Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore.

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model.

BAY 41-4109 is a member of a class of heteroaryl-pyrimidines that was recently identified as potent inhibitors of human hepatitis B virus (HBV) replication. We have investigated the antiviral activity of BAY 41-4109 (methyl (R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydro-pyrimidine-5-carboxylate) in HBV-transgenic mice (Tg [HBV1.3 fsX(-)3'5']). Bay 41-4109 was administered per os using different schedules (b.i.d. or t.i.d. for up to 28 days) and dosages ranging from 3 to 30 mg/kg. The compound reduced viral DNA in the liver and in the plasma dose-dependently with efficacy comparable to 3TC. In contrast to 3TC-treated mice, we found a reduction of cytoplasmic hepatitis B virus core antigen (HBcAg) in liver sections of BAY 41-4109-treated mice, which indicated a different mode of action. Pharmacokinetic studies in mice have shown rapid absorption, a bioavailability of 30% and dose-proportional plasma concentrations. We conclude that BAY 41-4109 is a new anti-HBV drug candidate.

Resistance mutations selected in vivo under therapy with anti-HIV drug HBY 097 differ from resistance pattern selected in vitro.

The quinoxaline derivative HBY 097, an orally active nonnucleoside inhibitor of HIV-1 reverse transcriptase (NNRTI), showed an efficient suppression of viral load in a dose-escalating phase I study with mean trough concentrations increasing from 137-1299 ug/l [Rübsamen-Waigmann et al., Lancet 349:1517]. Half-maximal inhibitory concentrations (IC50) for viruses grown from the patients at entry of the study were 0.1-3 nM, except for one patient who had a virus with reduced susceptibility to HBY 097 at entry (IC50: 160 nM). During therapy, only two patients developed a virus with a moderately increased IC50 (2.2 and 15 nM). This reduced susceptibility was associated with the known NNRTI-resistance mutation K ==> N at position 103, in contrast to resistance selection in vitro, which had yielded predominant mutations at positions 179 and 190. The Tyr mutation at position 181, inducing high resistance for other NNRTIs, was never observed. The resistant virus at study entry (IC50 = 160 nM) had a mutation at position 103 as well, combined with an AZT resistance mutation (K ==> R) at position 70, suggesting that nucleoside-resistance mutations may help increasing resistance to HBY 097. This is in line with our in vitro selection studies, where resistance mutations at the 'nucleoside sites' 74 and 75 increased the resistance phenotype of NNRTI mutations. Our findings highlight the crucial importance of IC50 determinations from cultured virus for determination of phenotypic resistance development during therapy and demonstrate that in vivo resistance development cannot be predicted from in vitro selection.

In vitro selection for different mutational patterns in the HIV-1 reverse transcriptase using high and low selective pressure of the nonnucleoside reverse transcriptase inhibitor HBY 097.

In vitro resistance of HIV-1 against high levels of HBY 097 ((S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydro-quinoxaline-2(1H)-thione) and other quinoxaline nonnucleoside reverse transcriptase inhibitors (NNRTIs) is characterized by a specific amino acid substitution in the reverse transcriptase (RT), Gly 190Glu. This change results in decreased RT polymerase activity and in reduced growth properties of the corresponding viral variant. Here we show that the appearance of the crippling mutation at codon 190 can be prevented by lowering the selective pressure exerted by HBY 097. Under low selective pressure an accumulation of other NNRTI-specific mutations is observed. Up to five NNRTI-specific substitutions were detected in some of these virus lineages. In addition, we report novel RT amino acid changes which were not observed previously, including Val106lle, Val106Leu, and Gly190Thr. HBY 097 selects for different mutational patterns under high and low selective pressure conditions, respectively. Thus, the type of mutations which appear in HIV-infected patients undergoing therapy may be determined by the levels of the selecting drug.

Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants.

The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4- dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTI-binding pocket, and five of six strains displayed the RT G190-->E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190-->Q) most likely evolved from preexisting G190-->E mutants. The negative charge introduced by the G190-->E substitution was maintained at that site of the pocket by simultaneous selection for V179-->D together with G190-->Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-->V/I and V75-->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddI, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74-->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside- and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNA-binding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of HIV-1 infection has yet to be determined.

Preclinical evaluation of HBY 097, a new nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 replication.

