The secondary amine/nitric oxide complex ion R(2)N[N(O)NO](-) as nucleophile and leaving group in S9N)Ar reactions.

Ions of structure R(2)N[N(O)NO](-) and their alkylation products have seen increasing use as nitric oxide (NO)-generating agents for biomedical research applications. Here we show that such diazeniumdiolate anions can readily displace halide from a variety of electrophilic aza- or nitroaromatic substrates to form O(2)-arylated derivatives of structure R(2)N-N(O)=N-OAr. The site of arylation and the cis arrangement of the oxygens were confirmed by X-ray crystallography. Displacement by various nucleophiles showed R(2)N[N(O)NO](-) to be a reasonably good leaving group, with rate constants for displacement by hydroxide, methoxide, and isopropylamine that were between those of chloride and fluoride in the S(N)Ar reactions we surveyed. The Meisenheimer intermediate could be spectrally observed. These O(2)-aryl diazeniumdiolates proved capable of reacting with the nucleophilic sulfur of the HIV-1 p7 nucleocapsid protein's zinc finger assembly to eject the zinc, disrupting a structural motif critical to viral replication and suggesting possible utility in the drug discovery realm.

Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action.

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

Inhibition of G-protein-coupled receptor function by disruption of transmembrane domain interactions.

G-protein-coupled receptors (GPCR) represent a superfamily of proteins that mediate the function of neurotransmitters and peptide hormones and are involved in viral entry and perception of light, smell, and taste. GPCRs are characterized by the presence of seven transmembrane domains (TMs). We demonstrate here that structural analogs of individual TMs of GPCRs can serve as potent and specific receptor antagonists. Peptides derived from the transmembrane regions of CXCR4 and CCR5 chemokine receptors specifically inhibited receptor signaling and the in vitro replication of human immunodeficiency virus-1 (HIV-1) at concentrations as low as 0.2 microM. Similarly, peptides mimicking the TMs of cholecystokinin receptor A, were found to abolish ligand binding and signaling through the receptor. Negative charges positioned at the extracellular termini of peptide antagonists appeared to be important for correct spontaneous insertion of the compounds into the cell membrane and for their activity. Targeting of the specific interactions between transmembrane domains of GPCRs is suggested as a general sequence-based method to disrupt receptor function for application in drug design and for structure-function studies of the receptors.

Thiazolothiazepine inhibitors of HIV-1 integrase.

A series of thiazolothiazepines were prepared and tested against purified human immunodeficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups are in general more potent against purified IN than those containing only one carbonyl group. Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears important for potency against IN, as its replacement with an oxygen or carbon abolished activity. Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed antiviral activity and inhibited IN within similar concentrations. These compounds inhibited IN when Mn(2+) or Mg(2+) was used as cofactor. None of these compounds showed detectable activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds for development as inhibitors of IN and HIV replication.

Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein.

BACKGROUND: The principal agent in the etiology of cervical cancer, i.e., human papillomavirus (HPV) type 16, encodes three oncoproteins, E5, E6, and E7. Structural and mutational studies have identified two potential zinc-finger domains as critical for E6 protein function. We investigated several assays to identify and characterize compounds that interfere with the binding of zinc to E6. METHODS: Thirty-six compounds were selected on the basis of their structure, which would facilitate their participation in sulfhydryl residue-specific redox reactions, and were tested for their ability to release zinc from E6 protein. The zinc-ejecting compounds were then tested for their ability to inhibit E6 binding to E6-associated protein (E6AP) and E6-binding protein (E6BP), two coactivators of E6-mediated cellular transformation. The binding of E6 to E6BP and E6AP was measured by use of surface plasmon resonance (a technique that monitors molecular interactions by measuring changes in refractive index) and by use of in vitro translation assays. The compounds were also tested for their effects on the viability of HPV-containing cell lines. RESULTS: Nine of the 36 tested compounds ejected zinc from E6. Two of the nine compounds inhibited the interaction of E6 with E6AP and E6BP, and one of these two, 4, 4'-dithiodimorpholine, selectively inhibited cell viability and induced higher levels of p53 protein (associated with the induction of apoptosis [programmed cell death]) in tumorigenic HPV-containing cells. CONCLUSION: We have described assay systems to identify compounds, such as 4,4'-dithiodimorpholine, that can potentially interfere with the biology and pathology of HPV. These assay systems may be useful in the development of drugs against cervical cancer, genital warts, and asymptomatic infections by genital HPVs.

