Independence of herpesvirus-induced T cell lymphoma from viral cyclin D homologue.

Cyclin D family members are cellular protooncogenes, and their viral homologues in the Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus type 8 [HHV-8]) and the closely related Herpesvirus saimiri have been implicated as putative cofactors of viral transformation and pathogenesis. KSHV is regularly found in Kaposi's sarcoma and in the primary effusion B cell lymphoma and Castleman's disease associated with immunosuppression and AIDS. H. saimiri strain C488 transforms human and marmoset T cells in vitro and causes polyclonal T cell lymphoma in New World monkeys. The viral cyclins stimulate cell cycle progression of quiescent fibroblasts, and they form active cyclin-dependent kinase (CDK)6 complexes of broad substrate specificity that can resist and downregulate cellular CDK inhibitors. This study shows that the viral cyclin of H. saimiri strain C488 is not required for viral replication, T cell transformation, and pathogenicity in New World primates.

Synthesis and activity of piperazine-containing antirhinoviral agents and crystal structure of SDZ 880-061 bound to human rhinovirus 14.

A series of antipicornaviral agents containing piperazinyl moieties was synthesized with the objective of obtaining a compound with a broad spectrum of antirhinovirus activity, high potency (< or = 0.003 microgram/ml), and low cytotoxicity (> or = 30 micrograms/ml). Five compounds of this series were evaluated in detail for efficacy against various HRV serotypes. The agent SDZ 880-061, containing the benzothiazine moiety SDZ 108-075, which is particularly active against HRV14, and the thiazolyl acetic acid ester group of SDZ 89-124, which is potent against HRV1B, indeed has a relatively broad antiviral spectrum. SDZ 880-061 inhibited 85% of 89 HRV serotypes tested at a concentration of < or = 3 micrograms/ml. The 3.0 A resolution X-ray structure of SDZ 880-061 bound to HRV14 has revealed the binding characteristics of this potent compound. It binds in the same pocket as other capsid-binding antiviral agents characterized to date, leaving the innermost portion of the pocket vacant. The binding causes similar, although less extensive, alterations of the HRV14 VP1 backbone conformation (residues 100 to 110, 151 to 159, and 213 to 224) compared to other antiviral agents analyzed structurally. Although the contacts between SDZ 880-061 and HRV14 are mostly of hydrophobic character, the inhibitor has three relatively short polar interactions with residues of VP1 that represent potential hydrogen bonds. The amount of solvent-accessible surface area of SDZ 880-061 buried in the complex (613 A2) is within the range of that observed in protein-protein interfaces. The observed influence of time of addition or removal of SDZ 880-061 on virus yield and on the infectious-center formation indicates that the compound primarily interferes with HRV14 cellular attachment. Since it is assumed that uncoating requires virion instability and/or flexibility, the finding that SDZ 880-061 has only a marginal effect on uncoating may be due to the fact that it does not completely fill the hydrophobic pocket.

In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages.

Because of the importance of macrophages in the pathogenesis of the disease caused by HIV, we investigated the efficacy of various anti-HIV drugs in human primary macrophages acutely or chronically infected by this virus. The results obtained for acutely infected macrophages show that dideoxynucleosides (AZT, ddI, and ddC), interferon-alpha and -gamma, mismatched double-stranded RNA, Tat inhibitor, phosphorothioate antisense, and inhibitors of HIV protease, all significantly inhibit virus replication at concentrations far below those toxic for the cells. However, in macrophages in which proviral DNA is already integrated (chronically infected macrophages), only the three inhibitors of HIV protease induced significant virus inhibition at concentrations 100 or more times higher than those effective in acutely infected macrophages. Treatment of macrophages with macrophage colony-stimulating factor does not affect the anti-HIV efficacy of protease inhibitors. These results suggest that therapeutic strategies with activity for macrophages, including inhibitors of HIV protease, are worth pursuing in patients with HIV infection.

Novel thiosemicarbazones derived from formyl- and acyldiazines: synthesis, effects on cell proliferation, and synergism with antiviral agents.

