Potential antiviral effects of pantethine against SARS-CoV-2.

SARS-CoV-2 interacts with cellular cholesterol during many stages of its replication cycle. Pantethine was reported to reduce total cholesterol levels and fatty acid synthesis and potentially alter different processes that might be involved in the SARS-CoV-2 replication cycle. Here, we explored the potential antiviral effects of pantethine in two in vitro experimental models of SARS-CoV-2 infection, in Vero E6 cells and in Calu-3a cells. Pantethine reduced the infection of cells by SARS-CoV-2 in both preinfection and postinfection treatment regimens. Accordingly, cellular expression of the viral spike and nucleocapsid proteins was substantially reduced, and we observed a significant reduction in viral copy numbers in the supernatant of cells treated with pantethine. In addition, pantethine inhibited the infection-induced increase in TMPRSS2 and HECT E3 ligase expression in infected cells as well as the increase in antiviral interferon-beta response and inflammatory gene expression in Calu-3a cells. Our results demonstrate that pantethine, which is well tolerated in humans, was very effective in controlling SARS-CoV-2 infection and might represent a new therapeutic drug that can be repurposed for the prevention or treatment of COVID-19 and long COVID syndrome.

GCN2 phosphorylates HIV-1 integrase and decreases HIV-1 replication by limiting viral integration.

GCN2 is a serine/threonine kinase involved in cellular stress response related to amino acid starvation. Previously, we showed that GCN2 interacts with HIV-1 integrase and is activated during HIV-1 infection. Herein, we identified HIV-1 integrase as a previously unknown substrate of GCN2 in vitro with a major site of phosphorylation at residue S255 located in the C-terminal domain of HIV-1 integrase. The underlying mechanism was investigated and it appeared that the integrase active site was required in order for GCN2 to target the integrase residue S255. Moreover, various integrases from other retroviruses (e.g. MLV, ASV) were also recognized as a substrate by GCN2. In cells, HIV-1 lentiviral particles harboring mutation at integrase position 255 were affected in their replication. Preventing phosphorylation resulted in an increase in infectivity that correlated with an increase in viral DNA integration. Infectivity of MLV was also higher in cells knocked-out for GCN2 suggesting a conserved mechanism to control viral replication. Altogether, our data suggest that GCN2 may constitute a general guardian of genome stability by regulating foreign DNA integration and as such be part of the antiviral armamentarium of the cell.

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

Structure-analysis of the HIV-1 integrase Y143C/R raltegravir resistance mutation in association with the secondary mutation T97A.

The HIV-1 integrase (IN) mutations Y143C/R are known as raltegravir (RAL) primary resistance mutations. In a previous study (S. Reigadas et al., PLoS One 5:e10311, 2010), we investigated the genetic pathway and the dynamics of emergence of the Y143C/R mutations in three patients failing RAL-containing regimens. In these patients, the Y143C/R mutation was associated with the T97A mutation. The aim of the present biochemical and molecular studies in vitro was to evaluate whether the secondary mutation, T97A, associated with the Y143C/R mutation could increase the level of resistance to RAL and impact IN activities. Site-directed mutagenesis experiments were performed with expression vectors harboring the region of the pol gene coding for IN. With a 3'-end processing assay, the 50% inhibitory concentrations (IC(50)) were 1.2 µM, 1.2 µM, 2.4 µM (fold change [FC], 2), and 20 µM (FC, 16.7) for IN wild type (WT), the IN T97A mutation, the IN Y143C/T97A mutation, and the IN Y143R/T97A mutation, respectively. FCs of 18 and 100 were observed with the strand transfer assay for IN Y143C/T97A and Y143R/T97A mutations, with IC(50) of 0.625 µM and 2.5 µM, respectively. In the strand transfer assay, the IN Y143C or R mutation combined with the secondary mutation T97A severely impaired susceptibility to RAL compared to results with the IN Y143C or R mutation alone. Assays without RAL suggested that the T97A mutation could rescue the catalytic activity which was impaired by the presence of the Y143C/R mutation. The combination of the T97A mutation with the primary RAL resistance mutations Y143C/R strongly reduces the susceptibility to RAL and rescues the catalytic defect due to the Y143C/R mutation. This result indicates that the emergence of the Y143C/R/T97A double-mutation pattern in patients is a signature of a high resistance level.

Therapeutic potential of peptide motifs against HIV-1 reverse transcriptase and integrase.

