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Current treatments of human immunodeficiency virus type 1 (HIV-1) infection consist in the combination of drugs targeting reverse transcriptase (RT) and protease (PR). Despite the multiple clinical benefits of this combination therapy, the emergence of resistance highlights the need for new anti-HIV agents. Agents able to interfere with additional steps of viral replication, such as integration of viral DNA in the host genome, would improve the antiviral potency of the treatment. In this regard, we have focused our interest on peptide-based compounds that have been shown to exhibit potential inhibition of RT and integrase (IN) activities in vitro and in vivo. Recently, the expansion of powerful technologies which allow the selection of peptides exhibiting high affinity for a target protein have provided a new approach to selecting potential anti-HIV drugs. Furthermore, efforts to characterize the protein-protein interactions involved in efficient reverse transcription and integration, as well as the determination of the enzyme structure, have generated a very useful source of data for the development of peptide inhibitors. Finally, while this class of compounds has long been considered as poor drug candidates, current knowledge on improving the stability and bioavailability of these agents would lead to the effective use of peptides in therapy.

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 IC50 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 IC50 of 40 nM.

Reverse transcription takes place in the cytoplasm of infected cells, although it has been demonstrated that retroviruses can also initiate reverse transcription prior to infection of target cells. In addition to partial reverse transcripts, full-length proviral molecules have been detected in the plasma and seminal fluid of HIV-1 seropositive patients. Intravirion endogenous reverse transcription appears to be directly correlated with an increased level of infectivity. Therefore, the ability of an inhibitor to reach and inhibit the replication complex in the core of the free-virion may constitute an important part of its capacity to suppress viral infection. In this work we tested the ability of some reverse transcriptase inhibitors to decrease viral infectivity in pretreated highly purified virions. Our results showed that Curie pyridinone [Dollé et al. (1995), J Med Chem 38: 4 679–4 686], a non nucleoside RT inhibitor, strongly inhibited the infectivity of extracellular HIV-1 particles. Other non nucleoside inhibitors (TIBO R82913, HEPT, nevirapine) tested in these conditions were unable to do so. Our data indicate that the effect of Curie pyridinone on intact virions may be related to its capacity to tightly bind the target RT. This approach may lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.

Three DNA polymerases (A, B and C) have been purified from the soluble cytoplasm of ungerminated embryos. Mainly on the basis of chromatographic, template-specificity and salt-inhibition evidence, we have characterized the three enzymes. Other physico-chemical and enzymic properties are described. From purified mitochondria we have purified a DNA polymerase that behaves like DNA polymerase B on chromatographic and template-specificity criteria. Only highly purified enzyme B from the soluble cytoplasm showed an exonuclease activity able to degrade 3′- or 5′-labelled polydeoxyribonucleotides, as well as a ‘proof-reading’ capacity.

A series of 22 derivatives of AZT substituted at the N-3 position of the thymine base were prepared and evaluated for anti-HIV activity in cell culture (Lai strain of HIV-1 in CEM-c113 cells). The AZT analogs bearing a N-3 amino group (7), a hydroxyalkyl chain (12f), and a phosphonomethyl (12k) substituent displayed activities in the 0.045−0.082 μM range. The analogs 12d, 12e, 12q, 15, and 19 were active at <0.5 μM concentration. Compound 18 in which two molecules of AZT are connected at N-3 via a two-carbon link and “dimer” 11 also displayed significant activity. To obtain information concerning the mechanism of RT inhibition by these AZT analogs, compounds 7, 12d, 12e, and 12q were incubated with recombinant HIV-1 RT in the presence of poly(A)-oligo[dT(12-18)] and poly(C)-oligo[dG(12-18)] template−primers. In contrast to AZT−TP (control), none of these nucleosides displayed any significant inhibition of RT in the recombinant enzyme assay, indicating that phosphorylation is a necessary prerequisite for activity.

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5'-monophosphate and adenosine 5'-monophosphate (AMP) when it is incubated in the presence of cytidine 5'-triphosphate (CTP), adenosine 5'-triphosphate, and Escherichia colitransfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When a 32PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3' end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3' end of which is found in a fragment having a sedimentation constant close to 5s.

Two DNA polymerases have been purified from the 105,000 × g supernatant of ungerminated wheat. The purification stages included: highspeed centrifugation, salt fractionation, DEAE-cellulose chromatography, Sephadex G-150 filtration and phosphocellulose chromatography. Several properties of the two enzymes (called A and B according to the order of elution from the phosphocellulose column) have been studied. Enzyme A has a sedimentation coefficient of about 7 S, utilizes activated DNA and synthetic polydeoxynucleotides as well as poly rA-dT12, while B has a sedimentation coefficient of about 6.2 and uses only activated DNA and synthetic polydeoxynucleotides as templates. Other parameters like KCl effect, MnCl2 effect, optimum pH, etc. allow us to distinguish clearly between both DNA polymerases.

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Diadenosine 5′,5″′-P1, P4-tetraphosphate (Ap4A) binding protein specifically binds Ap4A. The protein has been purified from Xenopus laevis oocytes and presents and estimated molecular weight of 100,000 by gek filtration. In the first stages of the purification, the Ap4A binding activity is found associated to DNA polymerase alpha-DNA primase, forming heterogeneous high molecular weight complexes. A monoclonal antibody has been prepared against the purified Ap4A binding protein. The antibody partially neutralizes the Ap4A binding activity. Using the immunoblot technique, it has been shown that the antibody is able to recognize either native or SDS-denaturated Ap4A binding protein. The monoclonal antibody immoreacted with a polypeptide of 90,000 which coincides with the molecular weight obtained by get chromatography and indicates that the native Ap4A binding protein from Xenopus oocytes is probably a monomeric protein.

DNA polymerase λ from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and ohioroquine inhibition is less pronounced. In the case of DNA polymerases α and β very little inhibition by ethidiuni bromide was observed. Intercalation of this dye in a poly dA-dT12–18 template-primer was studied spectrophotometrically under conditions simitar to those in the in vitro DNA polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase y activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT12–18 We conclude that the drug, most probably in the intercalated form, is ab'e to interact with the active site (s) of mitochondrial DNA polymerase.