Dmitry V. Yanvarev

Termination of translation in eukaryotes is mediated by the quaternary eRF1•eRF3•GTP•Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (Kd = 1.9 μM) and this interaction depends only minimally on the Mg2+ concentration; (ii) GTP binds to eRF3 (Kd = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.

Antiviral Properties, Metabolism, and Pharmacokinetics of a Novel Azolo-1,2,4-Triazine-Derived Inhibitor of Influenza A and B Virus Replication

ABSTRACT Influenza viruses of types A and B cause periodic pandemics in the human population. The antiviral drugs approved to combat influenza virus infections are currently limited. We have investigated an effective novel inhibitor of human influenza A and B viruses, triazavirine {2-methylthio-6-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazine-7(4Í)-one} (TZV). TZV suppressed the replication of influenza virus in cell culture and in chicken chorioallantoic membranes, and it protected mice from death caused by type A and B influenza viruses. TZV was also effective against a rimantadine-resistant influenza virus strain and against avian influenza A virus H5N1 strains. The pharmacokinetic parameters and bioavailability of TZV were calculated after the administration of TZV to rabbits. The TZV metabolite AMTZV {2-methylthio-6-amino-1,2,4-triazolo[5,1-s]-1,2,4-triazin(e)-7(4Í)-one} was discovered in ÍÅK 293T and Huh7 cell cultures, a liver homogenate, and rabbit blood after intragastric administration of TZV. AMTZV was nontoxic and inactive as an inhibitor of influenza virus in cell culture. Most likely, this metabolite is a product of TZV elimination.

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 t1/2 and Tmax 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 Cmax and Cmin. Such a combination of properties makes the compounds of this group promising for further studies as extended-release forms of AZT.

Specific features of HIV-1 integrase inhibition by bisphosphonate derivatives

The integration of viral DNA into the cell genome is one of the key steps in the replication cycle of human immunodeficiency virus type 1 (HIV-1). Therefore, the viral enzyme integrase (IN) catalyzing this process is of great interest as a target for new antiviral agents. We performed a structural-functional analysis of five different series of methylenebisphosphonates (BPs), PO3H2-C(R)(X)-PO3H2, as IN inhibitors with the goal of assessing structural elements required for the inhibitory activity. We found that IN is inhibited only by BP bearing a chlorobenzyl substituent R at the bridging carbon of the P-C-P backbone. These BP inhibited both IN-catalyzed reactions with similar efficacies. They were also active toward some INs with mutations characteristic for HIV-1 strains resistant to strand transfer inhibitors. The study of the mechanism of the IN inhibition by various BP showed that it is effected by the nature of the second substituent (X) at the bridging carbon. Among the tested compounds, only the BP with the amino group bound directly to the BP bridging carbon was found to be a noncompetitive inhibitor and, hence, it can be promising for further studies as potential inhibitor of the IN activity within the preintegration complex.

New Lipophilic Derivatives of AZT and d4T 5′-Phosphonates

Abstract 5′-Aminocarbonylphosphonyl and aminocarbonylmethylphosphonyl diesters of AZT and d4T were synthesized as potential anti-HIV agents.

[New phosphonoformic acid derivatives of 3'-azido-3'-deoxythymidine].

New 5′-alkyl ethoxy- and aminocarbonylphosphonates of 3′-azido-3′-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5′-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.

Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity

The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized.

AZT 5′-Cholinephosphate as an Anti-HIV Agent: The Study of Biochemical Properties and Metabolic Transformations Using Its 32P-Labelled Counterpart

Biochemical and metabolic transformations of 3′-azido-3′-deoxythymidine 5′-choline phosphate (1) were studied using its 32P-labelled counterpart for the evaluation of possible reasons for its enhanced anti-HIV activity. An effective synthesis of 32P-labelled 1 with a specific activity >1,000 Ci/mmol was developed by esterification of 32P-phosphoric acid with choline in the presence of BrCN followed by the coupling of the resulting choline phosphate with 3′-azido-3′-deoxythymidine (AZT). Chemical and enzymatic stabilities of 1 as well as the dynamics of penetration through HL-60 cell membranes were studied at the concentrations comparable to its antiviral concentrations. The products of intracellular transformations of the studied nucleotide were identified.

Data on synthesis of methylene bisphosphonates and screening of their inhibitory activity towards HIV reverse transcriptase

Inorganic pyrophosphate (PPi) mimetics designed on a basis of methylenediphosphonic acid backbone are promising inhibitors of two key HIV replication enzymes, IN [1] and RT [2]. Herein, we present chemical synthesis of eleven methylenebisphosphonates (BPs) with their NMR and HRMS analysis synthesized via five different ways. Also, we present data on inhibition of HIV RT catalyzed phosphorolysis and polymerization by synthesized BPs using two methods based on denaturing urea PAGE. Tests were also performed for thymidine analogue mutations reverse transcriptase (TAM RT), which was expressed and purified for that. Structure–activity relationships and inhibitory activity data of synthesized BPs are presented in “Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity” [2].

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 penetrationa cross HL-60 cell membranes was studied.