Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes.

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.

Synthesis and Immunological Evaluation of a Single Molecular Construct MUC1 Vaccine Containing l-Rhamnose Repeating Units.

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.

Synthesis and in Vitro Characterization of Trehalose-Based Inhibitors of Mycobacterial Trehalose 6-Phosphate Phosphatases.

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) µm] acting most strongly, followed by TNP [IC50 =(421±24) µm] and TEP [IC50 =(1959±261) µm]. The results also indicate significant differences in the analogue binding profile when comparing Mtb TPP, Mlt TPP, and Mtx TPP homologues.

Development of a Bioconjugate Platform for Modifying the Immune Response of Autoreactive Cytotoxic T Lymphocytes Involved in Type 1 Diabetes.

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.

Thermal and Photoinduced Copper-Promoted C-Se Bond Formation: Synthesis of 2-Alkyl-1,2-benzisoselenazol-3(2H)-ones and Evaluation against Mycobacterium tuberculosis.

2-Alkyl-1,2-benzisoselenazol-3(2H)-ones, represented by ebselen (1a), are being studied intensively for a range of medicinal applications. We describe both a new thermal and photoinduced copper-mediated cross-coupling between potassium selenocyanate (KSeCN) and N-substituted ortho-halobenzamides to form 2-alkyl-1,2-benzisoselenazol-3(2H)-ones containing a C-Se-N bond. The copper ligand (1,10-phenanthroline) facilitates C-Se bond formation during heating via a mechanism that likely involves atom transfer (AT), whereas, in the absence of ligand, photoinduced activation likely proceeds through a single electron transfer (SET) mechanism. A library of 15 2-alkyl-1,2-benzisoselenazol-3(2H)-ones was prepared. One member of the library was azide-containing derivative 1j that was competent to undergo a strain-promoted azide-alkyne cycloaddition. The library was evaluated for inhibition of Mycobacterium tuberculosis (Mtb) growth and Mtb Antigen 85C (Mtb Ag85C) activity. Compound 1f was most potent with a minimal inhibitory concentration (MIC) of 12.5 µg/mL and an Mtb Ag85C apparent IC50 of 8.8 µM.

Correction: Zwitterionic pyrrolidene-phosphonates: inhibitors of the glycoside hydrolase-like phosphorylase Streptomyces coelicolor GlgEI-V279S.

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.

Zwitterionic pyrrolidene-phosphonates: inhibitors of the glycoside hydrolase-like phosphorylase Streptomyces coelicolor GlgEI-V279S.

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.

Synthesis of a Liposomal MUC1 Glycopeptide-Based Immunotherapeutic and Evaluation of the Effect of l-Rhamnose Targeting on Cellular Immune Responses.

Generation of a CD8(+) response to extracellular antigen requires processing of the antigen by antigen presenting cells (APC) and cross-presentation to CD8(+) T cell receptors via MHC class I molecules. Cross-presentation is facilitated by efficient antigen uptake followed by immune-complex-mediated maturation of the APCs. We hypothesize that improved antigen uptake of a glycopeptide sequence containing a CD8(+) T cell epitope could be achieved by delivering it on a liposome surface decorated with an immune complex-targeting ligand, an l-Rhamnose (Rha) epitope. We synthesized a 20-amino-acid glycopeptide TSAPDT(GalNAc)RPAPGSTAPPAHGV from the variable number tandem repeat region of the tumor marker MUC1 containing an N-terminal azido moiety and a tumor-associated α-N-acetyl galactosamine (GalNAc) at the immunogenic DTR motif. The MUC1 antigen was attached to Pam3Cys, a Toll-like receptor-2 ligand via copper(I)-catalyzed azido-alkyne cycloaddition (CuAAc) chemistry. The Rha-decorated liposomal Pam3Cys-MUC1-Tn 4 vaccine was evaluated in groups of C57BL/6 mice. Some groups were previously immunized to generate anti-Rha antibodies. Anti-Rha antibody expressing mice that received the Rha liposomal vaccine showed higher cellular immunogenicity compared to the control group while maintaining a strong humoral response.

Synthesis and evaluation of new 2-aminothiophenes against Mycobacterium tuberculosis.

