Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design.

Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.

Structural basis for the activity and regulation of human α-ketoglutarate dehydrogenase revealed by Cryo-EM.

The human mitochondrial alpha-ketoglutarate (α-KG) dehydrogenase complex (hKGDHc) is a well-studied macromolecular enzyme that converts α-KG to succinyl-CoA and NADH. Abnormalities of the complex lead to several diseases, including neurodegenerative disorders. Despite its importance in human metabolism and diseases, structural information on hKGDHc is not well defined. Here, we report the 2.92 Å resolution cryo-electron microscopy (EM) structure of its E1 component 2-oxoglutarate dehydrogenase (OGDH). The density map comprised residues 129-1,023, which is nearly the full length of OGDH. The structure clearly shows the active site and Ca2+ binding site of OGDH. This structural information will improve our understanding of the structure and function of hKGDHc and benefit pharmaceutical and basic science targeting this enzyme complex.

Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection and Nanoparticle Assembly.

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.

Bioactive Exopolysaccharides Reveal Camellia oleifera Infected by the Fungus Exobasidium gracile Could Have a Functional Use.

Camellia oleifera is an important Chinese commercial crop. Camellia oleifera can display abnormal leaves due to infection by the parasitic fungus Exobasidium gracile. Exobasidium gracile was isolated from infected leaves and used in fermentation, and exopolysaccharides EP0-1 and EP0.5-1 were purified from the fermentation broth. EP0-1 was an alkaline polysaccharide consisting mainly of the linkages α-d-Manp(1→, →2)-α-d-Manp(1→ and →6)-α-d-Manp(1→, →3)-α-d-Glcp(1→ and→4)-α-d-Glcp(1→, terminal ß-d-Galf, (1→5)-ß-d-Galf, and terminal ß-D-GlcN(1→. EP0.5-1 was an acidic galactofuranose-containing polysaccharide. It contained the linkages of α-d-Manp(1→, →2)-α-d-Manp(1→, →6)-α-d-Manp(1→,→2, 6)-α-d-Manp(1→, →4)-α-d-Glcp(1→, and →4)-α-d-GlcUA(1→. Galactofuranose linkages were composed of terminal ß-d-Galf, (1→6)-ß-d-Galf and (1→2)-ß-d-Galf. Exobasidium gracile exopolysaccharides displayed significant immunoregulatory activity by activating macrophages. This research indicates that infected leaves from Camellia oleifera including the exopolysaccharides produced by the parasitic fungus Exobasidium gracile by are worth further investigation as a functional product.

Dissecting Multivalent Lectin-Carbohydrate Recognition Using Polyvalent Multifunctional Glycan-Quantum Dots.

Multivalent protein-carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein-glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN's higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC50 values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent Kd = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein-ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.

Size-dependent melting modes and behaviors of Ag nanoparticles: a molecular dynamics study.

The size-dependent melting behaviors and mechanisms of Ag nanoparticles (NPs) with diameters of 3.5-16 nm were investigated by molecular dynamics (MD). Two distinct melting modes, non-premelting and premelting with transition ranges of about 7-8 nm, for Ag NPs were demonstrated via the evolution of distribution and transition of atomic physical states during annealing. The small Ag NPs (3.5-7 nm) melt abruptly without a stable liquid shell before the melting point, which is characterized as non-premelting. A solid-solid crystal transformation is conducted through the migration of adatoms on the surface of Ag NPs with diameters of 3.5-6 nm before the initial melting, which is mainly responsible for slightly increasing the melting point of Ag NPs. On the other hand, surface premelting of Ag NPs with diameters of 8-16 nm propagates from the outer shell to the inner core with initial anisotropy and late isotropy as the temperature increases, and the close-packed facets {111} melt by a side-consumed way which is responsible for facets {111} melting in advance relative to the crystallographic plane {111}. Once a stable liquid shell is formed, its size-independent minimum thickness is obtained, and a three-layer structure of atomic physical states is set up. Lastly, the theory of point defect-pair (vacancy-interstitial) severing as the mechanism of formation and movement of the solid-liquid interface was also confirmed. Our study provides a basic understanding and theoretical guidance for the research, production and application of Ag NPs.

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions.

Nano-enabled bioanalytical approaches to ultrasensitive detection of low abundance single nucleotide polymorphisms.