The synthesis of a series of novel thiosemicarbazones (TSC's) derived from various alkyl diazinyl (3-pyridazinyl, 4-pyrimidinyl, 2-pyrazinyl) ketones and 3-pyridazinecarbaldehyde and their evaluation against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) as well as the determination of their cytotoxicity are described. In addition, the effects of combination of such TSC's with the well-known antiviral drugs acyclovir (ACV) and 3'-azido-3'-deoxythymidine (AZT) were studied. Under our experimental conditions, i.e. determination of virus-induced cytopathic effect upon infection of HUT78 cells with HSV-1 and upon infection of MT4 cells with HIV-1, no antiviral activity could be detected with any of the TSC's. However, pronounced effects on proliferation of these rapidly growing T4 lymphocyte cell lines were observed. Clear structure-activity relationships with regard to these cytotoxic effects could be established: compared to pyridine, pyrazine, or pyrimidine-derived TSC's most of the 3-pyridazinyl congeners investigated are less cytotoxic; introduction of a methyl group into C-6 of the pyridazine system or prolongation of the acyl moiety in these compounds has essentially no influence; all compounds bearing an N,N-dimethylamino or a cycloamino substituent are much more toxic than those with an NH2 or NHR substituent; the nature of R in the latter type of compounds has only moderate influence. It has been reported that combination of TSC's with the antiviral agent acyclovir (ACV) results in potentiation of this well-known drug. We evaluated the potential of our series of novel TSC's in combination with ACV for inhibition of HSV-1-induced cytopathic effect in HUT78 cells and in combination with 3'-azido-3'-deoxythymidine (AZT) for inhibition of HIV-1-induced cytopathic effect in MT4 cells. Only four compounds out of this series, all characterized by an unsubstituted NH2 group, exhibited moderate synergism with the above mentioned antiviral drugs. Our results do not support the previously expressed opinion that TSC's are selective antiviral agents. In our test systems no evidence for inhibition of virus-induced cytopathic effect was obtained. The TSC derivatives exhibited a broad range of cytotoxic effects, some at concentrations considerably below those reported to have antiviral efficacy. Several of our novel diazine-derived compounds proved advantageous over the previously described pyridine analogues with regard to cytotoxicity. Moderate synergism could be detected for relatively noncytotoxic TSC's with the antiviral drugs ACV (antiherpes) and AZT (anti-HIV).

Paracyclophanes: a novel class of water-soluble inhibitors of HIV proteinase.

A versatile synthesis of functionalized para- and metacyclophanes (macrocycles with one or more aromatic rings incorporated; ansa-compounds) has been developed. Cyclophanes constitute a novel building block for potent human immunodeficiency virus (HIV) protease inhibitors. The synthesis of the macrocyclic ring system was achieved by regio- and stereospecific ring opening of N-protected 4-amino-2,3-epoxy-5-phenylpentanoates with appropriate alpha, omega-diamines and consecutive ring closure under high dilution conditions. The resulting macrocyclic building blocks enabled further broad and flexible derivation. Paracyclophanes, containing oxyethylene substructures, were found to dissolve in phosphate-buffered saline at concentrations as high as 3 mg/mL at physiological pH. Several derivatives with Ki values lower than 10 nM and antiviral activities in the range of 15-50 nM have been obtained. The influence of the ring size and of the substitution pattern of the cyclophane moiety on enzyme inhibition, antiviral activity, and water solubility are discussed. Preliminary data on oral bioavailability in mice are given for selected compounds.

Phosphonoformate and phosphonoacetate derivatives of 5-substituted 2'-deoxyuridines: synthesis and antiviral activity.

The synthesis of potential "combined prodrugs" wherein phosphonoformate or phosphonoacetate was attached to the 5'-position of 2'-deoxyuridine, 2'-deoxythymidine, 5-iodo-2'-deoxyuridine (IDU), 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), or 5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or to the 3'-position of CEDU is described. The antiviral activities of these derivatives and of reference compounds were compared in Vero, HEp-2, and primary rabbit kidney cells against herpes simplex virus types 1 and 2 (HSV-1 and -2). The CEDU and BVDU analogues were also evaluated against systemic and intracutaneous HSV-1 infection in mice. The nature of the 5-substituent proved critical for antiviral activity, since only the 5-iodo-, 5-(2-bromovinyl)-, and 5-(2-chloroethyl)-substituted derivatives were inhibitory to the herpesviruses. Furthermore, the type specificity is determined by the nature of the 5-substituent: the IDU analogues were similarly inhibitory to HSV-1 and -2 whereas the CEDU and BVDU analogues inhibited HSV-2 replication only at considerably higher concentrations than HSV-1. In vivo, several derivatives were shown to possess significant antiviral activity; however, none surpassed its respective parent compound, CEDU or BVDU, in potency. It seems improbable, therefore, that a synergistic effect between PFA or PAA and the nucleoside analogue occurred. The extent of in vitro and in vivo activity of the CEDU and BVDU 5'-phosphonoformates and 5'-phosphonoacetates is most plausibly explained by the ease by which the "combined prodrugs" are hydrolyzed and the parent compound, CEDU and BVDU, respectively, is released.