HBY 097 [(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydroquinoxaline-2(1H)-thione] was selected from a series of quinoxalines as a nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (NNRTI). HBY 097 was shown to be a highly potent inhibitor of HIV-1 induced cell killing and HIV-1 replication in a variety of human cell lines as well as in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against a variety of clinical isolates of HIV-1 including different HIV-1 subtypes and viruses resistant to 3'-deoxy-3'-azidothymidine. Mutant reverse transcriptases which arise as a consequence of treatment with other nonnucleoside inhibitors of HIV-1 reverse transcriptase were still inhibited by HBY 097 at relatively low concentrations. An HIV-1MN variant resistant to inhibition by HBY 097 displayed in the reverse transcriptase gene a mutation causing a substitution at position 190 of a glutamic acid for a glycine residue (G190 --> E), which is characteristic for quinoxaline derivatives. The drug was demonstrated to possess a favorable toxicity profile and to show good oral bioavailability in both mice and dogs. As a consequence of its outstanding properties, HBY 097 was selected for further development and is at present undergoing clinical trials.

HIV protease inhibitor HOE/BAY 793, structure-activity relationships in a series of C2-symmetric diols.

A detailed structure-activity relationship of C2-symmetric diol inhibitors of HIV-1 protease leads to inhibitor 6 (HOE/BAY 793) which is outstanding in the inhibition of the enzyme and in the inhibition of viral replication in HIV infected cell culture (IC50: 0.3 nM; EC50: 3 nM). There are well defined steric requirements for the design of the side chains P1-P3 of the inhibitors. In addition, all three side chains need to be lipophilic. While the enzyme tolerates hydrophilic substituents in some cases, drastic reductions in anti-HIV activity are observed in cell culture, most likely due to insufficient cell penetration.

Resistance pattern of human immunodeficiency virus type 1 reverse transcriptase to quinoxaline S-2720.

The human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitor quinoxaline S-2720 showed a more-potent inhibitory effect on HIV-1-induced cytopathicity in CEM cells than either nevirapine, pyridinone L-697,661, bis-heteroarylpiperazine (BHAP) U-88204, TSAO ([2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4-amino-1",2"-oxathiole-2",2"-dioxide)-N3-ethylthymine, or 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4-benzodiazepin-2(I H)-one (TIBO) R82913. The quinoxaline derivative was also markedly more inhibitory to the mutant HIV-1 strains containing in their RT Ile-100, Asn-103, Ala-106, Lys-138, Cys-181, or His-188 substitutions than were the other HIV-1-specific RT inhibitors. Moreover, quinoxaline S-2720 totally prevented HIV-1 infection and emergence of drug-resistant mutant virus strains in CEM cell cultures at concentrations (i.e., 0.35 microM) that are 10- to 25-fold lower than those required for BHAP U-88204 and nevirapine to knock out the virus. Also, the concentration-response curve for S-2720 was markedly steeper than for BHAP and nevirapine, as reflected by the ratio of the 95% to the 50% antivirally effective concentration. Lower concentrations of quinoxaline dominantly lead to the appearance of the Ala-106 RT mutation, causing low-level resistance to the compound. At higher quinoxaline concentrations, the Glu-190 RT and/or the Cys-181 RT mutation is added to the Ala-106 mutation, whereas at the highest quinoxaline concentrations, the Ala-106 mutation tends to disappear from the virus pool, leaving the Glu-190 RT and Cys-181 RT mutations as the only mutations conferring high-level resistance to the compound.

Spectrum of activity of soluble intercellular adhesion molecule-1 against rhinovirus reference strains and field isolates.

The antiviral potency of soluble intercellular adhesion molecule-1 (ICAM-1; the major receptor for human rhinoviruses) was determined for a subset of American Type Culture Collection reference serotypes and field isolates of rhinovirus. The results indicate that soluble ICAM-1 exhibits a broad spectrum of activity against rhinoviruses and that field isolates have a sensitivity indistinguishable from that of laboratory strains.

Mutational analysis of residue 190 of human immunodeficiency virus type 1 reverse transcriptase.

