RESUMEN
Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.
Asunto(s)
Saponinas , Vacunas , Ratones , Animales , Liposomas , Porinas , Epítopos , Adyuvantes Inmunológicos , Pseudomonas aeruginosa , Inmunoglobulina G , ColesterolRESUMEN
C7/C8-cyclitols and C7N-aminocyclitols find applications in the pharmaceutical sector as α-glucosidase inhibitors and in the agricultural sector as fungicides and insecticides. In this study, we identified C7/C8-cyclitols and C7N-aminocyclitols as potential inhibitors of Streptomyces coelicolor (Sco) GlgEI-V279S based on the docking scores. The protein and the ligand (targets 11, 12, and 13) were prepared, the states were generated at pH 7.0 ± 2.0, and the ligands were docked into the active sites of the receptor via Glide™. The synthetic route to these targets was similar to our previously reported route used to obtain 4-âº-glucoside of valienamine (AGV), except the protecting group for target 12 was a p-bromobenzyl (PBB) ether to preserve the alkene upon deprotection. While compounds 11-13 did not inhibit Sco GlgEI-V279S at the concentrations evaluated, an X-ray crystal structure of the Sco GlgE1-V279S/13 complex was solved to a resolution of 2.73 Å. This structure allowed assessment differences and commonality with our previously reported inhibitors and was useful for identifying enzyme-compound interactions that may be important for future inhibitor development. The Asp 394 nucleophile formed a bidentate hydrogen bond interaction with the exocyclic oxygen atoms (C(3)-OH and C(7)-OH) similar to the observed interactions with the Sco GlgEI-V279S in a complex with AGV (PDB:7MGY). In addition, the data suggest replacing the cyclohexyl group with more isosteric and hydrogen bond-donating groups to increase binding interactions in the + 1 binding site.
RESUMEN
Patients receiving healthcare are at higher risk of acquiring healthcare-associated infections, which cause a significant number of illnesses and deaths. Most pathogens responsible for these infections are highly resistant to multiple antibiotics, prompting the need for discovery of new therapeutics to combat these evolved threats. We synthesized structural derivatives of (+)-puupehenone, a marine natural product, and observed growth inhibition of several clinically relevant Gram-positive bacteria, particularly Clostridioides difficile. The most potent compounds-(+)-puupehenone, 1, 15, 19, and 20-all inhibited C. difficile in the range of 2.0-4.0 µg/mL. Additionally, when present in the range of 1-8 µg/mL, a subset of active compounds-(+)-puupehenone, 1, 6, 15, and 20-greatly reduced the ability of C. difficile to produce exotoxins, which are required for disease in infected hosts. Our findings showcase a promising class of compounds for potential drug development against Gram-positive pathogens, such as C. difficile.
RESUMEN
Tuberculosis is a disease caused primarily by the organism Mycobacterium tuberculosis (Mtb), which claims about 1.5 million lives every year. A challenge that impedes the elimination of this pathogen is the ability of Mtb to remain dormant after primary infection, thus creating a reservoir for the disease in the population that reactivates under more ideal conditions. A better understanding of the physiology of dormant Mtb and therapeutics able to kill these phenotypically tolerant bacilli will be critical for completely eradicating Mtb. Our groups are focusing on characterizing the activity of derivatives of the marine natural product (+)-puupehenone (1). Recently, the Rohde group reported that puupehedione (2) and 15-α-methoxypuupehenol (3) exhibit enhanced activity in an in vitro multi-stress dormancy model of Mtb. To optimize the antimycobacterial activity of these terpenoids, novel 15-α-methoxy- and 15-α-acetoxy-puupehenol esters were prepared from (+)-puupehenone (1) accessed through a (+)-sclareolide-derived ß-hydroxyl aldehyde. For added diversity, various congeners related to (1) were also prepared from a common borono-sclareolide donor, which resulted in the synthesis of epi-puupehenol and the natural products (+)-chromazonarol and (+)-yahazunol. In total, we generated a library of 24 compounds, of which 14 were found to be active against Mtb, and the most active compounds retained the enhanced activity against dormant Mtb seen in the parent compound. Several of the 15-α-methoxy- and 15-α-acetoxy-puupehenol esters possessed potent activity against actively dividing and dormant Mtb. Intriguingly, the closely related triisobutyl derivative 16 showed similar activity to 1 in actively dividing Mtb but lost about 178-fold activity against dormant Mtb. However, the monopivaloyl compound 13 showed a modest 3- to 4-fold loss in activity in both actively dividing and dormant Mtb relative to the activity of 1 revealing the importance of the free OH at C19 supporting the potential role of quinone methide formation as critical for activity in dormant Mtb. Elucidating important structure-activity relationships and the mechanism of action of this natural product-inspired chemical series may yield insights into vulnerable drug targets in dormant bacilli and new therapeutics to more effectively target dormant Mtb.
