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PubMed1997

De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence.

The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.

Base Composition↗

PubMed1996

Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.

Animals↗

PubMed1990

Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme.

Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase. When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.

Cloning, Molecular↗

PubMed2001

The biological effects of structural variation at the meta position of the aromatic rings and at the end of the alkenyl chain in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors.

In an effort to elucidate a set of structure-activity relationships in the alkenyldiarylmethane (ADAM) series of non-nucleoside reverse transcriptase inhibitors, a number of modifications were made at two locations: (1) the meta positions of the two aromatic rings and (2) the end of the alkenyl chain. Forty-two new ADAMs were synthesized and evaluated for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cell culture and for inhibition of HIV-1 reverse transcriptase. The size of the aromatic substituents was found to affect anti-HIV activity, with optimal activity appearing with Cl, CH(3), and Br substituents and with diminished activity occurring with smaller (H and F) or larger (I and CF(3)) substituents. The substituents at the end of the alkenyl chain were also found to influence the antiviral activity, with maximal activity associated with methyl or ethyl ester groups and with diminished activity resulting from substitution with higher esters, amides, sulfides, sulfoxides, sulfones, thioesters, acetals, ketones, carbamates, ureas, and thioureas. Twelve of the new ADAMs displayed submicromolar EC(50) values for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cells. Selected ADAMs, 19 and 21, were compared to previously published ADAMs 15 and 17 for antiviral efficacy and activity against the HIV-1 reverse transcriptase enzyme. All four ADAMs were found to inhibit HIV-1 reverse transcriptase enzyme activity, to inhibit the replication of a variety of HIV-1 clinical isolates representing syncytium-inducing, nonsyncytium-inducing, and subtype representative isolates, and to inhibit HIV-1 replication in monocytes. Subsequent assessment against a panel of site-directed reverse transcriptase mutants in NL4-3 demonstrated no effect of the K103N mutation on antiviral efficacy and a slight enhancement (6- to 11-fold) in sensitivity to AZT-resistant viruses. Additionally, ADAMs 19 (44-fold) and 21 (29-fold) were more effective against the A98G mutation (found in association with nevirapine resistance in vitro), and ADAM 21 was 5-fold and 2-fold more potent against the Y181C inactivation mutation than the previously reported ADAMs 15 and 17, respectively. All four ADAMs were tested for efficacy against a multidrug-resistant virus derived from a highly experienced patient expressing resistance to the reverse transcriptase enzyme inhibitors AZT, ddI, 3TC, d4T, foscarnet, and nevirapine, as well as the protease inhibitors indinavir, saquinavir, and nelfinavir. ADAM 21 was 2-fold more potent than ADAM 15 and 6-fold more potent than ADAMs 17 and 19 at preventing virus replication. Thus, we have identified a novel series of reverse transcriptase inhibitors with a favorable profile of antiviral activity against the primary mutation involved in clinical failure of non-nucleoside reverse transcriptase inhibitors, K103N, and that retain activity against a multidrug-resistant virus.

Alkenes↗

PubMed2005

Analysis of interactions of DNA polymerase beta and reverse transcriptases of human immunodeficiency and mouse leukemia viruses with dNTP analogs containing a modified sugar residue.

Substrate properties of various morpholinonucleoside triphosphates in the reaction of DNA elongation catalyzed by DNA polymerase beta, reverse transcriptase of human immunodeficiency virus (HIV-1 RT), and reverse transcriptase of Moloney murine leukemia virus (M-MuLV RT) were compared. Morpholinonucleoside triphosphates were utilized by DNA polymerase beta and HIV-1 reverse transcriptase as substrates, which terminated further synthesis of DNA, but were virtually not utilized by M-MuLV reverse transcriptase. The kinetic parameters of morpholinoderivatives of cytosine (MorC) and uridine (MorU) were determined in the reaction of primer elongation catalyzed by DNA polymerase beta and HIV-1 reverse transcriptase. MorC was a more effective substrate of HIV-1 reverse transcriptase and significantly less effective substrate of DNA polymerase beta than MorU. The possible use of morpholinonucleoside triphosphates as selective inhibitors of HIV-1 reverse transcriptase is discussed.

Catalysis↗

PubMed2005

Effects of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis on HIV-1 reverse transcriptase.