S-2720 and other members of the quinoline/quinoxaline class of HIV-1-specific nonnucleoside reverse transcriptase inhibitors (NNRTIs) select for a glycine to glutamate substitution at residue 190 (Gly 190 Glu) of the reverse transcriptase (RT), when drug-resistant viruses are generated in cell culture. This mutation has not been described to appear upon selection for resistant viral variants using derivatives of any other class of NNRTIs. Notably, the RNA-dependent DNA polymerase activity of the Gly 190 Glu mutant enzyme is drastically diminished with respect to the wild-type RT. We describe here the effects of other amino acid substitutions at position 190 of the RT that were introduced by using site-directed mutagenesis. Polymerase activities and sensitivities to inhibition by a number of NNRTIs were determined for the different RT mutants. In general, an inverse correlation was found between the enzymatic activity and increasing length of the side chain, whereas the size of the residue and the level of resistance to NNRTIs appeared to be positively related. Double mutants, which contain the Gly 190 Glu mutation together with substitutions that confer resistance to other RT inhibitors, were all shown to possess severely diminished polymerase activity.

Activity of a novel quinoxaline derivative against human immunodeficiency virus type 1 reverse transcriptase and viral replication.

S-2720 [6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4- dihydroquinoxalin-2(1H)-thione], a quinoxaline derivative, was found to be a very potent inhibitor of both human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) activity and HIV-1 replication in tissue culture. Like other nonnucleoside RT inhibitors, S-2720 does not affect the HIV-2 RT. A S-2720-resistant virus was selected and shown to possess a mutation within the RT-coding region that has not previously been described. Notably, this mutation gives rise to a dramatic decrease in enzyme activity. S-2720, therefore, belongs to a new class of RT inhibitors that bind differently to the RT than other known nonnucleoside RT inhibitors. As no toxic effects were observed with S-2720 in mice, these quinoxaline derivatives deserve further evaluation to prove their potency as possible therapeutic agents for HIV-1 infection.

New antiviral compounds.

Several potent and selective antiviral agents against herpes virus infections have been developed. However, the majority of compounds against other viral diseases has not yet reached such high standard. Based on progress in molecular virology it can, however, be anticipated that similar concepts of selective inhibition will also be developed for other virus groups. In addition to virus-induced enzymes, viral proteins other than enzymes with specific activities will be identified. The identification of active sites will lead to the design of new and specific inhibitors. Moreover, studies on the mode of action of the huge number of known antiviral compounds may provide the basis for new and potent approaches to specific virus chemotherapy. New inhibitors of viral replication may also be derived from 2'-5'A and other mediators of the interferon induced antiviral state. However, since 2'-5'A does not enter cells, is rapidly degraded by phosphodiesterases, and affects viral and cellular protein synthesis, only analogs which do not have these disadvantages may qualify as antiviral drugs. In addition to refinements at the molecular level quantitative assays for a better evaluation of antiviral agents for clinical use are required. For clinical trials, rapid diagnosis, early initiation of treatment, and quantitative evaluation of the antiviral effects of a drug need to be developed. Moreover, new methods of drug delivery and/or drug targeting will improve potency and selectivity of antiviral compounds. Drug carriers have already successfully been used in cancer therapy (Poste and Fidler, 1981) they should be also applicable to virus chemotherapy. Finally, a better understanding of the pathogenesis and the natural course of viral diseases will contribute to the development of more effective and safe antiviral agents.

Purification and chemical characterization of the vitamin-B12-dependent 5-methyltetrahydrofolate: homocysteine methyltransferase from Escherichia coli B.

The transferase was isolated by means of hydrophobic chromatography and combination of ion-exchange and gel filtration at different pH values and ionic strengths. As judged by disc electrophoresis, the enzyme is homogeneous. Electrophoresis in the presence of sodium dodecylsulfate reveals only one band with Mr = 49500 +/- 10%. In gel filtration the native enzyme has a Mr of 200,000. The subunits can be crosslinked by iminothiolane followed by hydrogen peroxide oxidation. In sodium dodecylsulfate electrophoresis this results in a band pattern of integer multiples of 50,000 up to 20,000 but not higher. The high-Mr aggregates disappear on splitting the crosslinks by reduction. Thus the enzyme appears to be composed of four subunits identical or nearly identical in Mr. By the dansyl method, only phenylalanine and methionine were found as the amino-terminal residues, suggesting the existence of two different types of subunits.