RESUMEN
Pseudomonas aeruginosa is a highly prevalent gram-negative bacterium that is becoming more difficult to treat because of increasing antibiotic resistance. As chemotherapeutic treatment options diminish, there is an increased need for vaccines. However, the creation of an effective P. aeruginosa vaccine has been elusive despite intensive efforts. Thus, new paradigms for vaccine antigens should be explored to develop effective vaccines. In these studies, we have focused on the synthesis of two L-rhamnose-bearing epitopes common to glycoforms I and II of the outer core domain of Pseudomonas aeruginosa lipopolysaccharide, α-L-Rha-(1â6)-α-D-Glc-(1â4)-α-D-GalN-(Ala)-α-aminooxy (3) and α-L-Rha-(1â3)-ß-D-Glc-(1â3)-α-D-GalN-(Ala)-α-aminooxy (4), respectively. The target trisaccharides were both prepared starting from a suitably protected galactosamine glycoside, followed by successive deprotection and glycosylation with suitably protected D-glucose and L-rhamnose thioglycosides. Global deprotection resulted in the formation of targets 3 and 4 in 22 and 35% yield each. Care was required to modify basic reaction conditions to avoid early deprotection of the N-oxysuccinamido group. In summary, trisaccharides related to the L-rhamnose-bearing epitopes common to glycoforms I and II of the outer core domain of Pseudomonas aeruginosa lipopolysaccharide have been prepared as their aminooxy glycosides. The latter are expected to be useful in chemoselective oxime-based bioconjugation reactions to form Pseudomonas aeruginosa vaccines.
RESUMEN
Tetrahydrolipstatin (THL, 1a) has been shown to inhibit both mammalian and bacterial α/ß hydrolases. In the case of bacterial systems, THL is a known inhibitor of several Mycobacterium tuberculosis hydrolases involved in mycomembrane biosynthesis. Herein we report a highly efficient eight-step asymmetric synthesis of THL using a route that allows modification of the THL α-chain substituent to afford compounds 1a through 1e. The key transformation in the synthesis was use of a (TPP)CrCl/Co2(CO)8-catalyzed regioselective and stereospecific carbonylation on an advanced epoxide intermediate to yield a trans-ß-lactone. These compounds are modest inhibitors of Ag85A and Ag85C, two α/ß hydrolases of M. tuberculosis involved in the biosynthesis of the mycomembrane. Among these compounds, 10d showed the highest inhibitory effect on Ag85A (34 ± 22 µM) and Ag85C (66 ± 8 µM), and its X-ray structure was solved in complex with Ag85C to 2.5 Å resolution. In contrast, compound 1e exhibited the best-in-class MICs of 50 µM (25 µg/mL) and 16 µM (8.4 µg/mL) against M. smegmatis and M. tuberculosis H37Ra, respectively, using a microtiter assay plate. Combination of 1e with 13 well-established antibiotics synergistically enhanced the potency of few of these antibiotics in M. smegmatis and M. tuberculosis H37Ra. Compound 1e applied at concentrations 4-fold lower than its MIC enhanced the MIC of the synergistic antibiotic by 2-256-fold. In addition to observing synergy with first-line drugs, rifamycin and isoniazid, the MIC of vancomycin against M. tuberculosis H37Ra was 65 µg/mL; however, the MIC was lowered to 0.25 µg/mL in the presence of 2.1 µg/mL 1e demonstrating the potential of targeting mycobacterial hydrolases involved in mycomembrane and peptidoglycan biosynthesis.
