Final Report Summary - PGN-INNATE (Molecular recognition of bacterial cell wall peptidoglycans and related molecules by innate immune receptors) Peptidoglycan (PGN), a major component of bacterial cell walls, is increasingly recognized as an important danger signal being able to activate host immune responses. Danger signals are molecular fragments used by the immune system to detect pathogens or cell damage by triggering receptors of the innate immunity. As a result, activators of the innate immune system are potent immune modulators, which form the basis of designing new immune modulators that may be used either activate or suppress immune response. Such immunomodulators are urgently needed in many medical interventions, such as in vaccines or in aiding cancer immunotherapy. The investigations carried within the project are aimed at improving peptidoglycan based immunomodulators, which could be used as vaccine adjuvants or in other disease areas.During the project we focused on two major groups of PGN derivatives. First, we investigated Dap-type Peptidoglycan Monomer (PGM) isolated from the cell wall of the Gram-negative Brevibacterium divaricatum and its mannosylated derivative, ManPGM as well as their fragment, Peptidoglycan Pentapeptide, PP, and its mannosylated version, ManPP. Secondly, we investigated drug delivery formulations of adamantylated PGN derivatives, which are adamantylated tripeptides containing contain a minimal PGN fragment, L-Ala-D-isoGln with D and L chirality at the adamantly group attachment and their mannosylated conjugates. We investigated interactions of PGM and its mannosylated derivative, ManPGM and their pentapeptide fragments, PP and ManPP, with the soluble part of the whole length human Macrophage Mannose Receptor (hMMR) using NMR spectroscopy and molecular modelling. The parent compound containing a disaccharide-pentapeptide was found to possess weak binding affinity primarily via its GlcNAc residue to hMMR, while its pentapeptide fragment, PP, has shown marginal association. Upon mannosylation the pentapeptide fragment, ManPP gained stronger affinity to hMMR than PP or that of the mannosylated pentapeptide-disaccharide, ManPGM. Mapping of the binding epitope, docking calculations and molecular dynamics have shown that ManPP binds only via the mannosyl group, while multiple binding modes are involved in the association of ManPGM to a single lectin domain. Diminished immunostimulating properties of the mannosylated derivative compared with the parent compound, as observed in vivo in mice models, could be related to the weak association of ManPGM to hMMR, whereby the latter is involved in the clearing mannose containing glycoproteins and restoring homeostasis, thus downscaling the host immune response. We obtained a Toll-like Receptor 2 (TLR2) construct containing the immune active fragment binding Leucine Rich Repeat (LRR) domain. We investigated the interactions of the PGM with the LRR-TLR2 construct to identify binding events using ligand detected NMR spectroscopic methods. We applied Saturation Transfer Difference (STD) spectroscopy, WaterLogsy, spin-spin relaxation techniques and 2D NOESY experiments. We detected STD signals and strong, negative NOE cross peaks in the 2D NOESY spectrum for the PGM-LRR-TLR2 system, which indicated presence of a weak binding event. However, the pentapeptide fragment of PGM, PP, did not produce these signals with the LRR domain of TLR2, thus we concluded that the disaccharide part of PGM is likely necessary for binding. We developed a molecular mechanics-based description of the PGN derivatives, which is required for modelling their interactions with innate immune receptors and enable investigations of their conformational properties. Since the derivatives contain multiple non-standard fragments and linkages, definition of these molecules for a molecular mechanics simulation engine is not trivial. We generated library files containing molecular topology and parameters of the molecular mechanics energy terms in the AMBER SB14 GLYCAM H06 force field. We also recalculated partial atomic charges using ab initio methods. The resulting library files enabled modelling of conformational and dynamic properties of the PGN derivatives in aqueous solution or in organic solvents. In parallel, we carried out an NMR spectroscopic investigation of the conformational properties of the PGN derivatives in water and dimethyl-sulfoxide solvent. We determined NMR parameters that are related to geometrical descriptors providing information on the local and global molecular conformations. Specifically, we measured Nuclear Overhauser Effects, which are dependent on interproton distances; homo- and heteronuclear three bond vicinal coupling constants, which can be converted into torsion angles describing local arrangement of atoms around single bonds; temperature coefficients of amide group chemical shifts that are related to solvent exposure of hydrogen atoms and finally translational diffusion coefficients, which is affected by size and compactness of molecules, thus can be used to describe global conformational behaviour. Translational diffusion coefficients were also converted into hydrodynamic radii using the Einstein-Stokes equation.Using the developed the molecular mechanics parameter and topology files in the AMBER-GLYCAM force field, we performed multiple molecular dynamics (MD) simulations. We carried out Principal Component Analysis to access the convergence of the simulations, that is the completeness of the sampling of the conformational space during the simulations. We concluded that simulations with 1 µs length explored complementary conformations of the derivatives, thus longer time scales or simulations with enhanced sampling are required. For this reason, we also executed Gaussian Accelerated MD (GAMD) calculations. We compared molecular descriptors derived from NMR parameters with corresponding descriptors extracted from GAMD simulations and used them to validate the molecular mechanics force field developed for the PGN derivatives. Finally, we analysed the conformational behaviour of the PGN derivatives as seen in the MD simulations by performing cluster analysis, analysis of the hydrogen bonding patterns, salt bridges and Van Der Waals interactions. Furthermore, in order to help the design of improved PGN-based immunemodulators, it is of interest to investigate their delivery formulations. We studied interactions of PGN derivatives specifically designed for lipid incorporation with components of a liposome-based delivery systems using NMR spectroscopy at the molecular level. These derivatives contain a minimal PGN dipeptide fragment as a cargo, an adamantyl group that functions as an anchor to the lipid bilayer and a mannose group for active targeting of surface receptors on immune cells. We have found a surface association of the derivatives with the lipid bilayer in equilibrium with the aqueous solution. The lipid bilayer incorporation was close to 50% with stronger encapsulation efficiency for the adamantylated compounds than for the adamantylated mannosylated derivatives. The chirality of the adamantyl linker also influenced the incorporation efficiency to a lesser extent. The adamantylated derivatives were found to reside on the surface, while the derivatives with the mannose targeting group penetrate deeper in the bilayer. The data obtained indicates that the membrane encapsulation is primarily dominated by strong electrostatic interactions between charged termini of the compounds and the zwitterionic lipid molecules. On the basis of the findings, we suggested modifications to the derivatives to improve lipid incorporation efficiency and availability of the mannose-targeting group by cell surface receptors.We also launched studies on the immune stimulatory properties the adamantylated PGN derivatives alone and in a lipid nanoparticle-based delivery system using cell and molecular biological methods. Immune stimulation was detected by interleukine 6 and Rantes chemokine expression using in vitro cell assays on immortalized mouse bone-marrow derived macrophages. Macrophages in the in vitro cell assay were stimulated by lipopolysaccharide (LPS), which is commonly used for induction of inflammation and production of pro-inflammatory cytokines and chemokines. LPS is an activator of innate immune responses acting as danger signal for the Toll-like receptor 4.The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl centre, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchoring the PGN fragments into lipid nanoparticles. In summary, during the project we have gained new insights into the interactions of peptidoglycan with innate immune receptors as well as with lipid-based carriers. The understanding of the molecular details of the interactions will facilitate design of novel peptidoglycan based immune modulators and their delivery systems. Project website: https://nmr.science.unideb.hu/KF/PGN.html
