Conformational Properties of Aryl S-Glucosides in Solution.

The conformational study of saccharides and glycomimetics in solution is critical for a comprehensive understanding of their interactions with biological receptors and enabling the design of optimized glycomimetics. Here, we report a nuclear magnetic resonance (NMR) study centered on the conformational properties of the hydroxymethyl group and glycosidic bond of four series of aryl S-glucosides. We found that in acetyl-protected and free aryl S-ß-glucosides, the rotational equilibrium around the C5-C6 bond (hydroxymethyl group) exhibits a linear dependence on the electronic properties of the aglycone, namely, as the aryl's substituent electron-withdrawing character increases, the dominance of the gg rotamer declines and the gt contribution rises. Likewise, the conformational equilibrium around the glycosidic C1-S bond also depends on the aglycone's electronic properties, where glucosides carrying electron-poor aglycones exhibit stiffer glycosidic bonds in comparison to their electron-rich counterparts. In the case of the α anomers, the aglycone's effect over the glycosidic bond conformation is like that observed on their ß isomers; however, we observe no aglycone's influence over the hydroxymethyl group conformation in the α-glucosides.

Cetuximab-based PROteolysis targeting chimera for effectual downregulation of NSCLC with varied EGFR mutations.

PROteolysis Targeting Chimeras (PROTACs) showed tremendous therapeutic potential in degrading several oncoproteins including undruggable proteins. PROTACs are bifunctional molecules where one-part binds to target protein while the other end recruits protein degradation machinery. With the unveiling advancements in the field of PROTACs, we explored a combinatorial approach by developing antibody-based PROTAC (ABTAC) which may effectively degrade one of the key oncoprotein driving proliferation and progression of cancer - Epidermal growth factor receptor (EGFR). The objective of current research was to synthesize and characterize an EGFR degrading ABTAC for the treatment of non-small cell lung cancer (NSCLC). Cetuximab and pomalidomide (E3 ligase recruiting ligand) were conjugated using lysine conjugation and copper free azide-alkyne cycloaddition (CuAAC) click chemistry. Analytical characterization using reverse-phase liquid chromatography and mass spectrometry suggested conjugation of five E3-ligase inhibitor molecules/antibody. Nearly 10-30 folds reduction in IC50 was observed with ABTAC in HCC827 (EGFR sensitive) and H1650 (EGFR resistant) cells compared to cetuximab. Multicellular 3D spheroid assay strongly suggested that ABTAC induced significant apoptosis and also inhibited cell proliferation compared to control and antibody alone. Circular dichroism and surface plasmon resonance (SPR) confirmed minor alterations in the structure and receptor binding efficacy of the antibody post-conjugation.

Multivalent Targeting of the Asialoglycoprotein Receptor by Virus-Like Particles.

The asialoglycoprotein receptor (ASGPR) is expressed in high density on hepatocytes. Multivalent variants of galactosyl carbohydrates bind ASGPR with high affinity, enabling hepatic delivery of ligand-bound cargo. Virus-like particle (VLP) conjugates of a relatively high-affinity ligand were efficiently endocytosed by ASGPR-expressing cells in a manner strongly dependent on the nature and density of ligand display, with the best formulation using a nanomolar-, but not a picomolar-level, binder. Optimized particles were taken up by HepG2 cells with greater efficiency than competing small molecules or the natural multigalactosylated ligand, asialoorosomucoid. Upon systemic injection in mice, these VLPs were rapidly cleared to the liver and were found in association with sinusoidal endothelial cells, Kupffer cells, hepatocytes, dendritic cells, and other immune cells. Both ASGPR-targeted and nontargeted particles were distributed similarly to endothelial and Kupffer cells, but targeted particles were distributed to a greater number and fraction of hepatocytes. Thus, selective cellular trafficking in the liver is difficult to achieve: even with the most potent ASGPR targeting available, barrier cells take up much of the injected particles and hepatocytes are accessed only approximately twice as efficiently in the best case.

A Ferrier glycosylation/cis-dihydroxylation strategy to synthesize Leishmania spp. lipophosphoglycan-associated ßGal(1,4)Man disaccharide.

The Galß(1→4)Man disaccharide, found in the cell surface lipophosphoglycan (LPG) of Leishmania species, has been synthesized by a Ferrier glycosylation/cis-dihydroxylation strategy. This stereoselective method proved efficient for synthesizing the target saccharide in good yield. In addition, we prepared two clickable O-glycoside and phospho-glycoside versions of Galß(1→4)Man to enable conjugation to protein carriers for further immunological and antibody-binding studies.

