Inhibitors, reverse transcription

Boulm F., Freund F., Moreau S., Nielsen P.E., Gryaznov S., Toulme J.J., Litvak S. Modified (PNA, 2-O-methyl and phosphoramidate) anti-TAR anti-sense oligonucleotides as strong and specific inhibitors of in vitro HI.V.-l reverse transcription. Nucleic Acids Res. 1998 26 5492-5500. 

The total amplification achieved by PCR is described by the expression, (1 + )", where E is the average per-cycle efficiency and n is the total number of cycles. The amount of target sequence and the variable presence of inhibitors in clinical specimens influence both the efficiency and the kinetics of amplification. As seen in the preceding expression, small differences in the efficiency of amplification are exponentially compounded and lead to very large and unpredictable differences in product yield. The situation is even more complicated when the target is RNA. PCR must be preceded by reverse transcription to produce complementary DNA (cDNA), and the efficiency of this process is another variable that may influence product yield. 

The nonnucleoside reverse transcriptase inhibitors (NNRTIs), used in the treatment of AIDS, provide interesting examples of clinically relevant noncompetitive inhibitors. The causative agent of AIDS, HIV, belongs to a virus family that relies on an RNA-based genetic system. Replication of the vims requires reverse transcription of the viral genomic RNA into DNA, which is then incorporated into the genome of the infected host cell. Reverse transcription is catalyzed by a virally encoded nucleic acid polymerase, known as reverse transcriptase (RT). This enzyme is critical for viral replication inhibition of HIV RT is therefore an effective mechanism for abrogating infection in patients. 

Thiourea compounds have been observed to inhibit human immunodeficiency virus (HIV) reverse transcriptase, a viral enzyme that is responsible for the reverse transcription of the retroviral RNA to proviral DNA. Phenethylthiazoylthiourea (PETT) compounds were discovered as potent inhibitors of HIV type 1 and display certain structure-activity relationships among various substituents in their structure.199 207 Furthermore, thiourea derivatives have been found to be potent and selective viral inhibitors, antifungal and antibacterial compounds.208 215... 

Up to this point, GIPF expressions have been formulated for only one type of biological activity - the inhibition of reverse transcriptase (RT), the enzyme that promotes the reverse transcription of genomic RNA into double-stranded DNA, a key step in the replication of the human immunodeficiency virus, HIV [82, 87]. Analytical representations were obtained for the anti-HIV potencies of three families of RT inhibitors the correlation coefficients are between 0.930 and 0.952. We are currently investigating the effects of applying the GIPF approach to certain portions of the molecules rather than their entireties. This might reveal the source of the activity, or alternatively, indicate it to be delocalized. 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Although any of these seven steps could be a druggable target, most of the antiviral agents clinically employed for non-AIDS infections act on the synthesis or assembly of either purines or pyrimidines (steps 3 and 4). For AIDS, reverse transcriptase inhibitors block transcription of the HIV RNA genome into DNA, thereby preventing synthesis of viral mRNA and protein protease inhibitors act on the synthesis of late proteins (steps 5 and 6). 

FIGURE 34-3 T Schematic illustration of HIV replication and the site of action of the reverse transcriptase inhibitors [RTIs], These drugs interfere with the process of reverse transcription by inhibiting the enzyme that converts viral RNA [vRNA] to viral DNA (vDNA). See text for further discussion. 

HIV is an RNA virus, meaning the viral genome is encoded as RNA instead of DNA. One key step in the virus cycle is to synthesize double-stranded viral DNA using viral RNA as a template. A viral enzyme called reverse transcriptase (RT) accomplishes this task. The reverse transcription process may be blocked by nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs have already been discussed somewhat in Chapter 6. 

Zhang, H Domadula, G., Wu, Y., Havlir, D., Richman, D. D., and Pomerantz, R. J. (1996) Kinetic analysis of intravirion reverse transcription in the blood plasma of human immunodeficiency virus type 1-infected individuals direct assessment of resistance to reverse transcriptase inhibitors in vivo../. Virol. 70,628-634. 

Modified short DNA and RNA molecules with novel bond linkages between the bases have been designed with the aim to use the antisense approach by binding to the RNA template in the hTR subunit(s) to prevent or halt transcription and thereby act as competitive inhibitors of telomerase activity. Hence, the hTR RNA template is unavailable to hTERT for reverse transcription (52). The various types of sugar phosphodiester backbone modifications in these molecules are intended to confer certain desirable characteristics or properties, such as intracellular penetration, superior binding affinity, and therefore specificity, to the hTR RNA template and in order to enable intact delivery to their target. 

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