Human immunodeficiency virus type 1 pol gene mutations in an AIDS patient treated with multiple antiretroviral drugs.

Multiple mutations were found in the human immunodeficiency virus pol gene following treatment of an AIDS patient with antiretroviral drugs. After approximately 2.5 years of monthly alternating therapy with 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), most of the pol sequences amplified from the patient's peripheral blood mononuclear cell DNA contained known AZT resistance mutations at codons 41, 67, and 215 and a putative ddC resistance mutation at codon 69 as well as other novel mutations. These mutations persisted for 6 months after the patient was switched to 2',3'-dideoxyinosine monotherapy. Mutations known to be associated with 2',3'-dideoxyinosine resistance did not occur during this time. Antiviral susceptibility testing of point mutants, introduced into the genetic background of laboratory strain NL4-3, showed that the codon 41 mutation antagonized ddC resistance when present with the codon 69 mutation. However, this antagonism was not found with a chimeric mutant containing the patient's pol gene sequence from codons 25 to 218, implying that other mutations compensated for the antagonism. Thus, alternating therapy with AZT and ddC resulted in the selection of viruses resistant to both drugs.     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes.

In the present study, one has determined the relative role of plasma membrane equilibrative (Na+-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na+-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2'3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine) and ddI (2'3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative,nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes

In the present study, one has determined the relative role of plasma membrane equilibrative (Na⁺-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na⁺-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2' 3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine)and ddI (2' 3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

1H NMR study of the sugar pucker of 2',3'-dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity

The sugar ring conformations of 2',3'-dideoxyribosyladenine (ddA), 2',3'-dideoxyribosylcytosine (ddC), 2',3'-dideoxyribosylguanine (ddG), 2',3'-dideoxyribosylhypoxanthine (ddI), 3'-azido-2',3'-dideoxyribosylthymine (AZT), 3'-azido-2',3'-dideoxyribosyluracil (AZU) and 3'-fluoro-2',3'-dideoxyribosylthymine (FddT) have been investigated by 1H NMR spectroscopy. While the sugar ring in FddT exists almost totally in C2'-endo geometry, other nucleosides show equilibrium between sugar puckers of C3'-endo family (N-type) and C2'-endo family (S-type). For unsubstituted dideoxynucleosides C3'-endo conformer is favoured (congruent to 75%), whereas for AZT and AZU both the conformers have almost equal populations. Unlike X-ray diffraction studies, the NMR results do not support the suggestion that C3'-exo sugar puckers are desirable for the anti-HIV activity of these nucleosides.     

Resistance profiles of (+)2'-deoxy-3'-oxa-4'-thiocytidine and (-)2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine.

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) > (+)3TC > (-)3TC > PMPA > azidothymidine (AZT) > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T > ddA (ddI) > ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Analysis of Antiretroviral Nucleosides by Electrospray Ionization Mass Spectrometry and Collision Induced Dissociation

Abstract

Antiretroviral nucleoside drugs used against the human immunodeficiency virus (HIV) infection have been analyzed using negative ion electrospray ionization (ESI) mass spectrometry and collision-induced dissociation (CID-MS/MS). Mass fragmentation of azidothymidine (AZT), didanosine (ddI), dideoxycytidine (ddC) and dideoxythiacytidine (3TC) were obtained at different cone voltages and collision energies. Fragmentation of purines and pyrimidines occurred by different pathways. For purines (ddI), the fragmentation was similar to those found in endogenous nucleosides; mainly the pseudo molecular ion is present (M-H) and a cleavage through the glycosidic bond forming (B) was observed. For pyrimidines (AZT, ddC, 3TC), the fragmentation pathways were different from endogenous nucleosides; for AZT, the fragmentation occurred primarily through the elimination of the azido group in the 3′-position (M-H₂-N₃), whereas ddC and 3TC presented more complex fragmentation patterns. For ddC, fragmentation appeared to be dominated by a retro Diels-Alder mechanism (M-CONH). For 3TC, the sulfur atom in the sugar moiety provided greater stability to the charge, producing fragments where the charge resided initially in the dideoxyribose (M-C₂O₂H₆).     

Synthesis of 2',3'-dideoxy-2',3'-didehydro nucleosides via a serendipitous route

This paper describes a "green" synthesis of 2',3'-unsaturated 2',3'-dideoxynucleosides via an electrochemical reaction. Using this approach d4T, d4U, ddA and ddI can be synthesized in high yields.     

Synthesis of 2',3'-dideoxyinosine via radical deoxygenation

A synthetic method for 2',3'-dideoxyinosine (ddI) from inosine was established via radical deoxygenation of N1,5'-O-diprotected-2',3'-bis-S-methyl dithiocarbonate of inosine derivatives. The radical deoxygenation proceeded smoothly to give the desired dideoxy compounds in good yields using 1-ethylpiperidinium hypophosphite and triethylborane. Benzyl or p-methoxybenzyl protection of inosine at the N1, 5'-O-positions were effective for the ddI synthesis.     

2', 3'-Dideoxycytidine represses thymidine kinases 1 and 2 expression in T-lymphoid cells

In vitro culture of H9 human lymphoid cells in the presence of 5.0 microM dideoxycytidine (ddC), for about 40-45 days, selected cells (H9-ddC cells), which were resistant to the drug and cross-resistant to AZT (zidovudine) and 5-fluoro-2'-deoxyuridine (FdUR). The major mechanism of cross-resistance to AZT and FdUR in these cells was low cellular activity of thymidine kinase (TK). To explore molecular mechanisms of the reduced TK activity in H9-ddC cells, the mRNA expression of TK1 and TK2 and western blot analysis of TK1 protein were performed. RT-PCR analysis revealed that in H9-ddC cells the expression of both TK1 and TK2 mRNA was reduced to 27.1% and 79.4%, respectively. The reduced TK1 gene expression was confirmed by an absence of a detectable TK1 protein band in western blot of H9-ddC cells. These results demonstrate that long-term treatment of H9 cells in the presence of ddC down-regulated TK1 and TK2 gene expression and reduced the expression and activity of TK in the resistant cells.     

Synthesis of new homo and heterodimers of 2',3'-dideoxyinosine (ddI) using ester linkages

A series of new homo and heterodimers of ddI has been synthesized. A glutarate diester spacer was used to covalently couple ddI onto ddI, AZT or d4T.