Extension of the polyanionic cosalane pharmacophore as a strategy for increasing anti-HIV potency.

The anti-HIV agent cosalane inhibits both the binding of gp120 to CD4 as well as an undefined postattachment event prior to reverse transcription. Several cosalane analogues having an extended polyanionic "pharmacophore" were designed based on a hypothetical model of the binding of cosalane to CD4. The analogues were synthesized, and a number of them displayed anti-HIV activity. One of the new analogues was found to possess enhanced potency as an anti-HIV agent relative to cosalane itself. Although the new analogues inhibited both HIV-1 and HIV-2, they were more potent as inhibitors of HIV-1 than HIV-2. Mechanism of action studies indicated that the most potent of the new analogues inhibited fusion of the viral envelope with the cell membrane at lower concentrations than it inhibited attachment, suggesting inhibition of fusion as the primary mechanism of action.

Chicoric acid analogues as HIV-1 integrase inhibitors.

The present study was undertaken to examine structural features of L-chicoric acid (3) which are important for potency against purified HIV-1 integrase and for reported cytoprotective effects in cell-based systems. Through a progressive series of analogues, it was shown that enantiomeric D-chicoric acid (4) retains inhibitory potency against purified integrase equal to its L-counterpart and further that removal of either one or both carboxylic functionalities results in essentially no loss of inhibitory potency. Additionally, while two caffeoyl moieties are required, attachment of caffeoyl groups to the central linking structure can be achieved via amide or mixed amide/ester linkages. More remarkable is the finding that blockage of the catechol functionality through conversion to tetraacetate esters results in almost no loss of potency, contingent on the presence of at least one carboxyl group on the central linker. Taken as a whole, the work has resulted in the identification of new integrase inhibitors which may be regarded as bis-caffeoyl derivatives of glycidic acid and amino acids such as serine and beta-aminoalanine. The present study also examined the reported ability of chicoric acid to exert cytoprotective effects in HIV-infected cells. It was demonstrated in target and cell-based assays that the chicoric acids do not significantly inhibit other targets associated with HIV-1 replication, including reverse transcription, protease function, NCp7 zinc finger function, or replication of virus from latently infected cells. In CEM cells, for both the parent chicoric acid and selected analogues, antiviral activity was observable under specific assay conditions and with high dependence on the multiplicity of viral infection. However, against HIV-1- and HIV-2-infected MT-4 cells, the chicoric acids and their tetraacetylated esters exhibited antiviral activity (50% effective concentration (EC50) ranging from 1.7 to 20 microM and 50% inhibitory concentration (IC50) ranging from 40 to 60 microM).

Synthesis and biological properties of novel pyridinioalkanoyl thiolesters (PATE) as anti-HIV-1 agents that target the viral nucleocapsid protein zinc fingers.

Nucleocapsid p7 protein (NCp7) zinc finger domains of the human immunodeficiency virus type 1 (HIV-1) are being developed as antiviral targets due to their key roles in viral replication and their mutationally nonpermissive nature. On the basis of our experience with symmetrical disulfide benzamides (DIBAs; Rice et al. Science 1995, 270, 1194-1197), we synthesized and evaluated variants of these dimers, including sets of 4,4'- and 3,3'-disubstituted diphenyl sulfones and their monomeric benzisothiazolone derivatives (BITA). BITAs generally exhibited diminished antiviral potency when compared to their disulfide precursors. Novel, monomeric structures were created by linking haloalkanoyl groups to the benzamide ring through -NH-C(=O)- (amide) or -S-C(=O)- (thiolester) bridges. Amide-linked compounds generally lacked antiviral activity, while haloalkanoyl thiolesters and non-halogen-bearing analogues frequently exhibited acceptable antiviral potency, thus establishing thiolester benzamides per se as a new anti-HIV chemotype. Pyridinioalkanoyl thiolesters (PATEs) exhibited superior anti-HIV-1 activity with minimal cellular toxicity and appreciable water solubility. PATEs were shown to preferentially target the NCp7 Zn finger when tested against other molecular targets, thus identifying thiolester benzamides, and PATEs in particular, as novel NCp7 Zn finger inhibitors for in vivo studies.

Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory.

The reaction of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 (NCp7) with a variety of electrophilic agents was investigated by experimental measurements of Trp37 fluorescence decay and compared with theoretical measures of reactivity based on density-functional theory in the context of the hard and soft acids and bases principle. Statistically significant correlations were found between rates of reaction and the ability of these agents to function as soft electrophiles. Notably, the molecular property that correlated strongest was the ratio of electronegativity to hardness, chi2/eta, a quantity related to the capacity of an electrophile to promote a soft (covalent) reaction. Electronic and steric determinants of the reaction were also probed by Fukui function and frontier-orbital overlap analysis in combination with protein-ligand docking methods. This analysis identified selective ligand docking regions within the conserved zinc finger domains that promoted reaction. The Cys49 thiolate was found overall to be the NCp7 site most susceptible to electrophilic attack.

Small molecule inhibitor of HIV-1 cell fusion blocks chemokine receptor-mediated function.

The intersection of the HIV and the chemokine fields began with the observation that HIV entry into cells could be blocked by certain chemokines. Subsequent work showed that HIV entry is dependent on the presence of specific chemokine receptors. These observations led us to evaluate a series of compounds, ureido analogs of distamycin previously reported to block HIV entry into cells in vitro, for chemokine antagonist activity. One of the distamycin analogs, 2,2'[4,4'-[[aminocarbonyl]amino]bis[N,4'-di[pyrrole-2-carboxamide- 1,1'-dimethyl]]-6,8 napthalenedisulfonic acid] hexasodium salt (NSC 651016), is shown here to inhibit syncytia formation and cell fusion. Mechanistic studies showed that this inhibition was not due to conformational changes in gp120-gp41 induced by target cell CD4 and chemokine co-receptor and was therefore not due to interference with binding of HIV-1. Additional mechanistic studies demonstrated that NSC 651016 inhibited chemokine binding to specific chemokine receptors, induced CXCR4 and CCR5 receptor internalization, and inhibited chemokine-induced chemotaxis by macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and stromal-derived factor-1alpha but not monocyte chemotactic protein-1. Thus, we describe a novel compound that inhibits in vivo replication of HIV-1 by down-regulation of co-receptors. These data lead us to propose that NSC 651016 may have in vivo anti-inflammatory activity.

Inhibition of in vitro and in vivo HIV replication by a distamycin analogue that interferes with chemokine receptor function: a candidate for chemotherapeutic and microbicidal application.

Select chemokine receptors act as coreceptors for HIV-1 entry into human cells and represent targets for antiviral therapy. In this report we describe a distamycin analogue, 2,2'-[4, 4'-[[aminocarbonyl]amino]bis[N,4'-di[pryrrole-2-carboxamide- 1, 1'-dimethyl]]-6,8-naphthalenedisulfonic acid]hexasodium salt (NSC 651016), that selectively inhibited chemokine binding to CCR5, CCR3, CCR1, and CXCR4, but not to CXCR2 or CCR2b, and blocked chemokine-induced calcium flux. Inhibition was not due to nonspecific charge interactions at the cell surface, but was based on a specific competition for the ligand receptor interaction sites since the inhibitory effect was specific for some but not all chemoattractant receptors. NSC 651016 inhibited in vitro replication of a wide range of HIV-1 isolates, as well as HIV-2 and SIV, and exhibited in vivo anti-HIV-1 activity in a murine model. In contrast, a distamycin analogue with similar structure and charge and the monomeric form of NSC 651016 demonstrated no inhibitory effects. These data demonstrate that molecules which interfere with HIV-1 entry into cells by targeting specific chemokine coreceptors can provide a viable approach to anti-HIV-1 therapy. NSC 651016 represents an attractive candidate for the chemotherapeutic treatment of HIV-1 infection and as a microbicide to prevent the sexual transmisssion of HIV-1. Moreover, NSC 651016 can serve as a template for medicinal chemical modifications leading to more effective antivirals.

New alkenyldiarylmethanes with enhanced potencies as anti-HIV agents which act as non-nucleoside reverse transcriptase inhibitors.