Tuberculosis (TB) and its drug resistant forms kills more people than any other infectious disease. This fact emphasizes the need to identify new drugs to treat TB. 2-Aminothiophenes (2AT) have been reported to inhibit Pks13, a validated anti-TB drug target. We synthesized a library of 42 2AT compounds. Among these, compound 33 showed remarkable potency against Mycobacterium tuberculosis (Mtb) H37RV (MIC = 0.23 µM) and showed an impressive potency (MIC = 0.20-0.44 µM) against Mtb strains resistant to isoniazid, rifampicin and fluoroquinolones. The site of action for the compound 33 is presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based on structural similarity of the compound 33 with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies showing that the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies suggest 33 represents a promising anti-TB lead that exhibits activity well below toxicity to human monocytic cells.

Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S.

Streptomyces coelicolor (Sco) GlgEI is a glycoside hydrolase involved in α-glucan biosynthesis and can be used as a model enzyme for structure-based inhibitor design targeting Mycobacterium tuberculosis (Mtb) GlgE. The latter is a genetically validated drug target for the development of anti-Tuberculosis (TB) treatments. Inhibition of Mtb GlgE results in a lethal buildup of the GlgE substrate maltose-1-phosphate (M1P). However, Mtb GlgE is difficult to crystallize and affords lower resolution X-ray structures. Sco GlgEI-V279S on the other hand crystallizes readily, produces high resolution X-ray data, and has active site topology identical to Mtb GlgE. We report the X-ray structure of Sco GlgEI-V279S in complex with 2-deoxy-2,2-difluoro-α-maltosyl fluoride (α-MTF, 5) at 2.3 Å resolution. α-MTF was designed as a non-hydrolysable mimic of M1P to probe the active site of GlgE1 prior to covalent bond formation without disruption of catalytic residues. The α-MTF complex revealed hydrogen bonding between Glu423 and the C1F which provides evidence that Glu423 functions as proton donor during catalysis. Further, hydrogen bonding between Arg392 and the axial C2 difluoromethylene moiety of α-MTF was observed suggesting that the C2 position tolerates substitution with hydrogen bond acceptors. The key step in the synthesis of α-MDF was transformation of peracetylated 2-fluoro-maltal 1 into peracetylated 2,2-difluoro-α-maltosyl fluoride 2 in a single step via the use of Selectfluor®.

Correction: Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S.

Correction for 'Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S' by Sandeep Thanna et al., Org. Biomol. Chem., 2015, DOI: 10.1039/c5ob00867k.

Synthesis of a poly-hydroxypyrolidine-based inhibitor of Mycobacterium tuberculosis GlgE.

Long treatment times, poor drug compliance, and natural selection during treatment of Mycobacterium tuberculosis (Mtb) have given rise to extensively drug-resistant tuberculosis (XDR-TB). As a result, there is a need to identify new antituberculosis drug targets. Mtb GlgE is a maltosyl transferase involved in α-glucan biosynthesis. Mutation of GlgE in Mtb increases the concentration of maltose-1-phosphate (M1P), one substrate for GlgE, causing rapid cell death. We have designed 2,5-dideoxy-3-O-α-d-glucopyranosyl-2,5-imino-d-mannitol (9) to act as an inhibitor of GlgE. Compound 9 was synthesized using a convergent synthesis by coupling thioglycosyl donor 14 and 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-ß-d-fructopyranose (23) to form disaccharide 24. A reduction and intramolecular reductive amination transformed the intermediate disaccharide 24 to the desired pyrolidine 9. Compound 9 inhibited both Mtb GlgE and a variant of Streptomyces coelicolor (Sco) GlgEI with Ki = 237 ± 27 µM and Ki = 102 ± 7.52 µM, respectively. The results confirm that a Sco GlgE-V279S variant can be used as a model for Mtb GlgE. In conclusion, we designed a lead transition state inhibitor of GlgE, which will be instrumental in further elucidation of the enzymatic mechanism of Mtb GlgE.

Synthesis of α-L-rhamnosyl ceramide and evaluation of its binding with anti-rhamnose antibodies.

An α-L-rhamnosyl ceramide (1, α-L-RhaCer) has been prepared that was recognized by anti-L-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-L-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-L-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acetyl-1-(2,2,2-trichloroethanimidate)-α-L-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition-fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-L-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-ovalbumin (Rha-Ova) with Sigma Adjuvant System (SAS) and the anti-L-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-L-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies.

