Cryo-EM structure of the ancient eukaryotic ribosome from the human parasite Giardia lamblia.

Giardiasis is a disease caused by the protist Giardia lamblia. As no human vaccines have been approved so far against it, and resistance to current drugs is spreading, new strategies for combating giardiasis need to be developed. The G. lamblia ribosome may provide a promising therapeutic target due to its distinct sequence differences from ribosomes of most eukaryotes and prokaryotes. Here, we report the cryo-electron microscopy structure of the G. lamblia (WB strain) ribosome determined at 2.75 Å resolution. The ribosomal RNA is the shortest known among eukaryotes, and lacks nearly all the eukaryote-specific ribosomal RNA expansion segments. In contrast, the ribosomal proteins are typically eukaryotic with some species-specific insertions/extensions. Most typical inter-subunit bridges are maintained except for one missing contact site. Unique structural features are located mainly at the ribosome's periphery. These may be exploited as target sites for the design of new compounds that inhibit selectively the parasite's ribosomal activity.

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.

Structural Studies Reveal the Role of Helix 68 in the Elongation Step of Protein Biosynthesis.

The ribosome, a multicomponent assembly consisting of RNA and proteins, is a pivotal macromolecular machine that translates the genetic code into proteins. The large ribosomal subunit rRNA helix 68 (H68) is a key element in the protein synthesis process, as it coordinates the coupled movements of the actors involved in translocation, including the tRNAs and L1 stalk. Examination of cryo-electron microscopy (cryo-EM) structures of ribosomes incubated for various time durations at physiological temperatures led to the identification of functionally relevant H68 movements. These movements assist the transition of the L1 stalk between its open and closed states. H68 spatial flexibility and its significance to the protein synthesis process were confirmed through its effective targeting with antisense PNA oligomers. Our results suggest that H68 is actively involved in ribosome movements that are central to the elongation process. IMPORTANCE The mechanism that regulates the translocation step in ribosomes during protein synthesis is not fully understood. In this work, cryo-EM techniques used to image ribosomes from Staphylococcus aureus after incubation at physiological temperature allowed the identification of a conformation of the helix 68 that has never been observed so far. We then propose a mechanism in which such helix, switching between two different conformations, actively coordinates the translocation step, shedding light on the dynamics of ribosomal components. In addition, the relevance of helix 68 to ribosome function and its potential as an antibiotic target was proved by inhibiting Staphylococcus aureus ribosomes activity in vitro using oligomers with sequence complementarity.

Assessment of 6'- and 6'''-N-acylation of aminoglycosides as a strategy to overcome bacterial resistance.

Amongst the many synthetic aminoglycoside analogues that were developed to regain the efficacy of this class of antibiotics against resistant bacterial strains, the 1-N-acylated analogues are the most clinically used. In this study we demonstrate that 6'-N-acylation of the clinically used compound tobramycin and 6'''-N-acylation of paromomycin result in derivatives resistant to deactivation by 6'-aminoglycoside acetyltransferase (AAC(6')) which is widely found in aminoglycoside resistant bacteria. When tested against AAC(6')- or AAC(3)-expressing bacteria as well as pathogenic bacterial strains, some of the analogues demonstrated improved antibacterial activity compared to their parent antibiotics. Improvement of the biological performance of the N-acylated analogues was found to be highly dependent on the specific aminoglycoside and acyl group. Our study indicates that as for 1-N-acylation, 6'- and 6'''-N-acylation of aminoglycosides offer an additional promising direction in the search for aminoglycosides capable of overcoming infections by resistant bacteria.

Targeting anthracycline-resistant tumor cells with synthetic aloe-emodin glycosides.

The cytotoxic activity of aloe-emodin (AE), a natural anthranoid that readily permeates anthracycline-resistant tumor cells, was improved by the attachment of an amino-sugar unit to its anthraquinone core. The new class of AE glycosides (AEGs) showed a significant improvement in cytotoxicity-up to more than 2 orders of magnitude greater than those of AE and the clinically used anthracycline doxorubicin (DOX)-against several cancer cell lines with different levels of DOX resistance. Incubation with the synthetic AEGs induced cell death in less than one cell cycle, indicating that these compounds do not directly target the cell division mechanism. Confocal microscopy provided evidence that unlike DOX, AEGs accumulated in anthracycline-resistant tumor cells in which resistance is conferred by P-glycoprotein efflux pumps. The results of this study demonstrate that AEGs may serve as a promising scaffold for the development of cytotoxic agents capable of overcoming anthracycline resistance in tumor cells.