Asunto(s)
Mycobacterium tuberculosis , Animales , Antituberculosos/farmacología , Isoniazida , Pruebas de Sensibilidad Microbiana , OrlistatRESUMEN
Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-âº-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose-response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-âº-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)-OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)-OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Ciclohexenos/síntesis química , Glucósidos/síntesis química , Inhibidores de Glicósido Hidrolasas/síntesis química , Glicósido Hidrolasas/química , Hexosaminas/síntesis química , Streptomyces coelicolor/enzimología , Sustitución de Aminoácidos , Aminoácidos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Conformación de Carbohidratos , Dominio Catalítico , Cristalografía por Rayos X , Ciclohexenos/farmacología , Glucósidos/farmacología , Inhibidores de Glicósido Hidrolasas/farmacología , Glicósido Hidrolasas/genética , Hexosaminas/farmacología , Maltosa/química , Modelos Moleculares , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Mutación Puntual , Estereoisomerismo , Streptomyces coelicolor/genéticaRESUMEN
Ebselen (EBS) is an organo-selenium-containing compound that has anti-inflammatory, antitumor, and antibacterial properties. EBS is being explored as a possible treatment for reperfusion injury and stroke and is under clinical trials as a mimetic of lithium for the treatment of bipolar disorder [Mota et al. Synapse 2020, 74 (7), 1-6] and noise-induced hearing loss as a result of these actives [Martini et al. J. Psychiatr. Res. 2019, 109, 107-117. Slusarczyk et al. Neural Regener. Res. 2019, 17 (7), 1255-1261. Thangamani et al. PLoS One 2015, 10 (7), e0133877. Kil et al. Lancet 2017, 390 (10098), 969-979]. However, we wanted to characterize derivatives of EBS as neuroprotective, anti-neuroinflammatory, and antioxidant compounds. Recently, we have reported on a new thermal and photoinduced copper-mediated cross-coupling between potassium selenocyanate (KSeCN) and N-substituted ortho-halobenzamides to form ebselen derivatives with increased synthetic efficiency [Thanna et al. J. Org. Chem. 2017, 82 (7), 3844-3854]. Our synthesis allows for the varying of the remote benzene ring with various substituents or replacing that ring with heterocyclic rings such as pyridine, pyrrole, thiophene, etc. In this study, we synthesized seven new heterocyclic EBS derivatives to further diversify our EBS library. These 21 compounds were then evaluated for their neuroprotective properties, with four compounds showing an equal or better neuroprotective profile than EBS. Compounds 5, 9, 23, and 27 showed 73, 86, 80, 84% cell viability, respectively, at a 10 µM concentration. These studies were performed using human neuroblastoma SH-SY5Y cells in an oxygen and glucose deprivation (OGD) model of ischemia. At the same concentration, these compounds significantly inhibited lipopolysaccharide-induced nitric oxide and tumor necrosis factor alpha release from Human microglia clone 3 microglial cells. Compounds 9 and 27 showed significantly increased cell viability (84 and 80%, respectively) for SH-SY5Y cells exposed to microglia-activated media. These compounds showed only mild GPx-like reductive activity, with compounds 2, 7, 12, and 14 (115, 96, 95, and 82%, respectively) showing a higher percent rate of oxidation of NADPH in a coupled reaction assay compared to ebselen. This research highlights several derivatives of ebselen that show improved activity as neuroprotective agents over the parent compound.