Twenty-two new alkenyldiarylmethanes (ADAMs) were synthesized and evaluated for inhibition of HIV-1 replication. The most potent compound proved to be methyl 3',3"-dichloro-4',4"-dimethoxy-5', 5"-bis(methoxycarbonyl)-6,6-diphenyl-5-hexenoate (ADAM II), which displayed an EC50 of 13 nM for inhibition of the cytopathic effect of HIV-1RF in CEM-SS cells. ADAM II inhibited HIV-1 reverse transcriptase with an IC50 of 0.3 microM but was inactive as an inhibitor of HIV-1 attachment/fusion to cells, protease, integrase, and the nucleocapsid protein. Molecular target-based and cell-based assays revealed that ADAM II acted biologically as a nonnucleoside reverse transcriptase inhibitor (NNRTI). ADAM II inhibited replication of a wide variety of laboratory, clinical, and clade-representative isolates of HIV-1 in T cell lines and cultures of peripheral blood mononuclear cells or monocyte/macrophages. Mutations that conferred resistance to ADAM II clustered at residues 101, 103, 108, 139, 179, 181, and 188, which line the nonnucleoside binding pocket of HIV-1 reverse transcriptase. However, HIV-1 NL4-3 strain expressing a mutation at residue 100 of reverse transcriptase, and an AZT-resistant virus, displayed increased sensitivity to ADAM II. Thus, ADAM II could serve as an adjunct therapy to AZT and NNRTIs that select for L100I resistance mutations.

Efficacies of zinc-finger-active drugs against Giardia lamblia.

Twenty-nine of 34 (85%) Zn-finger-active compounds at 300 microM or less inhibited the growth of Giardia lamblia. The most active compound, disulfiram (Antabuse), was cidal at 1.23 +/- 0.32 microM. In the adult mouse model, significant in vivo activity was demonstrated by increased cure rates and decreased parasite burdens.

Anti-HIV agents that selectively target retroviral nucleocapsid protein zinc fingers without affecting cellular zinc finger proteins.

Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription. Selectivity of interactions of these agents with NCp7 was supported by molecular modeling analysis which (1) identified a common saddle-shaped nucleophilic region on the surfaces of both NCp7 Zn fingers, (2) indicated a strong correspondence between computationally docked positions for the agents tested and overlap of frontier orbitals within the nucleophilic loci of the NCp7 Zn fingers, and (3) revealed selective steric exclusion of the agents from the core of the GATA-1 Zn finger. Further modeling analysis suggests that the thiolate of Cys49 in the carboxy-terminal finger is the site most susceptible to electrophilic attack. These data provide the first experimental evidence and rationale for antiviral agents that selectively target retroviral nucleocapsid protein Zn fingers.

Inhibition of acute-, latent-, and chronic-phase human immunodeficiency virus type 1 (HIV-1) replication by a bistriazoloacridone analog that selectively inhibits HIV-1 transcription.

Nanomolar concentrations of temacrazine (1,4-bis[3-(6-oxo-6H-v-triazolo[4,5,1-de]acridin-5-yl)amino-propyl ]piperazine) were discovered to inhibit acute human immunodeficiency virus type 1 (HIV-1) infections and suppress the production of virus from chronically and latently infected cells containing integrated proviral DNA. This bistriazoloacridone derivative exerted its mechanism of antiviral action through selective inhibition of HIV-1 transcription during the postintegrative phase of virus replication. Mechanistic studies revealed that temacrazine blocked HIV-1 RNA formation without interference with the transcription of cellular genes or with events associated with the HIV-1 Tat and Rev regulatory proteins. Although temacrazine inhibited the in vitro 3' processing and strand transfer activities of HIV-1 integrase, with a 50% inhibitory concentration of approximately 50 nM, no evidence of an inhibitory effect on the intracellular integration of proviral DNA into the cellular genome during the early phase of infection could be detected. Furthermore, temacrazine did not interfere with virus attachment or fusion to host cells or the enzymatic activities of HIV-1 reverse transcriptase or protease, and the compound was not directly virucidal. Demonstration of in vivo anti-HIV-1 activity by temacrazine identifies bistriazoloacridones as a new class of pharmaceuticals that selectively blocks HIV-1 transcription.

Synthesis of a cosalane analog with an extended polyanionic pharmacophore conferring enhanced potency as an anti-HIV agent.

