Reverse transcriptase Nucleotide

The deterrnination of the presence of reverse transcriptase in vims-infected cells can be done using labeled nucleotide triphosphates. Reverse transcriptase is an enzyme capable of synthesizing DNA from RNA and it is thought to play an important role in vims-mediated cell modification. This enzyme is also a marker enzyme for HIV, the vims impHcated in causing acquired immunodeficiency syndrome (AIDS). The procedure utilizes radiolabeled nucleotides with nonlabeled substrates to synthesize tagged DNA. The degree of radioactive incorporation reflects the reverse transcriptase activity. 

Nucleosides, nucleotides, peptidomimetics, and reverse transcriptase inhibitors in HIV infection therapy 98CCC449. 

Meyer PR, Matsuura SE, Mian AM, So AG, Scott WA (1999) A mechanism of AZT resistance an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase. Mol Cell 4 35 3... 

The 2, 3 -dideoxynucleoside (ddN) analogues (Fig. 3) encompass a vast group of compounds that have been found active against HIV and HBV, although they have been primarily pursued for the treatment of HIV infections (AIDS). They are targeted at the HIV-associated reverse transcriptase (RT) and therefore also referred to as nucleoside reverse transcriptase inhibitors (NRTIs). They have to be distinguished from the nucleotide reverse transcriptase inhibitors (NtRTIs) such as adefovir (PMEA) and tenofovir (PMPA) (see above) which, like the NRTIs, act as chain... 

Nucleoside and nucleotide reverse transcriptase analogues (NRTI) lack a 3 hydroxyl group and as a result no additional nucleotides can be incorporated into the growing DNA chain. Two NRTI resistance mechanisms are identified impairment of the incorporation of the antiretroviral drug (discrimination) and removal of the analogue from the terminated DNA chain (excision) as reviewed in Chap. 3 (Arion et al. 1998 Meyer et al. 1999 Saralianos et al. 1999). 

Nucleoside reverse transcriptase inhibitor (NRTI)/nucleotide reverse transcriptase inhibitor (NtRI) A modified version of a naturally-occurring nucleoside or nucleotide that prevents human immunodeficiency virus (HIV) replication by interfering with the function of the viral reverse transcriptase enzyme. The nucleoside/nucleotide analog causes early termination of the proviral DNA chain. For activity, an NRTI requires three phosphorylation steps once inside the cell, whereas an NtRI has a phosphate group attached and needs only two phosphorylation steps inside the cell for activity. 

Dual nucleoside (nucleotide) reverse transcriptase inhibitor (NtRTI) backbones... 

Reverse transcriptase inhibitors are of two types those that are derivatives of purine- and pyrimidine-based nucleosides and nucleotides (NtRTIs) and those that are not nucleoside or nucleotide based (NNRTIs). 

Nucleoside (Nucleotide) reverse transcriptase inhibitors (NtRTIs) ... 

A study of the potency of the antibiotic daptomycin cited plasma protein binding of 92%, but it claimed only a 2-fold shift in potency in serum (expected 12-fold) [68]. This type of discrepancy is relatively common and can often reflect substantial binding to components in the "serum-free" media. In the cases of HIV-directed non-nucleotide reverse transcriptase inhibitors, this has been dealt with by measuring the unbound drug concentration in the "serum-free" medium and using that data to calculate the intrinsic, serum-free potency [69]. 

The current standard of care recommends the use of potent three-drug combinations, which typically involves two nucleoside/nucleotide reverse transcriptase inhibitors with either a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI). By attacking HIV infection with three drugs at one time, practitioners seek to avoid the emergence of resistance strains within the patient. 

The purpose of this study is to determine whether once-daily dosing of the lopinavir/ritonavir (Kaletra) tablet in combination with investigator-selected nucleoside/nucleotide reverse transcriptase inhibitors will reduce HIV viral load to very low levels in patients who have detectable viral loads with their current antiretroviral therapy. 

Study Design Treatment, randomized, open label, active control, parallel assignment, safety/efficacy study Official Title A Phase III, Randomized, Open-Label Study of Lopina-vir/Ritonavir Tablets 800/200mg Once-Daily Versus 400/100mg Twice-Daily When Co-administered With Nucleoside/Nucleotide Reverse Transcriptase Inhibitors in Antiretroviral-Experienced, HIV-1 Infected Subjects Primary Outcome Measures ... 

Abbreviation Bp, nucleotide base pairs cDNA, complementary DNA ChIP, chromatin Immunoprecipi-tation Cy5, cyanine 5-dCTP Cy3, cyanine 3-dCTP ESTs, expressed sequence tags FDR, false discovery rate MIAME, minimum information about a microarray experiment mRNA, RNA, messenger NIA, National Institutes of Aging RFUs, relative fluorescence units RT-PCR, reverse transcriptase polymerase chain reaction SAGE, serial analysis of gene expression SAM, significance analysis of microarrays... 

During the incubation, 2pL reverse transcriptase are added to the nucleotide mixture from step 1 and carefully mixed using a pipet. 

Since one of the catalytic actions of reverse transcriptase is DNA synthesis, analogues of pyrimidine and purine nucleotides inhibit this process. A drug, zidovudine, azi-... 

These substances are analogues of thymine (azidothymidine, stavudine), adenine (didanosine), cytosine (lami-vudine, zaldtabine), and guanine (car-bovir, a metabolite of abacavir). They have in common an abnormal sugar moiety. Like the natural nucleosides, they undergo triphosphorylation, giving rise to nucleotides that both inhibit reverse transcriptase and cause strand breakage following incorporation into viral DNA. 

Initiation of reverse transcription in HIV-infected cells relies on a critical RNA-RNA interaction between tRNA y s, which is preferentially packaged into the viral particle, and a specific viral RNA seqnence. The 3 -terminaI 18 nucleotides of tRNA y are complementary to the primer binding site (PBS) sequence located in the 5 -Iong terminal repeat (LTR) of the viral RNA genome (Figure 10.3). The UUU anticodon of the tRNA is complementary to and binds to an adenosine rich loop located 8 nucleotides upstream (5 ) of the PBS. This RNA-RNA duplex which is formed when tRNA y s binds to the PBS fits within the active site of HIV-1 reverse transcriptase, bnt mnitiple interactions between the viral RNA and tRNA y are necessary for efficient initiation of reverse transcription. This interaction nucleates the reverse transcription complex which contains viral RNA, reverse transcriptase, tRNA y pl , nncleocapsid p7, and Vpr (Viral protein R), as well as multiple host factors." ... 

DNA-directed DNA polymerases [EC 2.7.7.7], also called DNA nucleotidyltransferases (DNA-directed), are enzymes that catalyze the DNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . This enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. RNA-directed DNA polymerases [EC 2.7.7.49], also referred to as reverse transcriptases, DNA nucleotidyltransferases (RNA-directed), and revertases, are enzymes that catalyze the RNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . As was the case above, this enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. 

Adefovir dipivoxii is an orally-administered nucleotide analog reverse transcriptase inhibitor. However it is used for treatment of hepatitis B and failed as a treatment for HIV. 

NRTI Nucleoside (or nucleotide) transcriptase inhibitor NNRTI Non-nucleoside reverse transcriptase inhibitor PI Protease inhibitor... 

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