Asunto(s)
Fármacos Neuroprotectores , Compuestos de Organoselenio , Azoles/farmacología , Humanos , Isoindoles , Neuroprotección , Fármacos Neuroprotectores/farmacología , Compuestos de Organoselenio/farmacologíaRESUMEN
A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer , Células Dendríticas/inmunología , Mucina-1 , Ramnosa , Animales , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Femenino , Liposomas , Ratones , Mucina-1/química , Mucina-1/inmunología , Mucina-1/farmacología , Ramnosa/química , Ramnosa/inmunología , Ramnosa/farmacologíaRESUMEN
A focused library of 24 N-aryl urea derivatives was prepared and evaluated against serine esterases of Mycobacterium tuberculosis (Mtb) Rv3802c and Mtb Ag85C. The members of the library were evaluated for both selectivity and mode of inhibition. Furan-based urea derivative 6c was found to be the most potent non-covalent inhibitor of Rv3802c with a K i value of 5.2 ± 0.7 µM. On the other hand, triazole-based ureas 10a and 10b selectively inhibited Ag85C irreversibly with a k inact/K i value of 2.3 ± 0.3 and 5.5 ± 0.4 × 10-3 µM-1 min-1, respectively. The library was also evaluated for minimum inhibitory concentration (MIC) against two strains of Mtb, Mycobacterium smegmatis, and Mycobacterium abscessus. Compounds 4a and 4c were active against Mtb H37Rv mc26206 with MIC values of 3.12 and 1.5 µM, respectively. Closely related 4e showed similar activity against Mtb H37Rv mc26206 but also possessed activity against Mtb H37Ra, Mycobacterium smegmatis and Mycobacterium abscessus. Compounds 4a, 4c, and 4e all contained a common 1-(cyclohexylmethyl)-3-phenylurea motif. In summary, we identified a selective non-covalent inhibitor of Rv3802c and covalently irreversible inhibitors of Ag85C as well as the 1-(cyclohexylmethyl)-3-phenylurea motif which showed activity against a variety of mycobacteria.
RESUMEN
Type 1 diabetes (T1D) is an autoimmune disorder characterized by autoimmune cell mediated destruction of pancreatic beta cells. Pancreatic beta cells are the only source of insulin in the body. T1D patients then have to depend on insulin injections for their lifetime. Insulin injection can modulate the blood sugar levels, but insulin has little effect on the autoimmune process. Altered peptide ligands (APL) derived from known autoantigens in T1D are able to induce tolerance in autoreactive cells in T1D animal models, but are currently unable to elicit this protection in humans. There is a need to improve immunogenicity of the APLs, as these short peptides can be easily degraded by enzymes in the blood. GAD546-554 is a dominant epitope recognized by autoreactive T cells in the nonobese diabetic (NOD) mouse model that can cause destruction of beta cells. Alanine substitution at the eighth position of GAD546-554 peptide (APL9) induced tolerance in a GAD546-554 specific cytotoxic T lymphocyte clone. To improve the antigen presentation and endosomal escape of APL9, we developed a bioconjugate platform that consists of a liposome containing a bioconjugate of APL9 and toll-like receptor 2 ligand Pam3CysSK4 as well as an antibody against macrophage protein F4/80. APL9 bioconjugate liposome with F4/80 antibody was able to induce tolerance in a GAD 546-554 specific clone. Diabetic NOD splenocytes pretreated with APL9 bioconjugate were also not able to transfer diabetes into prediabetic NOD recipient mice. This work is beneficial to prevent T1D as an immunotherapy strategy to render autoreactive immune cells more tolerant of beta cells.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Péptidos/uso terapéutico , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Presentación de Antígeno/efectos de los fármacos , Diabetes Mellitus Tipo 1/inmunología , Femenino , Tolerancia Inmunológica/efectos de los fármacos , Factores Inmunológicos/síntesis química , Factores Inmunológicos/química , Ratones Endogámicos NOD , Péptidos/síntesis química , Péptidos/química , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Targeted delivery of antigens to antigen-presenting cells (APCs) by utilizing natural anticarbohydrate antibodies is a promising approach for selective uptake and enhanced antigen presentation. Previously, we reported that in the presence of a natural antibody, anti-rhamnose antibody (anti-Rha), the bacterial sugar rhamnose