Synthesis of a C-phosphonate mimic of maltose-1-phosphate and inhibition studies on Mycobacterium tuberculosis GlgE.

The emergence of extensively drug-resistant tuberculosis (XDR-TB) necessitates the need to identify new anti-tuberculosis drug targets as well as to better understand essential biosynthetic pathways. GlgE is a Mycobacterium tuberculosis (Mtb) encoded maltosyltransferase involved in α-glucan biosynthesis. Deletion of GlgE in Mtb results in the accumulation of M1P within cells leading to rapid death of the organism. To inhibit GlgE a maltose-C-phosphonate (MCP) 13 was designed to act as an isosteric non-hydrolysable mimic of M1P. MCP 13, the only known inhibitor of Mtb GlgE, was successfully synthesized using a Wittig olefination as a key step in transforming maltose to the desired product. MCP 13 inhibited Mtb GlgE with an IC50=230 ± 24 µM determined using a coupled enzyme assay which measures orthophosphate release. The requirement of M1P for the assay necessitated the development of an expedited synthetic route to M1P from an intermediate used in the MCP 13 synthesis. In conclusion, we designed a substrate analogue of M1P that is the first to exhibit Mtb GlgE inhibition.

Bio-Based Bisfuran: Synthesis, Crystal Structure and Low Molecular Weight Amorphous Polyester.

Discovery of renewable monomer feedstocks for fabrication of polymeric demand is critical in achieving sustainable materials. In the present work we have synthesized bisfuran diol (BFD) monomer from furfural, over four steps. BFD was examined via X-ray crystallography to understand the molecular arrangement in space, hydrogen bonding and packing of the molecules. This data was further used to compare BFD with structurally related Bisphenol A (BPA), and its known derivatives to predict the potential estrogenic or anti-estrogenic activities in BFD. Further, BFD was reacted with succinic acid to generate polyester material, bisfuran polyester (BFPE-1). MALDI characterization of BFPE-1 indicates low molecular weight polyester and thermal analysis reveals amorphous nature of the material.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

Design, synthesis, and X-ray analysis of a glycoconjugate bound to Mycobacterium tuberculosis antigen 85C.

Tuberculosis (TB) is a global health threat with nearly 500 000 new cases of multidrug-resistant TB estimated to occur every year, so new drugs are desperately needed. A number of current antimycobacterial drugs work by interfering with the biosynthesis of key components of the mycolylarabinogalactan (mAG). In light of this observation, other enzymes involved in the synthesis of the mAG should also serve as targets for antimycobacterial drug development. One potential target is the Antigen 85 (Ag85) complex, a family of mycolyltransferases that are responsible for the transfer of mycolic acids from trehalose monomycolate (TMM) to the arabinogalactan. Virtual thiophenyl-arabinoside conjugates were docked to antigen Ag85C (PDB code: 1va5 ) using Glide. Compounds with good docking scores were synthesized by a Gewald synthesis followed by linking to 5-thioarabinofuranosides. The resulting thiophenyl-thioarabinofuranosides were assayed for inhibition of mycoyltransferase activity using a 4-methylumbelliferyl butyrate fluorescence assay. The conjugates showed K(i) values ranging from 18.2 to 71.0 µM. The most potent inhibitor was soaked into crystals of Mycobacterium tuberculosis antigen 85C and the structure of the complex determined. The X-ray structure shows the compound bound within the active site of the enzyme with the thiophene moiety positioned in the putative α-chain binding site of TMM and the arabinofuranoside moiety within the known carbohydrate-binding site as exhibited for the Ag85B-trehalose crystal structure. Unexpectedly, no specific hydrogen bonding interactions are being formed between the arabinofuranoside and the carbohydrate-binding site of the active site suggesting that the binding of the arabinoside within this structure is driven by shape complementarily between the arabinosyl moiety and the carbohydrate binding site.

Synthesis and Evaluation of Marine Natural Product-Inspired Meroterpenoids with Selective Activity toward Dormant Mycobacterium tuberculosis.

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.

Synthesis of C7/C8-cyclitols and C7N-aminocyclitols from maltose and X-ray crystal structure of Streptomyces coelicolor GlgEI V279S in a complex with an amylostatin GXG-like derivative.

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-⍺-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.

Evaluation of Derivatives of (+)-Puupehenone against Clostridioides difficile and Other Gram-Positive Bacteria.

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.