Single nucleotide polymorphisms (SNPs) constitute the most common types of genetic variations in the human genome. A number of SNPs have been linked to the development of life threatening diseases including cancer, cardiovascular diseases and neurodegenerative diseases. The ability for ultrasensitive and accurate detection of low abundant disease-related SNPs in bodily fluids (e.g. blood, serum, etc.) holds a significant value in the development of non-invasive future biodiagnostic tools. Over the past two decades, nanomaterials have been utilized in a myriad of biosensing applications due to their ability of detecting extremely low quantities of biologically important biomarkers with high sensitivity and accuracy. Of particular interest is the application of such technologies in the detection of SNPs. The use of various nanomaterials, coupled with different powerful signal amplification strategies, has paved the way for a new generation of ultrasensitive SNP biodiagnostic assays. Over the past few years, several ultrasensitive SNP biosensors capable of detecting specific targets down to the ultra-low regimes (ca. aM and below) and therefore holding great promises for early clinical diagnosis of diseases have been developed. This mini review will highlight some of the most recent, significant advances in nanomaterial-based ultrasensitive SNP sensing technologies capable of detecting specific targets on the attomolar (10(-18) M) regime or below. In particular, the design of novel, powerful signal amplification strategies that hold the key to the ultrasensitivity is highlighted.

Probing the pH-dependency of DC-SIGN/R multivalent lectin-glycan interactions using polyvalent glycan-gold nanoparticles.

The dendritic cell tetrameric lectin, DC-SIGN, and its closely related endothelial cell lectin, DC-SIGNR (collectively abbreviated as DC-SIGN/R) play a key role in the binding and transmission of deadly viruses, including Ebola, HIV, HCV, and SARS-CoV-2. Their virus binding/release processes involve a gradually acidifying environment following the natural intracellular trafficking pathways. Therefore, understanding DC-SIGN/R's pH-dependent binding properties with glycan ligands is of great importance. We have recently developed densely glycosylated gold nanoparticles (glycan-GNPs) as a powerful new tool for probing DC-SIGN/R multivalent lectin-glycan interaction (MLGI) mechanisms. They can provide not only quantitative MLGI affinities but also important structural information, such as binding site orientation and binding modes. Herein, we further employ the glycan-GNP probes to investigate the pH dependency of DC-SIGN/R MLGI properties. We find that DC-SIGN/R MLGIs exhibit distinct pH dependence over the normal physiological (7.4) to lysosomal (∼4.6) pH range. DC-SIGN binds glycan-GNPs strongly and stably from pH 7.4 to ∼5.8, but the binding is weakened significantly as pH decreases to ≤5.4 and may be fully dissociated at pH 4.6. This behaviour is fully consistent with DC-SIGN's role as an endocytic recycling receptor. In contrast, DC-SIGNR's affinity with glycan-GNPs is enhanced with the decreasing pH from 7.4 to 5.4, peaking at pH 5.4, and then reduced as pH is further lowered. Interestingly, both DC-SIGN/R binding with glycan-GNPs are found to be partially reversible in a pH-dependent manner.

Aspirin-Mediated Acetylation of SIRT1 Maintains Intestinal Immune Homeostasis.

Aspirin, also named acetylsalicylate, can directly acetylate the side-chain of lysine in protein, which leads to the possibility of unexplained drug effects. Here, the study used isotopic-labeling aspirin-d3 with mass spectrometry analysis to discover that aspirin directly acetylates 10 HDACs proteins, including SIRT1, the most studied NAD+-dependent deacetylase. SIRT1 is also acetylated by aspirin in vitro. It is also identified that aspirin directly acetylates lysine 408 of SIRT1, which abolishes SIRT1 deacetylation activity by impairing the substrates binding affinity. Interestingly, the lysine 408 of SIRT1 can be acetylated by CBP acetyltransferase in cells without aspirin supplement. Aspirin can inhibit SIRT1 to increase the levels of acetylated p53 and promote p53-dependent apoptosis. Moreover, the knock-in mice of the acetylation-mimic mutant of SIRT1 show the decreased production of pro-inflammatory cytokines and maintain intestinal immune homeostasis. The study indicates the importance of the acetylated internal functional site of SIRT1 in maintaining intestinal immune homeostasis.

