Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k(pol): 22-fold (from 7.2 to 0.33 s(-1)) for K65R RT and 84-fold (from 7.2 to 0.086 s(-1)) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.
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PMID:A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions. 1504 78

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.
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PMID:Novel mechanism of inhibition of HIV-1 reverse transcriptase by a new non-nucleoside analog, KM-1. 1523 30

Genetic deficiencies in the purine catabolic enzyme adenosine deaminase (ADA) in humans results primarily in a severe lymphopenia and immunodeficiency that can lead to the death of affected individuals early in life. The metabolic basis of the immunodeficiency is likely related to the sensitivity of lymphocytes to the accumulation of the ADA substrates adenosine and 2'-deoxyadenosine. Investigations using ADA-deficient mice have provided compelling evidence to support the hypothesis that T and B cells are sensitive to increased concentrations of 2'-deoxyadenosine that kill cells through mechanisms that involve the accumulation of dATP and the induction of apoptosis. In addition to effects on the developing immune system, ADA-deficient humans exhibit phenotypes in other physiological systems including the renal, neural, skeletal, and pulmonary systems. ADA-deficient mice develop similar abnormalities that are dependent on the accumulation of adenosine and 2'-deoxyadenosine. Detailed analysis of the pulmonary insufficiency seen in ADA-deficient mice suggests that the accumulation of adenosine in the lung can directly access cellular signaling pathways that lead to the development and exacerbation of chronic lung disease. The ability of adenosine to regulate aspects of chronic lung disease is likely mediated by specific interactions with adenosine receptor subtypes on key regulatory cells. Thus, the examination of ADA deficiency has identified the importance of purinergic signaling during lymphoid development and in the regulation of aspects of chronic lung disease.
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PMID:Adenosine deaminase deficiency: metabolic basis of immune deficiency and pulmonary inflammation. 1570 18

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 microM(-1) s(-1), a value approximately 10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 microM(-1) s(-1), a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.
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PMID:Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase. 1700 53

Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.
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PMID:Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase. 1709 11

The intracellular metabolism of nucleoside reverse transcriptase inhibitors (NRTI) in mononuclear cells has been thoroughly studied, but that in red blood cells (RBC) has been disregarded. However, the phosphorylation of other analogous nucleosides (in particular, ribavirin) has been described previously. In this study, we investigated for the first time the phosphorylation of NRTI in human RBC. The presence of intracellular zidovudine (AZT) monophosphate, AZT triphosphate, lamivudine (3TC) triphosphate, and tenofovir (TFV) diphosphate, as well as endogenous dATP, dGTP, and dTTP, in RBC collected from human immunodeficiency virus-infected patients was examined. We observed evidence of a selective phosphorylation of 3TC, TFV, and endogenous purine deoxynucleosides to generate their triphosphate moieties. Conversely, no trace of AZT phosphate metabolites was found, and only faint dTTP signals were visible. A comparison of intracellular TFV diphosphate and 3TC triphosphate levels in RBC and peripheral blood mononuclear cells (PBMC) further highlighted the specificity of NRTI metabolism in each cell type. These findings raise the issue of RBC involvement in drug-drug interaction, drug pharmacokinetics, and drug-induced toxicity. Moreover, the typical preparation of PBMC samples by gradient density centrifugation does not prevent their contamination with RBC. We demonstrated that the presence of RBC within PBMC hampers an accurate determination of intracellular TFV diphosphate and dATP levels in clinical PBMC samples. Thus, we recommend removing RBC during PBMC preparation by using an ammonium chloride solution to enhance both the accuracy and the precision of intracellular drug monitoring.
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PMID:Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells. 1743 52

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (K(i) = 0.8 muM) and exhibits low inhibitory potency against host polymerases including DNA polymerase gamma. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 microM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.
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PMID:Design and profiling of GS-9148, a novel nucleotide analog active against nucleoside-resistant variants of human immunodeficiency virus type 1, and its orally bioavailable phosphonoamidate prodrug, GS-9131. 1805 82

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.
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PMID:Restoration of adenosine deaminase-deficient human thymocyte development in vitro by inhibition of deoxynucleoside kinases. 1901 8

Although the approved nucleoside reverse transcriptase (RT) inhibitors (NRTI) are integral components of therapy for human immunodeficiency virus type 1 (HIV-1) infection, they can have significant limitations, including the selection of NRTI-resistant HIV-1 and cellular toxicity. Accordingly, there is a critical need to develop new NRTI that have excellent activity and safety profiles and exhibit little or no cross-resistance with existing drugs. In this study, we report that the 3'-azido-2',3'-dideoxypurine nucleosides (ADPNs) 3'-azido-2',3'-dideoxyadenosine (3'-azido-ddA) and 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG) exert potent antiviral activity in primary human lymphocytes and HeLa and T-cell lines (50% inhibitory concentrations [IC50s] range from 0.19 to 2.1 microM for 3'-azido-ddG and from 0.36 to 10 microM for 3'-azido-ddA) and that their triphosphate forms are incorporated as efficiently as the natural dGTP or dATP substrates by HIV-1 RT. Importantly, both 3'-azido-ddA and 3'-azido-ddG retain activity against viruses containing K65R, L74V, or M184V (IC50 change of <2.0-fold) and against those containing three or more thymidine analog mutations (IC50 change of <3.5-fold). In addition, 3'-azido-ddG does not exhibit cytotoxicity in primary lymphocytes or epithelial or T-cell lines and does not decrease the mitochondrial DNA content of HepG2 cells. Furthermore, 3'-azido-ddG is efficiently phosphorylated to 3'-azido-ddGTP in human lymphocytes, with an intracellular half-life of the nucleoside triphosphate of 9 h. The present data suggest that additional preclinical studies are warranted to assess the potential of ADPNs for treatment of HIV-1 infection.
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PMID:Anti-human immunodeficiency virus activity, cross-resistance, cytotoxicity, and intracellular pharmacology of the 3'-azido-2',3'-dideoxypurine nucleosides. 1959 85

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg(72) were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg(72) were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.
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PMID:Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance. 1981 32


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