Inhibitors of HIV-1 proteinase containing 2-heterosubstituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, enzyme inhibition, and antiviral activity.

A convenient procedure for the synthesis of 2-heterosubstituted statine derivatives as novel building blocks in HIV-protease inhibitors has been developed. The synthesis starts with protected L-phenylalaninols, which were converted to gamma-amino alpha, beta-unsaturated esters in a one-pot procedure. A highly diastereoselective epoxidation of the N-protected (E)-enoates, followed by regioselective ring opening of the corresponding 2,3-epoxy esters with a variety of heteronucleophiles, resulted in 2-heterosubstituted statine derivatives. The overall stereo-chemical outcome of the transformations meets the required configuration of HIV-protease inhibitors. The short, synthetically flexible, and highly diastereoselective synthesis of 2-heterosubstituted statines has enabled a broad derivation, covering the S3, S2, and S1'-S3' sites of the enzyme. In a series of 46 derivatives, several potent inhibitors were obtained with Ki values as low as 3.4 nM and antiviral activity in the lower nanomolar-range. The structural parameters of the compounds which determine the potency of inhibition and selectivity for the viral enzyme are discussed.

Inhibitors of human immunodeficiency virus type 1 protease containing 2-aminobenzyl-substituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, activity, and oral bioavailability.

Systematic modifications of HIV protease inhibitor (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-valyl]-amino]-3-hydroxy-2-[(4- methoxybenzyl)amino]-5-phenylpentanoyl)-L-valine 2-(aminomethyl)- benzimidazole amide led to a novel series of inhibitors with shortened, modified carboxy terminus. Their synthesis, in vitro enzyme inhibitory data, and antiviral activities are reported. Of particular interest are derivatives featuring the (1S,2R)-1-amino-2-hydroxyindan moiety at the P2'-position since some of them exhibit substantial oral bioavailability in mice. The influence of aqueous solubility and structural parameters on the oral resorption of the inhibitors is discussed. Optimum enhancement of oral bioavailability was observed with L-tert-leucine in P2-position, resulting in the discovery of (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-tert-leucyl]- amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-phenylpentanoic acid (1S,2R)-1-amino-2-hydroxyindan amide which combines high antiviral activity (IC50 = 250 nM) with a good pharmacokinetic profile (AUC = 82.5 microM.h at a dose of 125 mg/kg po in mice).

2'-Fluorinated arabinonucleosides of 5-(2-haloalkyl)uracil: synthesis and antiviral activity.

The synthesis of 5-(2-fluoroethyl)-2'-deoxyuridine (FEDU, 4b), its 2'-fluoro analogue 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-(2-fluoroethyl)-1H,3H- pyrimidine-2,4-dione (FEFAU, 4k), and the 2'-fluoro analogue of the potent antiherpes virus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), 5-(2-chloroethyl)-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1H,3H-pyr imidine - 2,4-dione (CEFAU, 4i), is described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus (HSV) types 1 and 2 and varicella zoster virus (VZV). All compounds were shown to possess significant and selective antiviral activity. FEDU proved less potent than CEDU against VZV replication; however, it was more active against HSV-2. CEFAU showed marked activity against HSV-1, HSV-2, and VZV. The compound containing fluorine at both positions, FEFAU, exhibited the strongest antiviral potency against HSV-1, HSV-2, and VZV. It inhibited HSV-1 at a concentration of 0.03-0.2 microgram/mL, HSV-2 at 0.1-0.3 microgram/mL, and VZV at 0.03 microgram/mL. Neither FEDU nor CEFAU or FEFAU exerted a significant inhibitory effect on cell proliferation at a concentration of 100 micrograms/mL. Thus, the cytotoxicity of these compounds is as low as that of CEDU and compares favorably to that of previously described 2'-fluoroarabinosyl nucleoside analogues.

5-(Haloalkyl)-2'-deoxyuridines: a novel type of potent antiviral nucleoside analogue.