Sensitive, simultaneous quantitation of two unlabeled DNA targets using a magnetic nanoparticle-enzyme sandwich assay.

We report herein the development of a simple, sensitive colorimetric magnetic nanoparticle (MNP)-enzyme-based DNA sandwich assay that is suitable for simultaneous, label-free quantitation of two DNA targets down to 50 fM level. It can also effectively discriminate single-nucleotide polymorphisms (SNPs) in genes associated with human cancers (KRAS codon 12/13 SNPs). This assay uses a pair of specific DNA probes, one being covalently conjugated to an MNP for target capture and the other being linked to an enzyme for signal amplification, to sandwich a DNA target, allowing for convenient magnetic separation and subsequent efficient enzymatic signal amplification for high sensitivity. Careful optimization of the MNP surfaces and assay conditions greatly reduced the background, allowing for sensitive, specific detection of as little as 5 amol (50 fM in 100 µL) of target DNA. Moreover, this sensor is robust, it can effectively discriminate cancer-specific SNPs against the wild-type noncancer target, and it works efficiently in 10% human serum. Furthermore, this sensor can simultaneously quantitate two different DNA targets by using two pairs of unique capture- and signal-DNA probes specific for each target. This general, simple, and sensitive DNA sensor appears to be well-suited for a wide range of genetics-based biosensing and diagnostic applications.

Polyvalent Glycan Functionalized Quantum Nanorods as Mechanistic Probes for Shape-Selective Multivalent Lectin-Glycan Recognition.

Multivalent lectin-glycan interactions (MLGIs) are widespread in biology and hold the key to many therapeutic applications. However, the underlying structural and biophysical mechanisms for many MLGIs remain poorly understood, limiting our ability to design glycoconjugates to potently target specific MLGIs for therapeutic intervention. Glycosylated nanoparticles have emerged as a powerful biophysical probe for MLGIs, although how nanoparticle shape affects the MLGI molecular mechanisms remains largely unexplored. Herein, we have prepared fluorescent quantum nanorods (QRs), densely coated with α-1,2-manno-biose ligands (QR-DiMan), as multifunctional probes to investigate how scaffold geometry affects the MLGIs of a pair of closely related, tetrameric viral receptors, DC-SIGN and DC-SIGNR. We have previously shown that a DiMan-capped spherical quantum dot (QD-DiMan) gives weak cross-linking interactions with DC-SIGNR but strong simultaneous binding with DC-SIGN. Against the elongated QR-DiMan, DC-SIGN retains similarly strong simultaneous binding of all four binding sites with a single QR-DiMan (apparent K d ≈ 0.5 nM, ∼1.8 million-fold stronger than the corresponding monovalent binding), while DC-SIGNR gives both weak cross-linking and strong individual binding interactions, resulting in a larger binding affinity enhancement than that with QD-DiMan. S/TEM analysis of QR-DiMan-lectin assemblies reveals that DC-SIGNR's different binding modes arise from the different nanosurface curvatures of the QR scaffold. The glycan display at the spherical ends presents too high a steric barrier for DC-SIGNR to bind with all four binding sites; thus, it cross-links between two QR-DiMan to maximize binding multivalency, whereas the more planar character of the cylindrical center allows the glycans to bridge all binding sites in DC-SIGNR. This work thus establishes glycosylated QRs as a powerful biophysical probe for MLGIs not only to provide quantitative binding affinities and binding modes but also to demonstrate the specificity of multivalent lectins in discriminating different glycan displays in solution, dictated by the scaffold curvature.

Application of Lung-Targeted Lipid Nanoparticle-delivered mRNA of soluble PD-L1 via SORT Technology in Acute Respiratory Distress Syndrome.

