Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation.

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473-477, 499-502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.

Current medicinal chemistry strategies in the discovery of novel HIV-1 ribonuclease H inhibitors.

During HIV-1 genome replication, the viral reverse transcriptase-associated ribonuclease H (RT-associated RNase H) activity hydrolyzes the RNA strand of RNA/DNA heteroduplex intermediates. As of today, HIV-1 RNase H inhibitors (RHIs) remain at an investigational level, although none of them reached clinical trials. Therefore, RNase H remains as an attractive target for drug design and development. In this paper, we review the current status of medicinal chemistry strategies aimed at the discovery of novel RHIs, while discussing problems encountered in their characterization and further development, thereby providing an update on recent progress in the field.

Design, synthesis, and biological evaluation of novel double-winged galloyl derivatives as HIV-1 RNase H inhibitors.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains as the only enzyme encoded within the viral genome not clinically validated as an antiviral target. We have previously reported that the galloyl derivative II-25 had RNase H inhibitory activity in enzymatic assays but showed weak antiviral activity in phenotypic assays due its large polarity and poor membrane permeability. In this report, we report on a series of II-25 derivatives, obtained by addition of different hydrophobic moieties ("the wings") at the C-2 and C-3 positions of the piperazine ring that showed improved RNase H inhibitory activity. Six compounds showed strong inhibitory activity and were found to be more potent than ß-thujaplicinol in enzymatic assays. The most potent compound was IA-6 and exhibited the best inhibitory activity (IC50 = 0.067 ± 0.02 µM). IA-6 was around 11 and 30 times more potent than II-25 and ß-thujaplicinol, respectively. Molecular modeling studies predict a strong hydrophobic interaction between the furylmethylaminyl group of IA-6 and the side chain of His539, explaining the potent HIV-1 RNase H inhibition. Unfortunately, none of the derivatives showed significant antiviral activity in cell culture. It is worth emphasizing that most of the obtained compounds show low cytotoxicity (CC50 > 20 µM), which confirms the significance of identifying galloyl derivatives as valuable leads for further optimization.

Enzyme-support interactions and inactivation conditions determine Thermomyces lanuginosus lipase inactivation pathways: Functional and florescence studies.

Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.

Effect of protein load on stability of immobilized enzymes.

Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one. This negative effect of the enzyme crowding on enzyme stability was found in inactivations at pH 5, 7 or 9, but not in inactivations in the presence of organic solvents. The immobilization using ethanol to reduce the immobilization rate had no effect on the stability of the lowly loaded preparation, while the highly loaded enzyme biocatalysts increased their stabilities, becoming very similar to that of the lowly loaded preparation. Results suggested that CALB molecules immobilized on octyl agarose may be closely packed together due to the high immobilization rate and this produced some negative interactions between immobilized enzyme molecules during enzyme thermal inactivation. Slowing-down the immobilization rate may be a solution for this unexpected problem.

Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites.

Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.