Multiple clinical benefits have been obtained thanks to the combination of drugs targeting several steps of the HIV-1 replication. However, despite such combination therapy, complete eradication of the virus cannot be attained. Moreover, emergence of resistance observed under treatment and the lengthening life expectancy of treated patients highlight the need for new anti-HIV agents. Peptide-based compounds that exhibit anti RT and anti integrase activities were particularly described. Active peptides have been obtained from several ongoing approaches. The study of interaction between viral proteins inside the preintegration complex, and the growing knowledge of interactions between viral proteins and cellular partners, have generated a useful source of data for the development of peptide inhibitors. Recent data were also obtained from the observation that viral enzymes such as RT and integrase are fully active when they are in a dimeric (RT) or oligomeric state. Peptides derived from the interface of dimers are also of interest. The obtention of efficient small molecules as competitive oligomerization inhibitors is problematic, but anyway, improved cellular uptake and chemical modifications that were obtained in the past ten years allowed numerous peptide drugs to reach the clinic. Finally, a new promising class of peptide inhibitors is emerging called "shiftides", which interfere with the ability of IN to adopt an oligomeric active state.

HIV-1 integrase trafficking in S. cerevisiae: a useful model to dissect the microtubule network involvement of viral protein nuclear import.

Intracellular transport of karyophilic cargos comprises translocation to the nuclear envelope and subsequent nuclear import. Small cargos such as isolated proteins can reach the nuclear envelope by diffusion but movement of larger structures depends on active translocation, typically using microtubules. Centripetal transport ends at the perinuclear microtubule organizing centre called the spindle pole body (SPB) in yeast. Previously, we found by two hybrids that the karyophilic lentiviral-encoded integrase (IN) interacts with two yeast microtubule-associated proteins, Dyn2p (dynein light chain protein) and Stu2p, a centrosomal protein (de Soultrait et al., 2002). Thus, to investigate the hinge between cytoplasmic retrograde transport and nuclear import, we decided to analyse HIV-1 IN trafficking in yeast as the model, since each of these biological mechanisms is evolutionarily conserved in eukaryotic cells. Here, we found an accumulation of IN at the SPB in yeast via Stu2p colocalization. Disruption of the microtubule network by nocodazole or IN expression in a dynein 2-deficient yeast strain prevented IN accumulation in the nuclear periphery and additionally inhibited IN transport into the nucleus. By mutagenesis, we showed that trafficking of IN towards the SPB requires the C-terminus of the molecule. Taking our findings together, we proposed a model in which IN nuclear import seems to depend on an essential intermediate step in the SPB. We found that Dyn2p and Stu2p play an important role in driving IN toward MTOC and could optimize nuclear entry of the retroviral enzyme. Our results suggest a new hypothesis in keeping with the current HIV-1 intracellular trafficking model.

In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state.

HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

DNA aptamers derived from HIV-1 RNase H inhibitors are strong anti-integrase agents.

HIV-1 integrase, the retroviral-encoded enzyme involved in the integration of the retrotranscribed viral genome into the host nuclear DNA, is an attractive and still unexploited target. To date, very few inhibitors of this enzyme with a potential therapeutic value have been described. During the search for new HIV-1 targets, we recently described DNA oligodeoxynucleotide aptamers (ODN 93 and ODN 112) that are strong inhibitors of the RNase H activity associated with HIV-1 reverse transcriptase. The striking structural homology between RNase H and integrase led us to study the effect of the RNase H inhibitors on the integrase. Shorter DNA aptamers derived from ODNs 93 and 112 (ODNs 93del and 112del) were able to inhibit HIV-1 integrase in the nanomolar range. They had G-rich sequences able to form G-quartets stabilized by the presence of K(+). The presence of these ions increased the inhibitory efficiency of these agents dramatically. Inhibition of enzymatic activities by ODN 93del and ODN 112del was observed in a cell-free assay system using a recombinant integrase and HIV-1 replication was abolished in infected human cells. Moreover, cell fusion assays showed that these agents do not block viral cell entry at concentrations where viral replication is stopped.

HIV-1 integrase and RNase H activities as therapeutic targets.

The retroviruses are a large, diverse family of enveloped RNA viruses defined by their structure, composition and replicative properties. The hallmark of the family is its replicative strategy, essential steps of which include reverse transcription of the viral RNA and the subsequent integration of this DNA into the genome of the cell. These steps are performed by two viral-encoded enzymes, reverse transcriptase (RT), which possesses DNA polymerase and ribonuclease H (RNase H) activities, and integrase (IN). These enzymes are excellent targets for retroviral therapy since they are essential for viral replication. Numerous substances capable of inhibiting the DNA polymerase activity of HIV-1 RT are available, while few specific inhibitors of RNase H activity have been described. IN is absolutely necessary for stable and productive infection of cells. Some IN inhibitors have been recently reported and are available demonstrating the potential of IN as an antiviral target. This paper is an overview of the inhibitors of RNase H and IN and describes the most promising inhibitors.