Rationale: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by excessive immune response usually due to lung inflammation. Local immunosuppression is crucial for effective ARDS treatment. However, current methods are limited in their ability to target the lungs specifically. Methods: This study utilized lung-targeted lipid nanoparticles (LNPs) with 1,2-dioleoyl-3-trimethylammonium-propane (termed DOTAP-LNPs) to encapsulate chemically modified soluble programmed death ligand-1 (sPD-L1) mRNA and examined its physiological characteristics and therapeutic efficacy. A comparative analysis was performed between sPD-L1 mRNA delivered by DOTAP-LNPs, sPD-L1 mRNA delivered by regular LNPs (MC3-LNPs), and PD-L1-Fc recombinant protein administered systemically. Additionally, the survival rate of ARDS mice treated with different drugs was assessed. Results: Administration of sPD-L1 mRNA-LNPs to ARDS model mice significantly reduced leukocyte chemotaxis and protein accumulation in lung tissue, along with a decrease in pulmonary edema. Notably, in situ intervention using sPD-L1 mRNA-DOTAP-LNPs exhibited superior therapeutic effects compared to PD-L1-Fc recombinant protein and sPD-L1 mRNA encapsulated in MC3-LNPs. Importantly, treatment with sPD-L1 mRNA-DOTAP-LNPs improved the survival rate of ARDS model mice. Conclusion: This study demonstrates the feasibility of utilizing stable and reliable mRNA to express the immunosuppressive molecule sPD-L1 specifically in the lungs. The findings provide proof of concept for localized nanoparticle delivery and offer a novel therapeutic strategy for treating acute inflammation in ARDS.

Polyvalent Nano-Lectin Potently Neutralizes SARS-CoV-2 by Targeting Glycans on the Viral Spike Protein.

Mutations in spike (S) protein epitopes allow SARS-CoV-2 variants to evade antibody responses induced by infection and/or vaccination. In contrast, mutations in glycosylation sites across SARS-CoV-2 variants are very rare, making glycans a potential robust target for developing antivirals. However, this target has not been adequately exploited for SARS-CoV-2, mostly due to intrinsically weak monovalent protein-glycan interactions. We hypothesize that polyvalent nano-lectins with flexibly linked carbohydrate recognition domains (CRDs) can adjust their relative positions and bind multivalently to S protein glycans, potentially exerting potent antiviral activity. Herein, we displayed the CRDs of DC-SIGN, a dendritic cell lectin known to bind to diverse viruses, polyvalently onto 13 nm gold nanoparticles (named G13-CRD). G13-CRD bound strongly and specifically to target glycan-coated quantum dots with sub-nM Kd. Moreover, G13-CRD neutralized particles pseudotyped with the S proteins of Wuhan Hu-1, B.1, Delta variant and Omicron subvariant BA.1 with low nM EC50. In contrast, natural tetrameric DC-SIGN and its G13 conjugate were ineffective. Further, G13-CRD potently inhibited authentic SARS-CoV-2 B.1 and BA.1, with <10 pM and <10 nM EC50, respectively. These results identify G13-CRD as the 1st polyvalent nano-lectin with broad activity against SARS-CoV-2 variants that merits further exploration as a novel approach to antiviral therapy.

Tuning Ligands Ratio Allows for Controlling Gold Nanocluster Conformation and Activating a Nonantimicrobial Thiol Fragrance for Effective Treatment of MRSA-Induced Keratitis.

Bacterial keratitis is a serious ocular disease that affects millions of people worldwide each year, among which ≈25% are caused by Staphylococcus aureus. With the spread of bacterial resistance, refractory keratitis caused by methicillin-resistant S. aureus (MRSA) affects ≈120 000-190 000 people annually and is a significant cause of infectious blindness. Atomically precise gold nanoclusters (GNCs) recently emerged as promising antibacterial agents; although how the GNC structure and capping ligands control the antibacterial properties remains largely unexplored. In this study, by adjusting the ratio of a "bulky" thiol fragrance to a linear zwitterionic ligand, the GNC conformation is transformed from Au25 (SR)18 to Au23 (SR)16 species, simultaneously converting both inactive thiol ligands into potent antibacterial nanomaterials. Surprisingly, mixed-ligand capped Au23 (SR)16 GNCs exhibit superior antibacterial potency compared to their monoligand counterparts. The optimal GNC is highly potent against MRSA, showing >1024-fold lower minimum inhibitory concentration than the corresponding free ligands. Moreover, it displays excellent potency in treating MRSA-induced keratitis in mice with greatly accelerated corneal recovery (by approximately ninefold). Thus, this study establishes a feasible method to synthesize antibacterial GNCs by adjusting the ligand ratio to control GNC conformation and active non-antibacterial ligands, thereby greatly increasing the repertoires for combating multidrug-resistant bacterial infections.