It has been recently demonstrated that HIV-1 reverse transcriptase is the target of two diterpenes, (6 R)-6-hydroxydichotoma-3,14-diene-1,17-dial (compound 1) and (6 R)-6-acetoxydichotoma-3,14-diene-1,17-dial (compound 2), that inhibit HIV-1 replication in vitro. In this work, the effects of both diterpenes on the kinetic properties of the recombinant HIV-1 reverse transcriptase (RT) enzyme were evaluated. RNA-dependent DNA-polymerase (RDDP) activity assays demonstrated that both diterpenes behave as non-competitive inhibitors with respect to dTTP and uncompetitive inhibitors with respect to poly(rA).oligo(dT) template primers. The K(i) values obtained for compounds 1 and 2 were 10 and 35 microM, respectively. Neither of these diterpenes affected the DNA-dependent DNA-polymerase (DDDP) activity of the HIV-1 RT. The RDDP activities of AMV-RT and MMLV-RT enzymes were also inhibited by compounds 1 and 2. In contrast to the HIV-1 enzyme, the DDDP activities of AMV-RT and MMLV-RT enzymes were significantly reduced by compound 1. Taken together, our results demonstrate that compound 1 is a more effective inhibitor of the viral reverse transcriptases from HIV-1, AMV and MMLV than compound 2. The kinetic behavior analyses of the HIV-1 RT demonstrate that both diterpenes have similar mechanisms of inhibition of RDDP activity.

Anti-HIV Agents↗

PubMed1990

Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography.

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

Chelating Agents↗

PubMed1999

Rapid quantification of delavirdine, a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with fluorescence detection.

Delavirdine is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of delavirdine in human plasma suitable for drug monitoring in patients is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase, high-performance liquid chromatography coupled with fluorescence detection. The optimal excitation and emission wavelengths are 300 and 425 nm, respectively. The method has been validated over the range of 50-50 000 ng/ml using only 200 microl of plasma samples. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.4% for all quality control samples. The lower limit of quanititation is 50 ng/ml. Recovery of delavirdine from human plasma is 93.8%. Delavirdine is stable under various conditions, for example 1 h at 60 degrees C and one week at 4 degrees C. This validated assay is suited for use in pharmacokinetic studies with delavirdine and can readily be implemented in the setting of a hospital laboratory for the monitoring of delavirdine concentrations.

Anti-HIV Agents↗

PubMed1999

Quantitative determination of abacavir (1592U89), a novel nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection.

Abacavir is a novel nucleoside reverse transcriptase inhibitor for the treatment of HIV-1 infection. A simple and rapid high-performance liquid chromatographic method for the quantification of abacavir in human plasma suitable for pharmacokinetic research purposes is described. Sample pretreatment consists of protein precipitation with perchloric acid. The supernatant is injected directly into the chromatographic system after centrifugation. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 285 nm. The method has been validated over the range of 20-2000 ng/ml using a volume of 300 microl of plasma. The assay is linear over this concentration range as indicated by the F-test for lack-of-fit. Within- and between-day precisions are less than 7.5% for all quality control samples. The lower limit of quantitation is 20 ng/ml and the recovery of abacavir is 88.1% (+/-1.3%). Frequently coadministered drugs did not interfere with the described methodology. Abacavir is stable in human plasma under various relevant storage conditions, for example when stored for 51 days at -20 degrees C. This validated assay is suited for use in pharmacokinetic studies with abacavir in human plasma and can readily be implemented in the setting of a hospital laboratory for the monitoring of abacavir concentrations.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Quantitative determination of efavirenz (DMP 266), a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection.

Efavirenz is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the quantification of efavirenz in human plasma suitable for therapeutic drug monitoring in plasma is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 246 nm. The method has been validated over the range of 10 to 10,000 ng/ml using a volume of 250 microl of plasma. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.3% for all quality control samples. The lower limit of quantitation is 10 ng/ml and the recovery of efavirenz from human plasma is 106.4% (+/- 1.8%). Frequently co-administered drugs did not interfere with the described methodology. Efavirenz is stable under various relevant storage conditions, for example when stored for 24 h at room temperature. This validated assay is suited for use in pharmacokinetic studies with efavirenz and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz concentrations.

Acetonitriles↗

PubMed1995

Structure, function, and evolution of bacterial reverse transcriptase.

The discovery of retroelements in the prokaryotes raises intriguing questions concerning their roles in bacteria and the origin and evolution of reverse transcriptases. We first discuss a possible structure of bacterial reverse transcriptases on the basis of the known three-dimensional structure of HIV-1 reverse transcriptase, and how such a putative three-dimensional structure is able to recognize a single primer-template RNA molecule to initiate DNA chain elongation from the 2'-OH group of an internal G residue. This reaction leads to the production of a unique RNA-DNA complex called msDNA (multicopy single-stranded DNA) in which a single-stranded DNA branches out from an RNA molecule via a 2',5'-phosphodiester linkage. Second, the mobility of the bacterial retroelements called retrons, responsible for the production of msDNA, are discussed and compared with the mobility of group I and group II introns. Third, the original and evolution of bacterial reverse transcriptases are discussed in light of the question of whether the bacterial reverse transcriptases are older than eukaryotic reverse transcriptases.

Amino Acid Sequence↗

PubMed1983

The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin.

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.

Animals↗

PubMed1993

Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90.

Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.

