Comparative Study of Nanoparticle Blood Circulation after Forced Clearance of Own Erythrocytes (Mononuclear Phagocyte System-Cytoblockade) or Administration of Cytotoxic Doxorubicin- or Clodronate-Loaded Liposomes.

Recent developments in the field of nanomedicine have introduced a wide variety of nanomaterials that are capable of recognizing and killing tumor cells with increased specificity. A major limitation preventing the widespread introduction of nanomaterials into the clinical setting is their fast clearance from the bloodstream via the mononuclear phagocyte system (MPS). One of the most promising methods used to overcome this limitation is the MPS-cytoblockade, which forces the MPS to intensify the clearance of erythrocytes by injecting allogeneic anti-erythrocyte antibodies and, thus, significantly prolongs the circulation of nanoagents in the blood. However, on the way to the clinical application of this approach, the question arises whether the induced suppression of macrophage phagocytosis via the MPS-cytoblockade could pose health risks. Here, we show that highly cytotoxic doxorubicin- or clodronate-loaded liposomes, which are widely used for cancer therapy and biomedical research, induce a similar increase in the nanoparticle blood circulation half-life in mice as the MPS-cytoblockade, which only gently and temporarily saturates the macrophages with the organism's own erythrocytes. This result suggests that from the point of view of in vivo macrophage suppression, the MPS-cytoblockade should be less detrimental than the liposomal anti-cancer drugs that are already approved for clinical application while allowing for the substantial improvement in the nanoagent effectiveness.

Mechanistic insights into the slow peptide bond formation with D-amino acids in the ribosomal active site.

During protein synthesis, ribosomes discriminate chirality of amino acids and prevent incorporation of D-amino acids into nascent proteins by slowing down the rate of peptide bond formation. Despite this phenomenon being known for nearly forty years, no structures have ever been reported that would explain the poor reactivity of D-amino acids. Here we report a 3.7Å-resolution crystal structure of a bacterial ribosome in complex with a D-aminoacyl-tRNA analog bound to the A site. Although at this resolution we could not observe individual chemical groups, we could unambiguously define the positions of the D-amino acid side chain and the amino group based on chemical restraints. The structure reveals that similarly to L-amino acids, the D-amino acid binds the ribosome by inserting its side chain into the ribosomal A-site cleft. This binding mode does not allow optimal nucleophilic attack of the peptidyl-tRNA by the reactive α-amino group of a D-amino acid. Also, our structure suggests that the D-amino acid cannot participate in hydrogen-bonding with the P-site tRNA that is required for the efficient proton transfer during peptide bond formation. Overall, our work provides the first mechanistic insight into the ancient mechanism that helps living cells ensure the stereochemistry of protein synthesis.

Structure of Dirithromycin Bound to the Bacterial Ribosome Suggests New Ways for Rational Improvement of Macrolides.

Although macrolides are known as excellent antibacterials, their medical use has been significantly limited due to the spread of bacterial drug resistance. Therefore, it is necessary to develop new potent macrolides to combat the emergence of drug-resistant pathogens. One of the key steps in rational drug design is the identification of chemical groups that mediate binding of the drug to its target and their subsequent derivatization to strengthen drug-target interactions. In the case of macrolides, a few groups are known to be important for drug binding to the ribosome, such as desosamine. Search for new chemical moieties that improve the interactions of a macrolide with the 70S ribosome might be of crucial importance for the invention of new macrolides. For this purpose, here we studied a classic macrolide, dirithromycin, which has an extended (2-methoxyethoxy)-methyl side chain attached to the C-9/C-11 atoms of the macrolactone ring that can account for strong binding of dirithromycin to the 70S ribosome. By solving the crystal structure of the 70S ribosome in complex with dirithromycin, we found that its side chain interacts with the wall of the nascent peptide exit tunnel in an idiosyncratic fashion: its side chain forms a lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4. To our knowledge, the ability of this side chain to form a contact in the macrolide binding pocket has not been reported previously and potentially can open new avenues for further exploration by medicinal chemists developing next-generation macrolide antibiotics active against resistant pathogens.

Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel.

Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms often results in the rediscovery of known compounds, genome-mining programs allow researchers to harness much greater chemical diversity, and result in the discovery of new molecular scaffolds. Here we report the genome-guided identification of a new antibiotic, klebsazolicin (KLB), from Klebsiella pneumoniae that inhibits the growth of sensitive cells by targeting ribosomes. A ribosomally synthesized post-translationally modified peptide (RiPP), KLB is characterized by the presence of a unique N-terminal amidine ring that is essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosomes by interfering with translation elongation. Structural analysis of the ribosome-KLB complex showed that the compound binds in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramin-B. KLB adopts a compact conformation and largely obstructs the tunnel. Engineered KLB fragments were observed to retain in vitro activity, and thus have the potential to serve as a starting point for the development of new bioactive compounds.

Co-produced natural ketolides methymycin and pikromycin inhibit bacterial growth by preventing synthesis of a limited number of proteins.

Antibiotics methymycin (MTM) and pikromycin (PKM), co-produced by Streptomyces venezuelae, represent minimalist macrolide protein synthesis inhibitors. Unlike other macrolides, which carry several side chains, a single desosamine sugar is attached to the macrolactone ring of MTM and PKM. In addition, the macrolactone scaffold of MTM is smaller than in other macrolides. The unusual structure of MTM and PKM and their simultaneous secretion by S. venezuelae bring about the possibility that two compounds would bind to distinct ribosomal sites. However, by combining genetic, biochemical and crystallographic studies, we demonstrate that MTM and PKM inhibit translation by binding to overlapping sites in the ribosomal exit tunnel. Strikingly, while MTM and PKM readily arrest the growth of bacteria, ∼40% of cellular proteins continue to be synthesized even at saturating concentrations of the drugs. Gel electrophoretic analysis shows that compared to other ribosomal antibiotics, MTM and PKM prevent synthesis of a smaller number of cellular polypeptides illustrating a unique mode of action of these antibiotics.

Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state.

The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.

The Biological and Clinical Aspects of a Latent Tuberculosis Infection.

Tuberculosis (TB), caused by bacilli from the Mycobacterium tuberculosis complex, remains a serious global public health problem, representing one of the main causes of death from infectious diseases. About one quarter of the world's population is infected with Mtb and has a latent TB infection (LTBI). According to the World Health Organization (WHO), an LTBI is characterized by a lasting immune response to Mtb antigens without any TB symptoms. Current LTBI diagnoses and treatments are based on this simplified definition, although an LTBI involves a broad range of conditions, including when Mtb remains in the body in a persistent form and the immune response cannot be detected. The study of LTBIs has progressed in recent years; however, many biological and medical aspects of an LTBI are still under discussion. This review focuses on an LTBI as a broad spectrum of states, both of the human body, and of Mtb cells. The problems of phenotypic insusceptibility, diagnoses, chemoprophylaxis, and the necessity of treatment are discussed. We emphasize the complexity of an LTBI diagnosis and its treatment due to its ambiguous nature. We consider alternative ways of differentiating an LTBI from active TB, as well as predicting TB reactivation based on using mycobacterial "latency antigens" for interferon gamma release assay (IGRA) tests and the transcriptomic analysis of human blood cells.

Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.