DNA aptamers selected against the HIV-1 RNase H display in vitro antiviral activity.

The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.

Antiviral activity of 4-benzyl pyridinone derivatives as HIV-1 reverse transcriptase inhibitors.

In this overview, the antiviral properties of the Curie-pyridinone compounds, a new class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) developed as anti-HIV agents, are described. These compounds are hybrids between hydroxyethoxymethyl-phenylthiothymine (HEPT) and Merck pyridinones. Several structure-activity relationships (SAR) studies between HIV-1 reverse transcriptase (RT) and the Curie-pyridinones are described. The Curie-pyridinones are potent inhibitors of both HIV-1 replication in cell culture and of HIV-1 RT activity in vitro. They are specific to HIV-1 and do not inhibit the replication of HIV-2. The mechanism of inhibition is non-competitive with respect to the natural substrate dGTP. For these reasons, the Curie-pyridinones can be considered as non-nucleoside inhibitors of HIV-1 RT. Moreover, they have the unusual ability to reach the reverse transcription complex inside the extracellular virions and may therefore be useful as retrovirucides. This might lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.

Synthesis and antiviral activity of 4-benzyl pyridinone derivatives as potent and selective non-nucleoside human immunodeficiency virus type 1 reverse transcriptase inhibitors.

Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthesized and evaluated for their anti-HIV-l activities. Key transformations include metalation at the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine (5) and its coupling with benzyl bromide or benzaldehyde derivatives. Biological studies revealed that some of the new 4-benzylpyridinones show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 14, 19, and 27, which inhibit the replication of HIV-1 in CEM-SS cells, with IC(50) values ranging from 0.2 to 6 nM are the most active compounds in this series. Biochemical studies showed that compound 27 strongly inhibited the activity of a recombinant HIV-1 RT. Moreover, the infectivity of isolated HIV-1 particles was severely decreased after exposure to compound 27. Although cross resistance is frequently observed between non-nucleoside reverse transcriptase inhibitors, compound 27 was capable of inhibiting a virus resistant to nevirapine with an IC(50) of 40 nM.

Towards the selection of phosphorothioate aptamers optimizing in vitro selection steps with phosphorothioate nucleotides.

The high affinity of a given nucleic acid for a protein ligand can be used to isolate specific inhibitors of enzymes involved in pathological situations. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucleic acids inhibitors of HIV-1 replication have been isolated using the SELEX approach. However, phosphodiester oligodeoxynucleotides (PO-ODNs) were not used as antiviral agents because of their sensitivity to nucleases. Our goal in this work was to explore the possibility of selecting, from a fully substituted phosphorothioate library, oligonucleotides having both a strong affinity for HIV-1 reverse transcriptase (RT) and nuclease resistance. HIV-1 RT initiates in vivo reverse transcription from the 3' end of a host tRNALys. Although phosphorothioate ODNs (PS-ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN corresponding to the acceptor stem of tRNALys was able to inhibit specifically HIV-1 replication in HIV-1 infected cells, without showing cytotoxicity up to 10 microM. As the SELEX strategy requires 'in vitro' transcription and reverse transcription of the selected DNA, we have assayed the available PS precursors as a model system by using PS-dNTPs and rNTPs. We have also developed an experimental procedure to optimize the incorporation of four PS-dNTPs during the PCR step of the SELEX approach. In the course of this work, we have showed that the PS-dGTP is a strong inhibitor of thermostable DNA polymerases as well as of HIV-1 RT.

Synthesis and evaluation of oligo-1,3-thiazolecarboxamide derivatives as HIV-1 reverse transcriptase inhibitors.

A set of oligo-1,3-thiazolecarboxamide derivatives able to interact with the minor groove of nucleic acids was synthesized. These oligopeptides contained different numbers of thiazole units presenting dimethylaminopropyl or EDTA moieties on the C-terminus, and aminohexanoyl or EDTA moieties on the N-terminus. The inhibition of such compounds on HIV-1 reverse transcriptase activity was evaluated using different model template primer duplexes: DNA x DNA, RNA x DNA, DNA x RNA and RNA x RNA. The biological properties of the thiazolecarboxamide derivatives were compared to those of distamycin, another minor groove binder which contains three pyrrole rings. Similar to distamycin, the thiazole containing oligopeptides were good inhibitors of the reverse transcription reaction in the presence of DNA x DNA. But in contrast to distamycin, the oligothiazolide derivatives were able to inhibit reverse transcription in the presence of RNA x DNA or DNA x RNA template primers. Both distamycin and oligothiazolecarboxamides had low affinity for RNA x RNA duplexes. The inhibition obtained with the newly synthesized thiazolecarboxamides showed that these compounds were more powerful and versatile inhibitors of the RT-dependent polymerization than the natural minor groove binder distamycin.