Syntheses of 5-(2-haloethyl)-2'-deoxyuridines, 5-(3-chloropropyl)-2'-deoxyuridines, and 5-(2-chloroethyl)-2'-deoxycytidine are described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus types 1 and 2. All compounds were shown to possess significant and selective antiviral activity. The most potent derivative, 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), inhibited HSV-1 at concentrations below 0.1 microgram/mL. It exerted measurable inhibitory effects on cell proliferation only at concentrations higher than 100 micrograms/mL. In vivo CEDU reduced the mortality rate of HSV-1-infected mice at concentrations lower than 5 mg/kg per day when given intraperitoneally and orally. Thus, it proved to be more effective in this in vivo model than the reference compounds (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-[(2-hydroxyethoxy)methyl]guanine (ACV).

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Isolation and characterization of an arildone-resistant poliovirus 2 mutant with an altered capsid protein VP1.

Arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl] -3,5-heptanedione is a selective inhibitor of poliovirus uncoating. We have isolated an arildone-resistant poliovirus 2 variant and characterized it. In single cycle growth curve experiments the resistant virus was not influenced by 1 microgram/ml of arildone, which completely inhibited the replication of the sensitive virus. In neutralization experiments the rates of inactivation vs time of the parent and the resistant virus proved indistinguishable. The adsorption behavior of the mutant closely resembled that of the wild-type. Uncoating as measured with neutral red-sensitized arildone-resistant mutant virus was unaffected by arildone, in contrast to the uncoating of the sensitive parent virus. It has been previously shown that arildone stabilizes the poliovirus capsid from alkaline degradation. The arildone-resistant mutant, however, was not markedly stabilized by the drug under these conditions. No difference in thermostability between mutant and parent virus could be detected. In isoelectric focusing the sensitive virus exhibited 2 peaks, but in the presence of arildone only one peak was apparent. In contrast, the arildone-resistant virus remained unaffected by the presence of arildone. Analysis of the capsid proteins of the sensitive parent and the resistant virus in SDS-polyacrylamide gels revealed no difference in the patterns; however, peptide mapping showed clear differences in VP1.

In vivo efficacy of SDZ 35-682, a new picornavirus capsid-binding agent.

SDZ 35-682 is a potent and selective inhibitor of the replication of members of the picornavirus group. It belongs to the group of uncoating inhibitors binding to the hydrophobic pocket in the capsid of the virion. In cell culture it inhibits several rhinovirus serotypes and echovirus 9 at concentrations as low as 0.1 micrograms/ml. In the echovirus 9 animal model the protective effect of SDZ 35-682 was found to be dependent on both, dose of drug and duration of treatment. Significant protection of newborn mice from paralysis and death could be achieved by either a high dose (126 mg/kg) given only twice, at days 0 and 1 relative to echovirus 9 inoculation, or by a lower dose administered for 4 or 6 days. This finding might be explained by assuming a long half-life for SDZ 35-682. Though clinical usefulness of SDZ 35-682 may be limited by its relatively narrow antiviral spectrum it represents a novel potent and selective inhibitor of rhinovirus and echovirus 9 replication in cell culture and in the organism.

SDZ 35-682, a new picornavirus capsid-binding agent with potent antiviral activity.

SDZ 35-682 is a potent and selective inhibitor of the replication of members of the picornavirus group. It inhibits several rhinovirus serotypes and echovirus 9 at concentrations as low as 0.1 micrograms/ml, without exerting any effect on cell proliferation up to 30 micrograms/ml. As observed with other capsid-binding antipicornavirus compounds, there is a wide variation in sensitivity of the different serotypes within the rhinovirus group. The point of interference of SDZ 35-682 in a single cycle of virus growth is an early event taking place before 2 or 3 h of echo- or rhinovirus replication, respectively. By incorporation of neutral red into the viral capsid and measurement of acquisition of photoresistance it is shown that uncoating of echovirus 9 is inhibited by SDZ 35-682. In addition, efficiency of adsorption of echovirus 9 is reduced by SDZ 35-682. To demonstrate that SDZ 35-682, like other uncoating inhibitors of picornaviruses, binds to the hydrophobic pocket beneath the canyon floor co-crystallization with HRV 14 was performed. Considerable conformational changes occur in VP1 in the HRV 14/SDZ 35-682 complex. SDZ 35-682 is 19 A long from end to end and thus fills the entire hydrophobic pocket including its innermost end; it is less flexible than other long antiviral agents. It has been suggested that compounds filling the entire hydrophobic pocket will affect the uncoating process of the virion. Thus, inhibition of viral uncoating, as demonstrated with echovirus 9, probably is the predominant mode of action of SDZ 35-682.