A novel cosalane analog having an extended polyanionic pharmacophore was synthesized in order to target specific cationic residues on the surface of CD4. The design rationale is based on a hypothetical binding model of cosalane to the surface of the protein. The new analog displayed an EC50 of 0.55 microM as an inhibitor of the cytopathic effect of HIV-1RF in CEM-SS cells, which represents a significant increase in potency over cosalane itself (EC50 5.1 microM). Both cosalane and the new analog are inhibitors of viral entry into target cells.

Synthesis of a non-nucleoside reverse transcriptase inhibitor in the alkenyldiarylmethane (ADAM) series with optimized potency and therapeutic index.

A novel alkenyldiarylmethane (ADAM) analog has been synthesized with enhanced potency as an anti-HIV agent. The new compound (ADAM II) inhibits the cytopathic effect of HIV-1RF in CEM-SS cells with an EC50 of 13 nM, while it shows cytotoxicity with a CC50 of 31.6 microM, providing a therapeutic index of 2430. ADAM II is a non-nucleoside reverse transcriptase inhibitor, displaying an IC50 of 0.3 microM with poly(rC) oligo(dG) as the template/primer.

Zinc ejection as a new rationale for the use of cystamine and related disulfide-containing antiviral agents in the treatment of AIDS.

The highly conserved and mutationally intolerant retroviral zinc finger motif of the HIV-1 nucleocapsid protein (NC) is an attractive target for drug therapy due to its participation in multiple stages of the viral replication cycle. A literature search identified cystamine, thiamine disulfide, and disulfiram as compounds that have been shown to inhibit HIV-1 replication by poorly defined mechanisms and that have electrophilic functional groups that might react with the metal-coordinating sulfur atoms of the retroviral zinc fingers and cause zinc ejection. 1H NMR studies reveal that these compounds readily eject zinc from synthetic peptides with sequences corresponding to the HIV-1 NC zinc fingers, as well as from the intact HIV-1 NC protein. In contrast, the reduced forms of disulfiram and cystamine, diethyl dithiocarbamate and cysteamine, respectively, were found to be ineffective at zinc ejection, although cysteamine formed a transient complex with the zinc fingers. Studies with HIV-1-infected human T-cells and monocyte/macrophage cultures revealed that cystamine and cysteamine possess significant antiviral properties at nontoxic concentrations, which warrant their consideration as therapeutically useful anti-HIV agents.

Differential infection of CD34+ cell-derived dendritic cells and monocytes with lymphocyte-tropic and monocyte-tropic HIV-1 strains.

Monocytes and dendritic cells are infected by HIV-1 and subsequently produce virions that initiate further rounds of infection. Current methods for the isolation and study of dendritic cells are hampered by the low frequency of these cells and contamination with other cell types. A two-step culture method was devised to generate large numbers of either dendritic cells or monocytes from fetal liver CD34+ progenitors. CD34+ cells were first expanded with the growth factors granulocyte-macrophage CSF and stem cell factor to generate a population of intermediate progenitor cells with a relatively immature phenotype. To induce specific differentiation to dendritic cells, the cultures were switched to serum-free medium with the growth factors granulocyte-macrophage CSF, stem cell factor, TNF-alpha, and IL-4. The cells became highly positive for HLA class II Ags and the dendritic cell marker CD1a. Culture of the intermediate progenitors in serum-containing medium with macrophage CSF resulted in differentiation to adherent monocytes expressing high levels of CD14 with low CD1a expression. The intermediate progenitors were permissive for HIV infection by both monocyte- and lymphocyte-tropic strains. In contrast, differentiation to monocytes or dendritic cells resulted in restricted viral tropism. Dendritic cells efficiently replicated the lymphocyte-tropic virus HIV-1MN, but not the monocyte-tropic virus HIV-1ADA. As expected, monocytes only supported replication of HIV-1ADA. This two-step culture method allows for the production of large numbers of monocytes or dendritic cells from a common precursor pool for studying the development of tropism-associated events.

Suppression of retrovirial replication: inactivation of murine leukemia virus by compounds reacting with the zinc finger in the viral nucleocapsid protein.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two zinc fingers, consisting of the sequence C-X2-C-X4-H-X4-C. Rice et al. (Science 270:1194-1197, 1995) have described a series of compounds which inactivate HIV-1 particles and oxidize the sulfur atoms in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. In contrast, the compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV-1 by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses, and the lethality of all known mutations altering the zinc-binding residues, suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.