conjugated with liposomal cancer antigen MUC1-Tn enhances antigen presentation by APCs such as dendritic cells by targeting Fc gamma receptors. The idea was to utilize the natural human anti-Rha antibodies present in human serum for targeted delivery of cancer-specific antigens. Recently, we found that the IgG3 antibody isotype was the most prevalent anti-Rha antibody generated in mice immunized with rhamnose-Ficoll (Rha-Ficoll) antigen. In this manuscript, we have conjugated the murine IgG3-Fc with a MUC1-containing cancer vaccine and compared the humoral and cellular immune response to this vaccine with one targeted via the human anti-Rha antibody and to the MUC1 vaccine alone. This Fc approach enhanced antibody production and T-cell proliferation almost to the same level as using the anti-Rha antibody. These results suggest that targeting Fc directly to dendritic cells can be an alternative approach to human anti-Rha for generating effective antigen-primed T-cells.
RESUMEN
α,α'-Trehalose plays roles in the synthesis of several cell wall components involved in pathogenic mycobacteria virulence. Its absence in mammalian biochemistry makes trehalose-related biochemical processes potential targets for chemotherapy. The trehalose 6-phosphate synthase (TPS)/trehalose 6-phosphate phosphatase (TPP) pathway, also known as the OtsA/OtsB2 pathway, is the major pathway involved in the production of trehalose in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for Mtb survival. We describe the synthesis of α,α'-trehalose derivatives in the forms of the 6-phosphonic acid 4 (TMP), the 6-methylenephosphonic acid 5 (TEP), and the 6-N-phosphonamide 6 (TNP). These non-hydrolyzable substrate analogues of TPP were examined as inhibitors of Mtb, Mycobacterium lentiflavum (Mlt), and Mycobacterium triplex (Mtx) TPP. In all cases the compounds were most effective in inhibiting Mtx TPP, with TMP [IC50 =(288±32)â µm] acting most strongly, followed by TNP [IC50 =(421±24)â µm] and TEP [IC50 =(1959±261)â µm]. The results also indicate significant differences in the analogue binding profile when comparing Mtb TPP, Mlt TPP, and Mtx TPP homologues.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucosiltransferasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Trehalosa/farmacología , Conformación de Carbohidratos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Glucosiltransferasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Relación Estructura-Actividad , Trehalosa/síntesis química , Trehalosa/químicaRESUMEN
Utilizing natural antibodies to augment vaccine immunogenicity is a promising approach toward cancer immunotherapy. Anti-rhamnose (anti-Rha) antibodies are some of the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. It was shown previously that anti-Rha antibody generated in mice binds effectively with Rha-conjugated vaccine and is picked up by antigen presenting cells (APCs) through stimulatory Fc receptors. This leads to the effective uptake and processing of antigen and eventually presentation by major histocompatibility complex (MHC) molecules. In this article, we show that natural human anti-Rha antibodies can also be used in a similar mechanism and immunogenicity can be enhanced by targeting Rha-conjugated antigens. In doing so, we have purified human anti-Rha antibodies from human serum using a rhamnose affinity column. In vitro, human anti-Rha antibodies are shown to enhance the uptake of a model antigen, Rha-ovalbumin (Rha-Ova), by APCs. In vivo, they improved the priming of CD4+ T cells to Rha-Ova in comparison to non-anti-Rha human antibodies. Additionally, increased priming of both CD4+ and CD8+ T cells toward the cancer antigen MUC1-Tn was observed in mice that received human anti-Rha antibodies prior to vaccination with a rhamnose-modified MUC1-Tn cancer vaccine. The vaccine conjugate contained Pam3CysSK4, a Toll-like receptor (TLR) agonist linked via copper-free cycloaddition chemistry to a 20-amino-acid glycopeptide derived from the tumor marker MUC-1 containing the tumor-associated carbohydrate antigen α- N-acetyl galactosamine (GalNAc). The primed CD8+ T cells released IFN-γ and killed tumor cells. Therefore, we have confirmed that human anti-Rha antibodies can be effectively utilized as a targeting moiety for making an effective vaccine.
Asunto(s)
Anticuerpos/inmunología , Vacunas contra el Cáncer/inmunología , Inmunogenicidad Vacunal , Ramnosa/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Mucina-1/inmunología , Neoplasias/inmunología , Neoplasias/prevención & control , Ovalbúmina/inmunologíaRESUMEN
Oligosaccharide synthesis on organic solvent soluble, high molecular weight poly(2-hydroxyethylmethylacrylate) (pHEMA) is described. The pHEMA-bound oligosaccharide could be recovered after each reaction in 90-95% yield using a precipitation method. The methodology was used to synthesize a model tri-galactoside in 48% overall yield and a trisaccharide from the outer core domain of Pseudomonas aeruginosa lipopolysacchride (LPS) in 39% yield. The use of a photo-cleavable linker is also demonstrated to produce reducing-end protected oligosaccharides.
RESUMEN
The synthesis of a trisaccharide (common to glycoform I and II) and a tetrasaccharide (common to glycoform I) from the outer core domain of Pseudomonas aeruginosa lipopolysaccharide (LPS) using a novel hydroquinone-based reducing-end capping group is reported. This multifunctional capping group was utilized as purification handle and was stable toward many common transformations in oligosaccharide synthesis. The access to outer-core LPS antigens with a TBDPS-protected hydroquinone (TPH) at the reducing end will be useful for glycan array and therapeutic glycoconjugate synthesis.
Asunto(s)
Pseudomonas aeruginosa , Secuencia de Carbohidratos , Hidroquinonas , Lipopolisacáridos , Estructura Molecular , OligosacáridosRESUMEN
Correction for 'Zwitterionic pyrrolidene-phosphonates: inhibitors of the glycoside hydrolase-like phosphorylase Streptomyces coelicolor GlgEI-V279S' by Sri Kumar Veleti et al., Org. Biomol. Chem., 2017, 15, 3884-3891.
RESUMEN
We synthesized and evaluated new zwitterionic inhibitors against glycoside hydrolase-like phosphorylase Streptomyces coelicolor (Sco) GlgEI-V279S which plays a role in α-glucan biosynthesis. Sco GlgEI-V279S serves as a model enzyme for validated anti-tuberculosis (TB) target Mycobacterium tuberculosis (Mtb) GlgE. Pyrrolidine inhibitors 5 and 6 were designed based on transition state considerations and incorporate a phosphonate on the pyrrolidine moiety to expand the interaction network between the inhibitor and the enzyme active site. Compounds 5 and 6 inhibited Sco GlgEI-V279S with Ki = 45 ± 4 µM and 95 ± 16 µM, respectively, and crystal structures of Sco GlgE-V279S-5 and Sco GlgE-V279S-6 were obtained at a 3.2 Å and 2.5 Å resolution, respectively.
Asunto(s)
Glicósido Hidrolasas/antagonistas & inhibidores , Organofosfonatos/química , Fosforilasas/antagonistas & inhibidores , Pirroles/química , Pirroles/farmacología , Streptomyces coelicolor/enzimología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Fosforilasas/química , Conformación ProteicaRESUMEN
Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis (Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitor orientation that were unobserved in previous Ag85C ebselen structures. The kinact/KI values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein-inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein-inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Furthermore, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.