HIV proteinase inhibitors containing 2-aminobenzylstatine as a novel scissile bond replacement: biochemical and pharmacological characterization.

Derivation of the 2-aminobenzylstatine containing HIV-1 proteinase (PR) inhibitor I led to a series of compounds with considerably improved antiviral activity, the most potent derivatives inhibiting HIV-1 with IC50 values below 25 nM. This was achieved by the combination of several structural modifications, most prominently by introduction of a benzimidazole heterocycle into the inhibitor. The mode of action of the 2-aminobenzylstatine PR inhibitors was demonstrated to be inhibition of gag precursor processing. The antiviral efficacy of the PR inhibitors was demonstrated in various cell lines, in primary T4 lymphocytes and in monocytes. The most potent compound (XI) inhibited replication of several HIV-1 clinical isolates in primary cells with IC50 values of 8 to 23 nM. The analysis of the pharmacokinetic behaviour of compounds I and VII revealed blood half-lives in rodents in the range of about 1.5 h. Compound I also showed appreciable oral uptake in mice (18%), but yielded no detectable blood levels in rats after oral administration. Benzimidazole containing compounds like VII were not orally bioavailable to a significant extent, neither in mice nor in rats. Thus, while introduction of a benzimidazole group into the PR inhibitors was a successful structural modification with regard to antiviral activity in cell culture, it completely abolished oral bioavailability.

In vitro and in vivo antiviral activity of 2'-fluorinated arabinosides of 5-(2-haloalkyl)uracil.

5-(2-Fluoroethyl)-2'-deoxyuridine (FEDU), its 2'-fluoroarabinofuranosyl analog (FEFAU) and the 2'-fluoroarabinofuranosyl analog (CEFAU) of the potent anti-herpesvirus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU) were evaluated for activity against herpes simplex virus type 1 (HSV-1) and HSV-2 in vitro and in vivo. FEDU, FEFAU and CEFAU proved to be potent and selective anti-herpesvirus agents in vitro. Their potency is evident from their low minimum inhibitory concentrations for HSV-1 and HSV-2, and their selectivity is attested by the marginal inhibition of cell proliferation at relatively high concentrations, and by the high concentrations at which DNA-, RNA- or protein synthesis in normal uninfected host cells is inhibited. Their activity spectrum is broader than that of CEDU: in addition to being highly effective against HSV-1 replication, these derivatives, in particular FEFAU, inhibit HSV-2 replication at concentrations comparable to acyclovir (ACV). In the systemic and cutaneous HSV-1 infection models in mice, FEDU, FEFAU and CEFAU were markedly less potent than CEDU in suppressing the development of lesions and in reducing the mortality rate. In HSV-2 infections in mice and in guinea pigs FEDU, FEFAU and CEFAU were virtually ineffective. CEDU, however, exerted a protective effect in these animal models, albeit at relatively high concentrations.

Measurement of HIV-1 reverse transcriptase by a nonradioactive assay system.

Reverse transcriptase activity was measured by incorporation of dUMP linked to digoxigenin into a suitable template-primer molecule. Incorporation was monitored by using peroxidase-conjugated Fab fragments directed against digoxigenin. The standard assay measuring incorporation of radiolabeled nucleotides into acid-precipitable material was compared with this new immunochemical assay with regard to its usefulness for testing inhibitors of reverse transcriptase.

Purification and partial characterization of poliovirus protease 2A by means of a functional assay.

The purification of poliovirus protease 2A from infected cells by a functional assay is described. A small synthetic peptide was cleaved specifically by an esterase present in poliovirus-infected cells. Since the enzyme proved extremely unstable in crude extracts a rapid and efficient purification procedure had to be developed. By treatment with different detergents followed by high-speed centrifugation, the esterase activity was separated from inactivating cellular enzymes and was solubilized. Purification to more than 90% homogeneity could be achieved by a single chromatography step, namely, by gel filtration through Superose 12 under fast-protein liquid chromatography conditions. The esterase activity was associated with a protein of 17,000 daltons and copurified with poliovirus protein 2A. Furthermore, antibodies to 2A specifically precipitated the esterase activity. Thus, the esterase was identified as poliovirus protease 2A. Inhibition studies with known protease inhibitors revealed that 2A is probably a sulfhydryl protease. Of the metal ions tested, only zinc exerted significant inhibitory effects. The esterase activity was optimal near neutral pH and had an extremely short half-life at physiological temperatures.