HIV-1 protease inhibitors (PIs), such as darunavir (DRV), are the key component of antiretroviral therapy. However, HIV-1 often acquires resistance to PIs. Here, seven novel PIs were synthesized, by ...introducing single atom changes such as an exchange of a sulfur to an oxygen, scission of a single bond in P2'-cyclopropylaminobenzothiazole (or -oxazole), and/or P1-benzene ring with fluorine scan of mono- or bis-fluorine atoms around DRV's scaffold. X-ray structural analyses of the PIs complexed with wild-type Protease (PR
) and highly-multi-PI-resistance-associated PR
revealed that the PIs better adapt to structural plasticity in PR with resistance-associated amino acid substitutions by formation of optimal sulfur bond and adaptation of cyclopropyl ring in the S2'-subsite. Furthermore, these PIs displayed increased cell permeability and extreme anti-HIV-1 potency compared to DRV. Our work provides the basis for developing novel PIs with high potency against PI-resistant HIV-1 variants with a high genetic barrier.
Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain ...individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC
values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.
Nanoarchaeum equitans
is a species of hyperthermophilic archaea with the smallest genome size. Its alanyl-tRNA synthetase genes are split into AlaRS-α and AlaRS-β, encoding the respective subunits. ...In the current report, we surveyed
N. equitans
AlaRS-dependent alanylation of RNA minihelices, composed only of the acceptor stem and the T-arm of tRNA
Ala
. Combination of AlaRS-α and AlaRS-β showed a strong alanylation activity specific to a single G3:U70 base pair, known to mark a specific tRNA for charging with alanine. However, AlaRS-α alone had a weak but appreciable alanylation activity that was independent of the G3:U70 base pair. The shorter 16-mer RNA tetraloop substrate mimicking only the first four base pairs of the acceptor stem of tRNA
Ala
, but with C3:G70 base pair, was also successfully aminoacylated by AlaRS-α. The end of the acceptor stem, including CCA-3ʹ terminus and the discriminator A73, was able to function as a minimal structure for the recognition by the enzyme. Our findings imply that aminoacylation by
N. equitans
AlaRS-α may represent a vestige of a primitive aminoacylation system, before the appearance of the G3:U70 pair as an identity element for alanine.
Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) ...is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze–thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.
The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary ...seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of “multiple” functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.
TALEN is an artificial nuclease being applied for sequence-specific genome editing. For the plant genome editing, a pair of TALEN genes is expressed in the cells, and a binary plasmid for ...Agrobacterium-mediated transformation should be assembled. We developed a novel procedure using the Gateway-assisted plasmids, named Emerald-Gateway TALEN system. We constructed entry vectors, pPlat plasmids, for construction of a desired TALEN gene using Platinum Gate TALEN kit. We also created destination plasmid, pDual35SGw1301, which allowed two TALEN genes to both DNA strands to recruit using Gateway technology. Resultant TALEN genes were evaluated by the single-strand annealing (SSA) assay in E. coli cells. By this assay, the TALENs recognized the corresponding targets in the divided luciferase gene, and induced a specific recombination to generate an active luciferase gene. Using the TALEN genes constructed, we created a transformant potato cells in which a site-specific mutation occurred at the target site of the GBSS gene. This suggested that our system worked effectively and was applicable as a convenient tool for the plant genome editing.
During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 ...nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.
Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) ...is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze–thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.
Darunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug ...resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIV
). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIV
are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIV
and rHIV
, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIV
also developed high-level DRV resistance. However, wild-type HIV
(rHIV
) failed to acquire V32I and did not develop DRV resistance. Compared to rHIV
, rHIV
was highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIV
with DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1's DRV resistance development and elucidate, at least in part, a mechanism of DRV's high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution.
Darunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1's drug resistance. However, the mechanism(s) of the DRV's high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all
-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIV
), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIV
, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1's resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.
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