Interaction of oligonucleotides conjugated to substituted chromones and coumarins with HIV-1 reverse transcriptase.

Ten different pyranone-related substituents (chromones or coumarins) were covalently linked to the 5' end of various oligonucleotides (ODN). The interaction of these compounds with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was analyzed. A different behavior was found to depend on the structure of the oligonucleotide derivatives. Some compounds activated the enzyme at relatively low concentrations (0.1-0.5 microM), followed by an inhibition of the activity at higher concentrations (5-20 microM), whereas others behave just as inhibitors. Because the presence of some coumarin or chromone derivatives conjugated to ODNs enhanced the interaction with the reverse transcriptase, we analyzed the capacity of such ODN derivatives to be used as primers. The introduction of substituent I, a chromone derivative, the 2-[(3-(aminopropyl)amino]-8-isopropyl-5-methyl-4-oxo-4H-1-benzopyran-3-c arbaldehyde], and II, a coumarin derivative, the 1-(3-aminopropoxy)-2-ethyl-3H-naphto[2,1-b]pyran-3-one, into the 5' end of a noncomplementary ODN allowed these compounds to be used as primers. In the case of complementary primers, the presence of conjugated derivatives enhanced the affinity with Km values that were two to three orders of magnitude lower than that of a complementary primer of the same length. After addition of a ddT-unit to the 3'-terminal end of the ODN, some of these primers became very effective inhibitors of RT with Ki values in the nanomolar range.

Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNALys3.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates the synthesis of DNA from the 3' end of its specific primer, tRNALys3. The regions of tRNALys3 in close contact with RT are well known, while a precise knowledge of the RT regions interacting with tRNALys3 is not yet available. To address this question we cross-linked the heterodimeric p66/p51 RT to tRNALys3 using cis-aquahydroxydiammino-platinum. Ribonucleoprotein complexes of molecular masses higher than the p66 subunit were obtained. After RNase A digestion of the RT-tRNA complex, a labeled oligoribonucleotide (ORN) was mainly found associated to the p66 subunit. This labeled p66-ORN complex was then proteolyzed with Staphylococcus aureus V8 protease. A highly purified radioactive peptide was obtained after two chromatographic purification steps. Its N-terminal sequence corresponded with amino acid residues 241VQPI244. Using the crystallographic structure of HIV-1 RT, this peptide was localized at the beta14-sheet end, near to the hairpin formed by beta12 and beta13-sheets ("primer grip") and the alphaH-helix. The so called "VQPI peptide" is in the border of the thumb and the palm subdomains of the p66 subunit. This study palliates the absence of a three- dimensional structure of the RT-tRNA complex and led to a peptide in interaction with tRNALys3 present in all HIV-1 RT isolates.

Structural constraints in the HIV-1 reverse transcriptase-primer/template complex for the initiation of DNA synthesis from primer tRNALys3.

The topography and functional implications of the complex formed in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNALys3 were studied in this work. On the basis of previous results showing the high affinity both of the native primer, tRNALys3, as well as that of mismatched short oligonucleotide primers for HIV-1 RT, we synthesized chimeric primers containing tRNALys3 linked to U and T residues of different lengths. We found that the affinity of the oligonucleotide primers for HIV-1 RT is dramatically increased when linked to primer tRNA. Our results also show that in the tRNA.RT complex, before annealing tRNALys3 to the retroviral RNA genome, the 3'-terminal nucleotide of tRNALys3 is positioned at a distance of one nucleotide unit away from the template in the active polymerization site of the enzyme.

p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.

Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.

A new series of pyridinone derivatives as potent non-nucleoside human immunodeficiency virus type 1 specific reverse transcriptase inhibitors.

4-(Arylthio)-pyridin-2(1H)-ones variously substituted in their 3-, 5-, and 6-positions have been synthesized as a new series of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)-pyridinone hybrid molecules. Biological studies revealed that some of them show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 16 and 7c, the most active ones, inhibit the replication of HIV-1 at 3 and 6 nM, respectively.