site specificity of the oxidation process. There are a number of programs available that can be used to predict whether a given small molecule will bind to a macromolecular receptor. DOCK has been used for the prediction of small molecules that bind to and inhibit a variety of enzymes. Future developments in this area should include utilization of the continuing improvements in ligand docking programs, such as, conformationally flexible searching, minimization of ligand/receptor complexes during searches, and more sophisticated scoring algorithms.

Fusion proteins of cytochrome P450(cam) with putidaredoxin (Pd) and putidaredoxin reductase (PdR), the two proteins required to transfer electrons from NADH to P450(cam), were constructed by fusing cDNAs encoding the three proteins in the expression vector pCWori(+). Several fusion proteins, in which the order of the three protein domains and the linkers between them were varied, were expressed in Escherichia coli, purified, and characterized, The highest activity (k(cat) = 30 min(-1)) was obtained with a PdR-Pd-P450(cam) construct in which the peptides TDGTASS and PLEL were used, respectively, to Link the PdR to the Pd and the Pd to the P450(cam) domains, Oxygen and NADH consumption is tightly coupled to substrate oxidation in the fusion proteins, The rate-limiting step in the catalytic turnover of these fusion proteins is electron transfer from Pd to P450(cam). This is indicated by high rates of electron transfer from the PdR and Pd domains to exogenous electron accepters, by an increase in the activity of the P450(cam) domain upon addition of exogenous Pd, and by the high activity of wild-type P450(cam) when incubated with a PdR-Pd fusion protein. E. cold cells expressing the PdR-Pd-P450(cam) fusion protein efficiently oxidize camphor to 5-exo-hydroxycamphor and 5-oxocamphor. E. coli cells expressing the triple fusion protein thus constitute the first heterologous self-sufficient catalytic system for the oxidation of camphor and other substrates by P450(cam).

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTComputer-Assisted, Structure-Based Prediction of Substrates for Cytochrome P450camJames J. De Voss and Paul R. Ortiz de MontellanoCite this: J. Am. Chem. Soc. 1995, 117, 14, 4185–4186Publication Date (Print):April 1, 1995Publication History Published online1 May 2002Published inissue 1 April 1995https://pubs.acs.org/doi/10.1021/ja00119a041https://doi.org/10.1021/ja00119a041research-articleACS PublicationsRequest reuse permissionsArticle Views72Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Starting from D-(-)-ribose, a set of synthetic routes sharing common intermediates has been developed and exemplified in [5'-H-2(2)]-, [4'-H-2]-, [1'-H-2]-, (5'R)-[5'-H-2]-, and (5'S)-[5'-H-2]-N-4-benzoylcytidine and, by deoxygenation, their corresponding 2'-deoxynucleosides. These syntheses provide convenient access to millimolar quantities of deuterium/tritium-labeled natural or unnatural nucleosides for direct use or automated oligonucleotide synthesis.

The proteases expressed by the HIV-1 and HIV-2 viruses process the polyproteins encoded by the viral genomes into the mature proteins required for virion replication and assembly. Eight analogs of haloperidol have been synthesized that cause time-dependent inactivation of the HIV-1 protease and, in six cases, HIV-2 protease. The IC50 values for the analogues are comparable to that of haloperidol itself. Enzyme inactivation is due to the presence of an epoxide in two of the analogues and carbonyl-conjugated double or triple bonds in the others. Irreversible inactivation is confirmed by the failure to recover activity when one of the inhibitors is removed from the medium. At pH 8.0, the agents inactivate the HIV-1 protease 4-80 times more rapidly than the HIV-2 protease. Faster inactivation of the HIV-1 protease is consistent with alkylation of cysteine residues because the HIV-1 protease has four such residues whereas the HIV-2 protease has none. Inactivation of the HIV-2 protease requires modification of non-cysteine residues. The similarities in the rates of inactivation of the HIV-2 protease by six agents that have intrinsically different reactivities toward nucleophiles suggest that the rate-limiting step in the inactivation process is not the alkylation reaction itself. At least five of the agents inhibit polyprotein processing in an ex vivo cell assay system, but they are also toxic to the cells.

In efforts to synthesize haloperidol analogues with improved properties as HIV protease inhibitors, methods were sought to introduce mono- and dialkylamino groups into the two aromatic rings of the parent structure. We report here that the reaction of haloperidol with alkylamines in the presence of a strong base (NaNH2) regiospecifically introduces the alkylamino group into the chlorophenyl ring, whereas the same reaction in the presence of a weak base (K2CO3) results in exclusive replacement of the fluorine of the fluorophenyl ring. Different amine functions can be introduced at the two rings by sequential reactions in the presence of, respectively, a strong and a weak base. The reaction catalyzed by NaNH2 involves, at least in part, benzyne formation, whereas the reaction catalyzed by K2CO3 involves direct nucleophilic addition to the aromatic ring. The regiospecificity of the reaction is due to conjugation of the fluorophenyl ring to a ketone group. The fluorophenyl ring is activated by the ketone towards nucleophilic aromatic substitution but is deactivated by the same function when it is converted by strong base to the enolate anion. Carbonyl conjugation of one of two haloaryl groups appears to be a general strategy for regiospecific introduction of alkylamino functions into complex aromatic molecules. The alkylamino derivatives actually prepared are comparable to haloperidol as inhibitors of the HIV-1 protease.

Ring expansion of the readily available cyclic haloketals and halo-O,S-ketals provides a versatile method for the synthesis of medium rings containing sulfur and/or oxygen atoms.

Haloperidol ketals and ethanedithioketals of interest as HIV-1 protease inhibitors were synthesized by addition of organolithium and organomagnesium reagents to ketone precursors already containing the ketal or thioketal functionality. Addition of Grignard reagents to the thioketal containing ketone was enhanced remarkably, and to the ketal containing ketone moderately, by the addition of magnesium chloride. The effect of magnesium chloride is attributed to its ability to competitively prevent chelation of the Grignard reagent and proton abstraction from the 4-oxopiperidine ring. The biological activities of the ketals and thioketals indicate that the thioketal function conveys greater ability to inhibit the HIV-1 protease than the ketal function.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSpecific abstraction of the 5'S- and 4'-deoxyribosyl hydrogen atoms from DNA by calicheamicin .gamma.1IJon J. Hangeland, James J. De Voss, Julie A. Heath, Craig A. Townsend, Wei Dong Ding, Joseph S. Ashcroft, and George A. EllestadCite this: J. Am. Chem. Soc. 1992, 114, 23, 9200–9202Publication Date (Print):November 1, 1992Publication History Published online1 May 2002Published inissue 1 November 1992https://pubs.acs.org/doi/10.1021/ja00049a069https://doi.org/10.1021/ja00049a069research-articleACS PublicationsRequest reuse permissionsArticle Views241Altmetric-Citations96LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts