Bioinspired Pyrano[2,3-f]chromen-8-ones: Ring C-Opened Analogues of Calanolide A: Synthesis and Anti-HIV-1 Evaluation.

We have designed and synthesized a series of bioinspired pyrano[2,3-f]coumarin-based Calanolide A analogs with anti-HIV activity. The design of these new calanolide analogs involved incorporating nitrogen heterocycles or aromatic groups in lieu of ring C, effectively mimicking and preserving their bioactive properties. Three directions for the synthesis were explored: reaction of 5-hydroxy-2,2-dimethyl-10-propyl-2H,8H-pyrano[2,3-f]chromen-8-one with (i) 1,2,4-triazines, (ii) sulfonylation followed by Suzuki cross-coupling with (het)aryl boronic acids, and (iii) aminomethylation by Mannich reaction. Antiviral assay of the synthesized compounds showed that compound 4 has moderate activity against HIV-1 on enzymes and poor activity on the cell model. A molecular docking study demonstrates a good correlation between in silico and in vitro HIV-1 reverse transcriptase (RT) activity of the compounds when docked to the nonnucleoside RT inhibitor binding site, and alternative binding modes of the considered analogs of Calanolide A were established.

Dual-targeted anti-CMV/anti-HIV-1 heterodimers.

Despite the development of efficient anti-human immunodeficiency virus-1 (HIV-1) therapy, HIV-1 associated pathogens remain a major clinical problem. Human cytomegalovirus (CMV) is among the most common HIV-1 copathogens and one of the main causes of persistent immune activation associated with dysregulation of the immune system, cerebrovascular and cardiovascular pathologies, and premature aging. Here, we report on the development of dual-targeted drugs with activity against both HIV-1 and CMV. We synthesized seven compounds that constitute conjugates of molecules that suppress both pathogens. We showed that all seven compounds exhibit low cytotoxicity and efficiently inhibited both viruses in cell lines. Furthermore, we chose a representative compound and demonstrated that it efficiently suppressed replication of HIV-1 and CMV in human lymphoid tissue ex vivo coinfected with both viruses. Further development of such compounds may lead to the development of dual-targeted anti-CMV/HIV-1 drugs.

DEAD-box RNA Helicase DDX3: Functional Properties and Development of DDX3 Inhibitors as Antiviral and Anticancer Drugs.

This short review is focused on enzymatic properties of human ATP-dependent RNA helicase DDX3 and the development of antiviral and anticancer drugs targeting cellular helicases. DDX3 belongs to the DEAD-box proteins, a large family of RNA helicases that participate in all aspects of cellular processes, such as cell cycle progression, apoptosis, innate immune response, viral replication, and tumorigenesis. DDX3 has a variety of functions in the life cycle of different viruses. DDX3 helicase is required to facilitate both the Rev-mediated export of unspliced/partially spliced human immunodeficiency virus (HIV) RNA from nucleus and Tat-dependent translation of viral genes. DDX3 silencing blocks the replication of HIV, HCV, and some other viruses. On the other hand, DDX displays antiviral effect against Dengue virus and hepatitis B virus through the stimulation of interferon beta production. The role of DDX3 in different types of cancer is rather controversial. DDX3 acts as an oncogene in one type of cancer, but demonstrates tumor suppressor properties in other types. The human DDX3 helicase is now considered as a new attractive target for the development of novel pharmaceutical drugs. The most interesting inhibitors of DDX3 helicase and the mechanisms of their actions as antiviral or anticancer drugs are discussed in this short review.

Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation.

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

Synthesis and in vitro Biological Evaluation of Novel Thymidine Analogs Containing 1H-1,2,3-Triazolyl, 1H-Tetrazolyl, and 2H-Tetrazolyl Fragments.

3'-Azidothymidine (AZT) reacts with 1-propargyl-5-R-1H- and 2-propargyl-5-R-2H-tetrazoles (R = H, Me, CH2COOEt, CH2CON(CH3)2, Ph, 2-CH3-C6H4, or 4-NO2-C6H4) via the Cu(I)-catalyzed asymmetric [3 + 2] cycloaddition to give 3'-modified thymidine analogs incorporating 1H-1,2,3-triazolyl, 1H-, and 2H-tetrazolyl fragments in 41-76% yield. The structures of the obtained compounds have been elucidated by means of HRESI+-MS, 1H and 13 C{1H} NMR, and single crystal X-ray diffraction {for 3'-[4-(1H-5-N,N-dimethylaminocarbonylmethyltetrazol-1-yl)-1H-1,2,3-triazol-1-yl]thymidine 10d}. In vitro biological evaluation of the prepared compounds has been performed; they have exhibited low activity against phenotypic HIV-1899A. Moderate anti-influenza activity against influenza virus A/Puerto Rico/8/34 (H1N1) strain has been observed in the cases of 3'-(4-(1H-tetrazol-1-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 10a (IC50 39.6 µg/mL), 3'-(4-(2H-5-ethoxycarbonyltetrazol-2-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 11c (IC50 31.6 µg/mL), and 3'-(4-(2H-5-(4-nitrophenyl)-tetrazol-2-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 11g (IC50 46.4 µg/mL). The tested compounds possess very low cytotoxicity towards MDCK and MT4 cells as well as tumor human cervical carcinoma HeLa and promyelocytic leukemia HL-60 cells.

Versatile synthesis of oxime-containing acyclic nucleoside phosphonates--synthetic solutions and antiviral activity.

New oxime-containing acyclic nucleoside phosphonates 9-{2-[(phosphonomethyl)oximino]ethyl}adenine (1), -guanine (2) and 9-{2-[(phosphonomethyl)oximino]propyl}adenine (3) with wide spectrum activity against different types of viruses were synthesized. The key intermediate, diethyl aminooxymethylphosphonate, was obtained by the Mitsunobu reaction. Modified conditions for the by-product separation (without chromatography and distillation) allowed us to obtain 85% yield of the aminooxy intermediate. The impact of DBU and Cs2CO3 on the N(9)/N(7) product ratio for adenine and guanine alkylation was studied. A convenient procedure for aminooxy group detection was found. The synthesized phosphonates were tested and they appeared to display moderate activity against different types of viruses (HIV, herpes viruses in cell cultures, and hepatitis C virus in the replicon system) without toxicity up to 1000 µM.

Crystal structure of ammonium 3'-azido-3'-de-oxy-thymidine-5'-amino-carbonyl-phospho-nate hemi-hydrate: an anti-HIV agent.

The asymmetric unit of the title compound, NH4 (+)·C11H14N6O7P(-)·0.5H2O, contains one 3'-azido-3'-de-oxy-thymidine-5'amino-carbonyl-phospho-nate (ACP-AZT) anion, half of an NH4 (+) cation lying on a twofold rotation axis and in another position, occupied with equal probabilities of 0.5, an NH4 (+) cation and a water mol-ecule. The amide group of the ACP-AZT anion is disordered (occupancy ratio 0.5:0.5), with one part forming an N-H⋯O (involving C=O⋯H4N(+)) hydrogen bond and the other an O-H⋯N (involving C-NH2⋯OH2) hydrogen bond with the components of the split NH4 (+)/H2O position. The pseudorotation parameters of ACP-AZT set it apart from previously studied AZT and thymidine. In the crystal, the various components are linked by N-H⋯O, O-H⋯O, N-H⋯N, C-H⋯O and C-H⋯N hydrogen bonds, forming a three-dimensional framework.

New dinucleoside phosphonate derivatives as prodrugs of 3'-azido-3'-deoxythymidine and ß-L-2',3'-dideoxy-3'-thiacytidine: synthesis and anti-HIV properties.

[Formula: see text]New phosphonate homodimers of 3'-azido-3'-deoxythymidine (AZT) and a phosphonate heterodimer of ß-L-2',3'-dideoxy-3'-thiacytidine (3TC) and AZT were synthesized. The compounds demonstrated moderate anti-HIV activity. Stability of the compounds in human blood serum was studied. A correlation between anti-HIV activity and stability was defined.

Synthesis and anti-HIV properties of new carbamate prodrugs of AZT.

A series of new 5'-O-carbamate prodrugs of AZT have been prepared. The stability in biological media, anti-HIV properties and pharmacokinetic parameters in dogs were evaluated. The compounds display moderate anti-HIV activity in cell culture. After oral administration of carbamate IV in dogs, both intact prodrug IV and released AZT were discovered in dog blood. Pharmacokinetic parameters of the compound IV were estimated. Half-life (T(1/2)) of AZT released after oral administration of IV in dogs was close to that after administration of AZT itself, and time to the maximum concentration (T(max)) of AZT released from IV was two and three times longer compared with that of AZT and H-phosphonate AZT, respectively. Acute toxicity was more than five times less if compared with AZT. As a result, we consider this series of carbamate derivatives of AZT as perspective for development of anti-HIV agents.

5'-phosphonate derivatives of 2',3'-dideoxy-3'-thiacytidine as new anti-HIV prodrugs.

Two new phosphonate 3TC prodrugs were synthesized and studied in MT-4 cells as inhibitors of HIV replication. Their pharmacokinetic parameters were evaluated following intragastric administration in rabbits and oral administration in dogs. Both compounds were much less toxic than parent 3TC in cell cultures and could generate the active nucleoside in laboratory animals.

Antiherpetic properties of acyclovir 5'-hydrogenphosphonate and the mutation analysis of herpes virus resistant strains.

In this study, we continued to study antiherpetic properties of acyclovir 5'-hydrogenphosphonate (Hp-ACV) in cell cultures and animal models. Hp-ACV was shown to inhibit the development of herpetic infection in mice induced by the HSV-1/L(2) strain. The compound suppressed replication of both ACV-sensitive HSV-1/L(2) and ACV-resistant HSV-1/L(2)/R strains in Vero cell culture. Viral population resistant to Hp-ACV (HSV-1/L(2)/R(Hp-ACV)) was developed much slower than ACV-resistant population. The analysis of Hp-ACV-resistant clones isolated from the HSV-1/L(2)/R(Hp-ACV) population demonstrated their partial cross-resistance to ACV. The mutations determining the resistance of HSV-1 clones to Hp-ACV were partly overlapped with mutations defining ACV resistance but did not always coincide. HSV-1/L(2)/R(Hp-ACV) herpes virus thymidine kinase is shortened from the C-terminus by 100 amino acid residues in length.

5'-aminocarbonyl phosphonates as new zidovudine depot forms: antiviral properties, intracellular transformations, and pharmacokinetic parameters.

The main disadvantages of 3'-azido-3'-deoxythymidine (zidovudine, AZT), the most common anti-HIV drug, are toxicity and a short half-life in the organism. The introduction of an H-phosphonate group into the AZT 5' position resulted in significant improvement of its therapeutic properties and allowed a new anti-HIV drug, Nikavir (AZT H-phosphonate). In this work, we described a new group of AZT derivatives, namely, AZT 5'-aminocarbonylphosphonates. The synthesized compounds displayed antiviral properties in cell cultures infected with HIV-1 and the capacity to release the active nucleoside in animals (rabbits and dogs) in a dose-dependent manner. The compounds were less toxic in MT-4 and HL-60 cell cultures and experimental animals compared with AZT. Major metabolites found in MT-4 cells after their incubation with AZT 5'-aminocarbonylphosphonate 1 were AZT and AZT 5'-phosphate (25 and 55%, respectively). Among the tested compounds, phosphonate 1 was the most effective AZT donor, and its longest t(1/2) and T(max) values in the line phosphonate 1--AZT H-phosphonate--AZT imply that compound 1 is an extended depot form of AZT. Although bioavailability of AZT after oral administration of phosphonate 1 was lower than those of AZT H-phosphonate and AZT (8 against 14 and 49%), we expect that this reduction would not cause essential decrease of antiviral activity but noticeably decrease toxicity as a result of gradual accumulation of AZT in blood and the absence of sharp difference between C(max) and C(min). Such a combination of properties makes the compounds of this group promising for further studies as extended-release forms of AZT.

Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system.

Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA-oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp.

5'-carbamoylphosphonyl-[6-3H]-AZT as a tool for studying metabolic transformations of the nonradioactive counterpart, an inhibitor of HIV replication.

An effective synthesis of 5'-carbamoylphosphonyl-[6-3H]-AZT was developed from [6-3H]-AZT. For the synthesized compound, chemical and enzymatic stability were determined and its penetration across HL-60 cell membranes was studied.

Cell metabolism of acyclovir phosphonate derivatives and antiherpesvirus activity of their combinations with alpha2-interferon.

The combinational use of acyclovir (ACV) phosphonate esters and alpha(2)-interferon was shown to produce a synergistic effect on inhibition of HSV-1 replication in Vero cell cultures. Unlike other acyclovir phosphonate derivatives studied earlier, ACV H-phosphonate is not an ACV prodrug. On penetrating into the cells, it may be directly converted into ACV monophosphate escaping dephosphonylation-phosphorylation steps.

New triphosphate conjugates bearing reporter groups: labeling of DNA fragments for microarray analysis.

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.

Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins.

The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form (NS5BDelta55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BDelta55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BDelta55 protein was expressed in Rosetta(DE3) Escherichia coli strain bearing an additional pRARE plasmid encoding extra copies of rare tRNAs. The yield of the target enzyme exceeded by a factor of 29 the yield of NS5BDelta55 protein expressed from the parental pET-21d-5BDelta55 plasmid (5 mg/L). The increase in the protein yield could be explained by facilitated protein translation initiation, resulted from disruption of the stable secondary mRNA structure. The pET-21d-2c-5BDelta55 plasmid yielded one third amount of the protein when expressed in BL-21(DE3) strain, indicating that the pRARE plasmid is required for a high-level expression of NS5BDelta55 protein. The 29-fold enhancement of the protein yield was accompanied by only a 2.5-fold increase of the corresponding mRNA level. The expression of another HCV NS5A protein His-tagged at the C-terminus in the developed system yielded a similar amount of the protein (4 mg/L), whereas its N-terminally His-tagged counterpart was obtained in a 30 mg/L yield. The NS5A protein purified under denaturing conditions and renatured in solution inhibited the HCV RdRp and was a substrate for human casein kinase II.

Hypophosphoric acid is a unique substrate of pyrophosphorolysis catalyzed by HIV-1 reverse transcriptase.

Pyrophosphate analogues, namely, pyrophosphorous, hypophosphoric, and hypophosphorous acids, were evaluated as inhibitors in elongation reactions and substrates in pyrophosphorolysis reaction catalyzed by HIV-1 reverse transcriptase and DNA polymerase I (the Klenow fragment). The substrate efficacy of hypophosphoric acid in pyrophosphorolysis reaction exceeded that of pyrophosphate for both enzymes by more than ten times. The product of the reaction was a dNTP analogue bearing a hypophosphate in the beta,gamma-position. Pyrophosphorous and hypophosphorous acids were neither inhibitors nor substrates for the enzymes. Kinetic parameters of the pyrophosphorolysis reaction catalyzed by HIV reverse transcriptase in the presence of hypophosphoric acid were evaluated. The dTMP analogue bearing a hypophosphate in the beta,gamma-position was synthesized and its substrate properties in elongation reaction catalyzed by HIV-1 reverse transcriptase were similar to those of natural dTTP. Hypophosphoric acid was capable of removing ddTMP, ddTMP(3'N3), and ddTMP(3'NH2) from the 3'-end of primers with an equal efficacy.

Uncharged AZT and D4T derivatives of phosphonoformic and phosphonoacetic acids as anti-HIV pronucleosides.

Two series of new lipophilic phosphonoformate and phosphonoacetate derivatives of AZT and d4T were synthesized and evaluated as anti-HIV agents. The efficacy of some of the synthesized compounds in cell cultures infected with HIV-1 was higher than that of the parent nucleosides and only slightly correlated to their stability in the phosphate buffer and human blood serum. The synthesized phosphonates are most probably prodrug forms of the corresponding nucleosides.

Phosphorodiamides as prodrugs for antiviral nucleosides.

New phosphorodiamides of modified nucleoside monophosphates were synthesized and their antiviral properties were evaluated.