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PubMedDate not supplied

Pyridazines 82. Synthesis of pyridazino [3,4-b][1,5]benzodiazepin-5-ones and their biological evaluation as non-nucleoside HIV reverse transcriptase inhibitors.

Starting from 3,6-dichloro-N-(2-chloro-5-nitrophenyl)-pyridazine-4-carboxamide (7) a series of 6,11-dialkylated pyridazino- [3,4-b][1,5]benzodiazepin-5-ones with a 3-chloro-8-nitro, 8-amino, 8-acetylamino, or 8-chloro substitution pattern was prepared via N-alkyl-3-alkylamino-6-chloro-N-(2-chloro-5-nitrophenyl) -pyridazine-4-carboxamides. The new compounds were screened as non-nucleoside reverse transcriptase inhibitors. The influence of the substitution pattern in compounds 10-13 on inhibitory potency is discussed.

Anti-HIV Agents↗
PubMed1991

Inhibition of HIV-reverse transcriptase activity by asterriquinone and its analogues.

Asterriquinone (ARQ; 2,5-bis-[1'-(1", 1"-dimethyl-2"-propenyl)- indol-3'-yl]-3,6-dihydroxy-1,4-benzoquinone) and its three analogues [i.e., 3,6-dihydroxy-2-[2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl]-5-[1', 7'- (1",1"-dimethylpropano)-indol-3'-yl]-1,4-benzoquinone (B1-4), 3,6-dihydroxy-2-[2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl]-5-indol-3'-yl-1,4-benzoquinone (C1-1) and 3,6-dihydroxy-2,5-diindol-3'-yl-1,4-benzoquinone (D-1)] were found to be strong inhibitors of the activity of reverse transcriptase from human immunodeficiency virus type-1. Under the reaction conditions employed, the enzyme activity was inhibited by more than 70% in the presence of 10 microM each of these compounds. The mode of inhibition by these compounds was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values of HIV-1 reverse transcriptase were determined to be 2.3, 1.5, 0.1 and 0.3 microM for ARQ, B1-4, C1-1 and D-1, respectively.

Aspergillus↗
PubMed1992

HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.

An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system.

Antigens, Viral↗
PubMed1992

Conformational changes of HIV reverse transcriptase subunits on formation of the heterodimer: correlation with kcat and Km.

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) is initially expressed as a 66-kDa protein and is subsequently proteolytically processed in vivo to form a 66-kDa/51-kDa heterodimer. Comparison of circular dichroism spectra of the 66-kDa, 51-kDa, and heterodimeric forms of RT indicates that the conversion is accompanied by dramatic changes in subunit conformation. The mean residue ellipticity per subunit at 220 nm decreases from -10.7 x 10(3) deg cm2 dmol-1 for the 66-kDa protein to -6 x 10(3) deg cm2 dmol-1 for the heterodimer. The same loss of ellipticity is observed whether the heterodimer is produced by proteolysis or by mixing a separately-expressed cloned 51-kDa subunit with the 66-kDa protein. Comparison with the spectrum of the cloned 51-kDa protein suggests that much of the conformational change arises from formation of the 51-kDa subunit but substantial changes occur in the remaining 66-kDa subunit as well. A kinetic analysis was performed to correlate these conformational changes with changes in enzyme function. Application of an integrated Michaelis-Menten equation to the catalysis of poly(dT) formation using a d(pT)20-poly(rA) primer-template shows that the kcat for the heterodimer is approximately half that of the 66 kDa enzyme, decreasing from 2.9 to 1.2 nucleotides/s upon formation of the heterodimer. However, km values for the primer-template decrease from 0.54 to 0.12 microM upon heterodimer formation. Thus, kcat/Km is 2-fold larger for the heterodimer, giving it a distinct catalytic advantage at undersaturating concentrations of enzyme and primer-template.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed2001

Novel inhibitors of HIV-reverse transcriptase catalyzed DNA strand transfer: can we alter the infidelity of the enzyme?

Reverse transcription of viral RNA by the enzyme HIV1-reverse transcriptase (HIV1-RT) involves two DNA-strand transfers (DNA-ST). We report here a time-dependent inactivation of DNA-strand transfer by specific DNA-strand transfer inhibitors that do not affect the polymerase activity of the enzyme. Irreversible inhibitors of this type may be useful in characterizing the sites on the enzyme responsible for DNA-strand transfer.