Bictegravir nanomicelles and anionic pullulan loaded vaginal film: Dual mechanistic pre-exposure prophylaxis (PrEP) for HIV.

Locally delivered pre-exposure prophylaxis (PrEP) has proven to be a promising strategy to combat Human immunodeficiency virus (HIV) transmission but several findings encountered toxicities or proved to be marginally effective in clinical settings. Therefore, innovative, multifunctional, and safer alternatives are being progressively investigated. Herein, we explored negatively charged carbohydrate, anionic pullulan (AP) as a rapidly soluble film-former and novel anti-HIV agent. Additionally, Bictegravir (BCT), an HIV integrase inhibitor was co-delivered in the form of nanomicelles for sustained antiviral activity. BCT-loaded PLGA-PEG polymeric nanomicelles (BN) were incorporated into PVA/pullulan-based film matrix comprising of 2 % w/v AP (BN-AP film). In cell-based assays, biocompatibility and TEER values for BN-AP films were similar to control while the commercial vaginal contraceptive film (VCF®) showed severe cytotoxicity and drastically reduced the tight junction integrity. Rapid disintegration of BN-AP film with >85 % drug release was observed in simulated vaginal and seminal fluid. Most importantly, AP and BN-AP film significantly inhibited HIV-1 replication with IC50 at as low as 91 µg/mL and 0.708 nM, respectively. Therefore, this study entails successful development of BN-AP film that functioned as an effective, biocompatible dual-acting PrEP formulation.

Anodic Reactivity of Alkyl S-Glucosides.

A voltammetric study of a series of alkyl and aryl S-glucosides unveiled the reactivity patterns of alkyl S-glucosides toward anodic oxidation and found noteworthy differences with the trends followed by aryl derivatives. The oxidation potential of alkyl S-glucosides, estimated herein from square-wave voltammetry peak potentials (Ep), depends on the steric properties of the aglycone. Glucosides substituted with bulky groups exhibit Ep values at voltages more positive than the values of those carrying small aglycones. This relationship, observed in all analyzed alkyl series, is evidenced by good linear correlations between Ep and Taft's steric parameters (ES) of the respective alkyl substituents. Moreover, the role of the aglycone's steric properties as a primary reactivity modulator is backed by poor correlations between Ep and the radical stabilization energies (RSEs) of the aglycone-derived thiyl radicals (RS•). In contrast, aryl glucosides' Ep values exhibit excellent correlations with the aryl substituents' Hammett parameters (σ+) and the ArS• RSEs, evidencing the inherent stability of the reactive radical intermediate as the primary factor controlling aryl glucoside's electrochemical reactivity. The reactivity differences between alkyl and aryl S-glucosides also extend to the protective group's effect on Ep. Alkyl S-glucosides' reactivity proved to be more sensitive to protective group exchange.

Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

Glycan-Modified Virus-like Particles Evoke T Helper Type 1-like Immune Responses.

Dendritic cells (DCs) are highly effective antigen-presenting cells that shape immune responses. Vaccines that deliver antigen to the DCs can harness their power. DC surface lectins recognize glycans not typically present on host tissue to facilitate antigen uptake and presentation. Vaccines that target these surface lectins should offer improved antigen delivery, but their efficacy will depend on how lectin targeting influences the T cell subtypes that result. We examined how antigen structure influences uptake and signaling from the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin or CD209). Virus-like particles (VLPs) were engineered from bacteriophage Qß to present an array of mannoside ligands. The VLPs were taken up by DCs and efficiently trafficked to endosomes. The signaling that ensued depended on the ligand displayed on the VLP: only those particles densely functionalized with an aryl mannoside, Qß-Man540, elicited DC maturation and induced the expression of the proinflammatory cytokines characteristic of a T helper type 1 (TH1)-like immune response. This effect was traced to differential binding to DC-SIGN at the acidic pH of the endosome. Mice immunized with a VLP bearing the aryl mannoside, and a peptide antigen (Qß-Ova-Man540) had antigen-specific responses, including the production of CD4+ T cells producing the activating cytokines interferon-γ and tumor necrosis factor-α. A TH1 response is critical for intracellular pathogens (e.g., viruses) and cancer; thus, our data highlight the value of targeting DC lectins for antigen delivery and validate the utility of DC-targeted VLPs as vaccine vehicles that induce cellular immunity.

Virus-like Particle Display of the α-Gal Carbohydrate for Vaccination against Leishmania Infection.

Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the α-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of α-Gal than the former. A polyvalent α-Gal conjugate on the immunogenic Qß virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 α-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-α-Gal antibodies. As expected, α-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Qß-α-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the infection and proliferation of parasites in the liver and spleen as probed by qPCR. The α-Gal epitope may therefore be considered as a vaccine candidate to block human cutaneous and visceral leishmaniasis.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

Virus-like Particle Display of the α-Gal Epitope for the Diagnostic Assessment of Chagas Disease.

The α-Gal antigen [Galα(1,3)Galß(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.

Amblyomma sculptum tick saliva: α-Gal identification, antibody response and possible association with red meat allergy in Brazil.

The anaphylaxis response is frequently associated with food allergies, representing a significant public health hazard. Recently, exposure to tick bites and production of specific IgE against α-galactosyl (α-Gal)-containing epitopes has been correlated to red meat allergy. However, this association and the source of terminal, non-reducing α-Gal-containing epitopes have not previously been established in Brazil. Here, we employed the α-1,3-galactosyltransferase knockout mouse (α1,3-GalT-KO) model and bacteriophage Qß-virus like particles (Qß-VLPs) displaying Galα1,3Galß1,4GlcNAc (Galα3LN) epitopes to investigate the presence of α-Gal-containing epitopes in the saliva of Amblyomma sculptum, a species of the Amblyomma cajennense complex, which represents the main tick that infests humans in Brazil. We confirmed that the α-1,3-galactosyltransferase knockout animals produce significant levels of anti-α-Gal antibodies against the Galα1,3Galß1,4GlcNAc epitopes displayed on Qß-virus like particles. The injection of A. sculptum saliva or exposure to feeding ticks was also found to induce both IgG and IgE anti-α-Gal antibodies in α-1,3-galactosyltransferase knockout mice, thus indicating the presence of α-Gal-containing epitopes in the tick saliva. The presence of α-Gal-containing epitopes was confirmed by ELISA and immunoblotting following removal of terminal α-Gal epitopes by α-galactosidase treatment. These results suggest for the first known time that bites from the A. sculptum tick may be associated with the unknown etiology of allergic reactions to red meat in Brazil.

Evaluation of a focused virtual library of heterobifunctional ligands for Clostridium difficile toxins.

A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.

Stereochemical properties of glucosyl sulfoxides in solution.

A series of alkyl ß-glucosyl sulfoxides were synthesized and characterized in order to study their stereochemical properties. The dependence of the aglycon, solvent and absolute configuration of the sulfinyl group on the conformational properties around the glucosidic and C5-C6 (hydroxymethyl group) bonds were studied. The results for R(S) sulfoxides show linear correlations between the rotamer populations of the hydroxymethyl group and the corresponding Taft's steric parameter (E(S)) of the alkyl group attached to the sulfinyl group in polar and apolar solvents, an increase in the absolute value of E(S) leading to an increase in the gt population. In addition, NOE experiments reveal that as the bulkiness of the alkyl group increases the population of the g- rotamer increases, the latter stabilized by the exo-anomeric effect. These results are in complete agreement with the participation of the exo-anomeric effect in both conformational properties of R(S) sulfoxides. Sulfoxides with the S(S) configuration show different behavior to their R(S) epimers; thus, an increase in the E(S) value of the alkyl group leads to similar or lower gt populations in apolar solvents and to increases in gt in polar solvents. Their NOE studies reveal a conformational equilibrium (in polar and apolar solvents) between g- and g+, dependent on the size of the alkyl group R attached to the sulfinyl group. All these results for both epimers support the general hypothesis that the exo-anomeric effect modifies the conformation of the hydroxymethyl group, fulfills the stereoelectronic requirements, and shows dependence on the solvent.

Cytotoxic effects of C-glycosides in HOS and HeLa cell lines.

Fifty-two C-glycosides were synthesized and their in-vitro antiproliferative activity screened against human cervical carcinoma (HeLa) and osteosarcoma (HOS) cell lines. Nine of them had growth inhibitions (GI(50) values) below 10 microM, the C-glucopyranoside 38 being the most active against HeLa (5.4 microM) and the dichlorocyclopropyl derivative 42 against HOS (1.6 microM). Some preliminary structure-activity relationships were established.

Antiproliferation and apoptosis induced by C-glycosides in human leukemia cancer cells.

A large series of alkyl C-glycosides was synthesized from D-glucal or D-galactal. These compounds were screened against the human promyelocytic leukemia cell line (HL60), showing significant activity and apoptosis. Up to 13 C-glucopyranosides, but no C-galacto- or C-mannopyranosides, exhibited inhibitory concentrations (IC(50) values) below 20 microM, five of them in the range 4-8 microM. Preliminary structure-activity relationships were established.