Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.NWO15.002.Metals in Catalysis, Biomimetics & Inorganic MaterialsBio-organic Synthesi

Effective Melanoma Immunotherapy in Mice by the Skin-Depigmenting Agent Monobenzone and the Adjuvants Imiquimod and CpG

Background: Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity. Methodology and Principal Findings: We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16. F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation. Conclusions: MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B-and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clini

Synthesis of Unsymmetrical Difluoromethylene Bisphosphonates

We demonstrate the use of the symmetrical diethyl(dimethyl)difluoromethylene bisphosphonate reagent for the synthesis of terminal and unsymmetrical difluoromethylene bisphosphonates, close analogues of biologically important molecules. The difference in reactivity of the methyl and ethyl groups in the symmetrical diethyl(dimthyl)difluoromethylene bisphosphonate is exploited in a stepwise demethylation-condensation sequence to functionalize either side of the reagent to allow the generation of a series of close bioisosteres of natural pyrophosphate molecules, including ADPr, CDP-glycerol and CDP-ribitol.</p

(Automated) Synthesis of Well-defined Staphylococcus Aureus Wall Teichoic Acid Fragments

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; β-GlcNAc at the RboP C4; β-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains

A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene

International audienceDNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler’s ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain’s interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1’s auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation

Stereoselectivity in the Lewis Acid Mediated Reduction of Ketofuranoses

The Lewis acid mediated reduction of ribose-, arabinose-, xylose-, and lyxose-derived methyl and phenyl ketofuranoses with triethylsilane as nucleophile was found to proceed with good to excellent stereoselectivity to provide the 1,2-<i>cis</i> addition products. The methyl ketoses reacted in a more stereoselective manner than their phenyl counterparts. The stereochemical outcome of the reactions parallels the relative stability of the oxocarbenium ion conformers involved, as assessed by calculating the free energy surface maps of their complete conformational space. The Lewis acid mediated reduction allows for a direct synthesis of <i>C</i>-glycosides with predictable stereochemistry