Base Sequence↗

PubMed1998

The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor.

Small RNA pseudoknots, selected to bind human immunodeficiency virus type 1 (HIV-1) reverse transcriptase tightly, are potent inhibitors of reverse transcriptase. The co-crystal structure of reverse transcriptase complexed with a 33 nucleotide RNA pseudoknot has been determined by fitting the ligand into a high quality, 4-fold averaged 4.8 A resolution electron density map. The RNA is kinked between stems S1 and S2, thereby optimizing its contacts with subunits of the heterodimer. Its binding site extends along the cleft that lies between the polymerase and RNase H active sites, partially overlaps with that observed for duplex DNA and presumably overlaps some portion of the tRNA site. Stem S2 and loop L1 stabilize the 'closed' conformation of the polymerase through extensive electrostatic interactions with several basic residues in helix I of the p66 thumb and in the p66 fingers domain. Presumably, this RNA ligand inhibits reverse transcriptase by binding to a site that partly overlaps the primer-template binding site.

Crystallization↗

PubMed2002

Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography.

A rapid (less than 30 min), sensitive, and specific liquid chromatography method for simultaneous assay of nine antiretroviral drugs in human plasma is described. This technique allows therapeutic drug monitoring of six approved protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two approved non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Assays were performed after diethyl ether liquid-liquid extraction from 250-microL plasma samples. Chromatographic separation was achieved on an X-TERRA (Waters; Saint Quentin, France) column using a 58% water (with 3 mmol/L pyrrolidine) and 42% acetonitrile mobile phase. Three ultraviolet wavelengths were used for detection with a diode array detector. This method allowed quantitative assay of all nine antiretroviral drugs within a concentration range of 25 ng/mL to 9000 ng/mL. The method has been validated extensively and has been in routine use in our laboratory for several months for drug monitoring in plasma samples from patients treated with antiretroviral drugs.

Chromatography, High Pressure Liquid↗

PubMed1995

tRNAs as primer of reverse transcriptases.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).

Binding Sites↗

PubMed1992

Inhibitory effects of flavonoids on Moloney murine leukemia virus reverse transcriptase activity.

Several flavonoids were tested for their effects on Moloney murine leukemia virus reverse transcriptase activity. Four groups of flavonoids, namely flavones, flavanones, flavonols, and flavanonols, were studied, and it was found that flavonols and flavanonols were very active in this regard while flavones and flavanones displayed very low activity. Among the flavonoids tested, fisetin, quercetin, myricetin, kaempferol, morin, (+/-)-taxifolin, (+)-catechin, and (-)-epicatechin were shown to be highly effective in inhibiting the reverse transcriptase activity. Structure-activity relationship analysis of these flavonoids revealed that the simultaneous presence of free hydroxyl groups at positions 3 and 4' enhanced the reverse transcriptase inhibitory activity. Replacement of the 3-hydroxyl group with a monosaccharide or of the 4'-hydroxyl group with a methyl group reduced inhibitory activity. The double bond at position 2 and 3 of the flavonoid's pyrone ring is not essential for inhibiting reverse transcriptase activity. The flavonoids studied demonstrated ability to inhibit the reverse transcriptase activity using either (rA)n(dT)12-18 or (rC)n(dG)12-18 as template-primers.

DNA Polymerase I↗

PubMed2001

Conformational analysis of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, based on quantum mechanical calculations.

The structure and the conformational behavior of the HIV-1 reverse transcriptase inhibitor, 11-cyclopropyl-5,11dihydro-4-methyl-6H-dipyrido[3,2-b2',3'-e][1,4]diazepin-6-one (nevirapine), is investigated by semiempirical (MNDO, AMI and PM3) method, ab initio at the HF/3-21G and HF/6-31G** levels and density functional theory at the B3LYP/6-31G** level. The fully optimized structure and rotational potential of the nitrogen and carbon bond in the cyclopropyl ring were examined in detail. A similar geometrical minimum is obtained from all methods which shows an almost identical structure to the geometry of the molecule in the complex structure with HIV-1 reverse transcriptase. To get some information on the structure in solution, NMR chemical shift calculations were also performed by a density functional theory at the B3LYP/6-31G** level, using GIAO approximation. The calculated 1H-NMR and 13C-NMR spectra for the energy minimum geometry agree well with the experimental results, which indicated that the geometry of nevirapine in solution is very similar to that of the molecule in the inhibition complex. Furthermore, the obtained results are compared to the conformational studies of other non-nucleoside reverse transcriptase inhibitors and reveal a common agreement of the non-nucleoside reverse transcriptase inhibitors. The specific butterfly-like shape and conformational flexibility within the side chain of the non-nucleoside reverse transcriptase inhibitors play an important role inducing conformational change of HIV-1 reverse transcriptase structure and are essential for the association at the inhibition pocket.

Anti-HIV Agents↗