Base Sequence↗
PubMed1998

Allosteric inhibitors against HIV-1 reverse transcriptase: design and synthesis of MKC-442 analogues having an omega-functionalized acyclic structure.

Based on X-ray crystallographic analysis of MKC-442/human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) complex, analogues in which the N1-substituent is replaced with omega-functionalized alkyl groups were designed to improve the affinity for the enzyme. Synthesis of these compounds was carried out starting from MKC-442 by a sequence of reactions (N3-protection, removal of N1-ethoxymethyl group, alkylation, and N3-deprotection). The compounds were evaluated for anti-HIV activity. Structure-activity relationships are discussed in terms of the possible interaction with the enzyme.

Allosteric Site↗
PubMed1989

Differential inhibition of HIV-reverse transcriptase and various DNA and RNA polymerases by some catechin derivatives.

The two components of Camellia sinensis (green tea) (i.e., (-)-epigallocatechin gallate and (-)-epicatechin gallate were found to differentially inhibit the activities of reverse transcriptase and cellular DNA and RNA polymerases. The strongest inhibition by these compounds was observed with reverse transcriptase. DNA polymerases alpha and beta were also strongly inhibited. The mode of the inhibition of reverse transcriptase and other DNA polymerases was competitive with respect to the template.primer, whereas the mode the inhibition of RNA polymerase was competitive with respect to the nucleotide substrate.

Catechin↗
PubMed1993

Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase.

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1987

Conformational properties of 3'-azido-3'deoxy-thymidine (AZT), an inhibitor of HIV reverse transcriptase.

The low-energy conformations of 3'-azido-3'-deoxy-thymidine, (AZT), an inhibitor of retroviral reverse transcriptase, have been studied by molecular mechanics techniques. A force-field has been developed for the azido group by quantum-mechanical methods, and used in the analysis. The global low-energy structure of AZT has C3'-endo sugar pucker, an anti glycosidic angle, and a g+ C4'-C5' conformation. It is concluded that the AZT molecule has conformational properties that are very similar to those of standard deoxypyrimidines.

Antiviral Agents↗
PubMed1999

Collective motions in HIV-1 reverse transcriptase: examination of flexibility and enzyme function.

In order to study the inferences of structure for mechanism, the collective motions of the retroviral reverse transcriptase HIV-1 RT (RT) are examined using the Gaussian network model (GNM) of proteins. This model is particularly suitable for elucidating the global dynamic characteristics of large proteins such as the presently investigated heterodimeric RT comprising a total of 982 residues. Local packing density and coordination order of amino acid residues is inspected by the GNM to determine the type and range of motions, both at the residue level and on a global scale, such as the correlated movements of entire subdomains. Of the two subunits, p66 and p51, forming the RT, only p66 has a DNA-binding cleft and a functional polymerase active site. This difference in the structure of the two subunits is shown here to be reflected in their dynamic characteristics: only p66 has the potential to undergo large-scale cooperative motions in the heterodimer, while p51 is essentially rigid. Taken together, the global motion of the RT heterodimer is comprised of movements of the p66 thumb subdomain perpendicular to those of the p66 fingers, accompanied by anticorrelated fluctuations of the RNase H domain and p51 thumb, thus providing information about the details of one processivity mechanism. A few clusters of residues, generally distant in sequence but close in space, are identified in the p66 palm and connection subdomains, which form the hinge-bending regions that control the highly concerted motion of the subdomains. These regions include the catalytically active site and the non-nucleoside inhibitor binding pocket of p66 polymerase, as well as sites whose mutations have been shown to impair enzyme activity. It is easily conceivable that this hinge region, indicated by GNM analysis to play a critical role in modulating the global motion, is locked into an inactive conformation upon binding of an inhibitor. Comparative analysis of the dynamic characteristics of the unliganded and liganded dimers indicates severe repression of the mobility of the p66 thumb in RT's global mode, upon binding of non-nucleoside inhibitors.

Binding Sites↗
PubMedDate not supplied

An efficient, scalable synthesis of the HIV reverse transcriptase inhibitor Ziagen (1592U89).

Ziagen, (1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]- 2-cyclopentene-1-methanol, was synthesized from (1S,4R)-azabicyclo[2.2.1]hept-5-en-3-one by efficient processes which bypass problematic steps in earlier routes. 2-Amino-4,6-dichloro-5-formamidopyrimidine is a key intermediate which makes possible an efficient construction of the purine from a chiral cyclopentenyl precursor.

Anti-HIV Agents↗
PubMed1990

Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses.

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.

Antibodies, Viral↗