Structure−Activity Relationships of [2‘,5‘-Bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]- 3‘-spiro-5‘ ‘-(4‘ ‘-amino-1‘ ‘,2‘ ‘-oxathiole-2‘ ‘,2‘ ‘-dioxide)thymine Derivatives as Inhibitors of HIV-1 Reverse Transcriptase Dimerization

Abstract

The polymerase activity of HIV-1 reverse transcriptase (RT) is entirely dependent on the heterodimeric structure of the enzyme. Accordingly, RT dimerization represents a target for the development of a new therapeutic class of HIV inhibitors. We previously demonstrated that the N-3-ethyl derivative of 2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5' '-(4' '-amino-1' ',2' '-oxathiole-2' ',2' '-dioxide)thymine (TSAO-T) destabilizes the inter-subunit interactions of HIV-1 RT [Sluis-Cremer, N.; Dmietrinko, G. I.; Balzarini, J.; Camarasa, M.-J.; Parniak, M. A. Biochemistry 2000, 39, 1427-1433]. In the current study, we evaluated the ability of 64 TSAO-T derivatives to inhibit RT dimerization using a novel screening assay. Five derivatives were identified with improved activity compared to TSAO-T. Four of these harbored hydrophilic or aromatic substituents at the N3 position. Furthermore, a good correlation between the ability of the TSAO-T derivatives to inhibit RT dimerization and the enzyme's polymerase activity was also observed. This study provides an important framework for the rational design of more potent inhibitors of RT dimerization.

Mutagenesis of Human Immunodeficiency Virus Reverse Transcriptase p51 Subunit Defines Residues Contributing to Vinylogous Urea Inhibition of Ribonuclease H Activity

“…As shown in supplemental Fig. S3b, equivalent levels of DNA synthesis were evident for all enzymes, differing only in the ratio of the strand transfer intermediate (STI 40 ) to strand transfer product (STP 60 ). Thereafter, RNase H activity generates a series of hydrolysis products resulting from cleavage 17, 14, and 11 nt (R 17 , R 147 and R 11 , respectively) from the 5Ј terminus of the Cy5-labeled donor RNA template.…”

“…Data of Fig. 2b indicate that Ala substitutions of p51 ␣-helix I residues Val-276 and Cys-280 -Arg-284 reduce accumulation of the R 11 polymerization-independent hydrolysis product, resulting in accumulation of STI 40 and reduced levels of STP 60 . In the case of mutant L283A, accumulation of intermediate-sized hydrolysis products reflects RT stalling shortly after initiation of DNA synthesis and concomitant cleavage of the RNA/DNA hybrid.…”

“…However, although wild type RT catalyzes polymerization-dependent (R 17 product) and -independent RNase H activities (R 14 and R 11 products), p66/ p51⌬13 RT, as previously reported (21), lacks polymerizationindependent activity and, as a consequence, failed to support DNA strand transfer. Surprisingly, although polymerizationindependent RNase H activity of p66/p51⌬5 and p66/p51⌬9 RT generated significant levels of R 14 hydrolysis product, STI 40 accumulates at the expense of STP 60 . At the same time, Table 4 indicates that these subtle alterations to polymerization-independent RNase H activity induce ϳ3-fold increased sensitivity of p66/p51⌬5 RT to DNTP (IC 50 0.35 Ϯ 0.06 M), and ϳ10-fold increased sensitivity of mutant p66/p51⌬9 (IC 50 0.11 Ϯ 0.01 M).…”

“…We, therefore, asked whether reduced strand transfer function affected inhibitor sensitivity using p66/p51 heterodimers whose p51 subunit contained short C-terminal deletions. Accumulation of STI 40 in Fig. 3 indicates that all mutants retained RNA-dependent DNA polymerase activity.…”

Structure-Activity Analysis of Vinylogous Urea Inhibitors of Human Immunodeficiency Virus-Encoded Ribonuclease H

“…Inhibition of RT function is achieved either directly, by incorporating chain-terminating nucleoside derivatives (nucleoside RT inhibitors [NRTIs]), or indirectly, by nonnucleoside RT inhibitors (NNRTIs) that occupy a hydrophobic pocket at the base of the p66 thumb to interrupt the chemical step of DNA synthesis (25) and to influence the binding orientation and binding position of RT on substrates (1,20). Although they are not presently in clinical use, the 1-[2Ј ,5Ј -bis-O -(t -butyldimethylsilyl)-␤ -Dpentofuranosyl]-3Ј-spiro-5Љ-(4Љ-amino-1Љ,2Љ-oxathiole-2Љ,2Љ-dioxide)pyrimidine (TSAO) derivatives targeting the interface of the p66-p51 RT heterodimer (5,29) and nucleoside-competing RT inhibitors (15) are also showing promise as anti-HIV drugs. However, the absolute dependence on RT-encoded RNase H activity for virus replication (32) suggests that inhibitors targeting this critical function could be added to the armament of antiviral drugs, especially if synergy with the NRTI-NNRTI combinations currently in clinical use could be achieved.…”