Estudios computacionales de mecanismos moleculares de la inmunidad innata

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 20-12-2022Antimicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity, which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of bacterial lipopolysaccharide (LPS), and the phospholipid composition of the membrane, inflecting the membrane permeability to antibiotics. We have applied coarse-grained molecular dynamics simulations to capture the role of the phospholipid composition and lipid A structure in the membrane properties and morphology of ESKAPE Gram-negative bacterial vesicles. Moreover, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf were used to unveil their implications for membrane disruption. This study opens a promising starting point for understanding the molecular keys of bacterial membranes and promoting the discovery of new antimicrobials to overcome AMR...La resistencia a los antimicrobianos (AMR) es una emergencia sanitaria mundial. Los patógenos ESKAPE incluyen las familias bacterianas más resistentes a antibióticos y son altamente virulentas. En particular, las bacterias Gram negativas destacan por la complejidad de su pared celular, que presenta una fuerte resistencia frente a los antibióticos. Un elemento clave para la AMR es la estructura química del lipopolisacárido bacteriano (LPS) y la composición de los fosfolípidos de la membrana bacteriana, que influyen en su permeabilidad a los antibióticos. Se han empleado simulaciones de dinámica molecular de grano grueso para captar el papel de la composición de los fosfolípidos y la estructura del LPS en las propiedades y morfología de modelos de vesículas bacterianas Gram negativas ESKAPE. Además, se han empleado los péptidos antimicrobianos Cecropin B1, JB95 y PTCDA1-kf para desvelar su mecanismo disrupción de la membrana bacteriana. Este estudio abre un prometedor punto de partida para comprender las claves moleculares de la resistencia en membranas bacterianas y acelerar el descubrimiento de nuevos antibióticos para hacer frente a la AMR...Fac. de FarmaciaTRUEunpu

Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA

New glucosamine-based TLR4 agonists: design, synthesis, mechanism of action, and in vivo activity as vaccine adjuvants

20 p.-15 fig.-1 graph. abst.We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.The authors acknowledge the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie, project BactiVax (www.bactivax.eu) grant agreement no. 860325; the consortium CINMPIS; the project of excellence CHRONOS, CHRonical multifactorial disorders explored by NOvel integrated Strategies of the Department of Biotechnology and Biosciences; the Agencia Estatal de Investigacion (Spain) for project PID2021-126130OB-I00 (N.G.A.A.), PID2020-113588RB-I00 (S.M.-S.), PRE2018-086249 (A.M.-R), PRE2021-097247 (M.M.-T.); and project FEDER MINECO, the EM-platform at the CIC bioGUNE for support in cryo-EM imaging. J.J.-B. also thanks funding by CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Perkin-Elmer Italia is also acknowledged for providing the cell imaging reagents.Peer reviewe

Computational approaches to the dynamics and activation mechanism of Toll-like receptor 4

1 p.Toll-like receptors (TLRs) are pattern recognition receptors involved in innate immunity. In particular, TLR4 binds to lipopolysaccharides (LPS), a membrane constituent of Gram-negative bacteria and, together with MD-2 protein, forms a heterodimeric complex which leads to the activation of the innate immune system response. TLR4 activation has been associated with certain autoimmune diseases, noninfectious inflammatory disorders, and neuropathic pain,suggesting a wide range of possible clinical settings for the application of TLR4 antagonists,while TLR4 agonists would be useful as adjuvants in vaccine development and in cancer immunotherapy.[1,2] Specific molecular features of extracellular, transmembrane, and cytoplasmic domains of TLR4 are crucial for coordinating the complex innate immune signaling pathway. Although structural and biochemical data is currently available for the independent TLR4 domains, this only provides a partial fragmented view, because full-length proteins are flexible entities and dynamics play a key role in their functionality. Therefore, many structural and dynamical features of the TLR4 mode of action remain largely unknown.[3]Computational studies of the different independent domains composing the TLR4 were undertaken, using ab-initio calculations, homology modeling, protein-protein docking, all-atom molecular dynamics simulations, and thermodynamics calculations, to understand the differential domain organization of TLR4. From the information gathered from our independent TLR4 domains studies, we have modeled, by all-atom MD simulations, the structural assembly of plausible full-length TLR4 models embedded into realistic plasma membranes, with different chemical compositions, accounting for the active (agonist) state of the TLR4. We have also applied computational techniques to characterize, at the atomic level, the molecular recognition processes by reported TLR4 modulators, thus proposing a mechanism for their biological activity. These observations unveil relevant molecular aspects involved in the mechanism of receptor activation, and adaptor recruitment in the innate immune pathways, and will promote the discovery of new TLR4 modulators and probes.Peer reviewe

Modeling of transmembrane domain and full-length TLRs in membrane models

36 p.-5 fig.Toll-like receptors (TLRs), classified as pattern recognition receptors, have a primordial role in the activation of the innate immunity. In particular, TLR4 binds to lipopolysaccharides (LPS), a membrane constituent of Gram-negative bacteria, and, together with Myeloid Differentiation factor 2 (MD-2) protein, forms a heterodimeric complex which leads to the activation of the innate immune system response. Identification of TLRs has sparked great interest in the therapeutic manipulation of the innate immune system. In particular, TLR4 antagonists may be useful for the treatment of septic shock, certain autoimmune diseases, noninfectious inflammatory disorders, and neuropathic pain, and TLR4 agonists are under development as vaccine adjuvants in antitumoral treatments. Therefore, TLR4 has risen as a promising therapeutic target, and its modulation constitutes a highly relevant and active research area. Deep structural understanding of TLR4 signaling may help in the design and discovery of TLR4-modulating molecules with desirable therapeutic properties. Computational studies of the different independent domains composing the TLR4 were undertaken, to understand the differential domain organization of TLR4 in aqueous and membrane environments, including Liquid-disordered (Ld) and Liquid-ordered (Lo) membrane models, to account for the TLR4 recruitment in lipid rafts over activation. We modeled, by means of all-atom Molecular Dynamics (MD) simulations, the structural assembly of plausible full-length TLR4 models embedded into a realistic plasma membrane, accounting for the active (agonist) state of the TLR4, thus providing an analysis at both atomic/molecular and thermodynamic levels of the TLR4 assembly and biological activity. Our results unveil relevant molecular aspects involved in the mechanism of receptor activation, and adaptor recruitment in the innate immune pathways, and will promote the discovery of new TLR4 modulators and probes.This work was financially supported by the Spanish Ministry for Science and Innovation (grants PID2020-113588RB-I00, PRE2018-086249 for A.M.R. and PRE2021-097247 for M.M.T.).Peer reviewe

Immune evasion through Toll-like receptor 4: The role of the core oligosaccharides from α2-Proteobacteria atypical lipopolysaccharides

Lipopolysaccharides (LPS) are major players in bacterial infection through the recognition by Toll-like receptor 4 (TLR4). The LPS chemical structure, including the oligosaccharide core and the lipid A moiety, can be strongly influenced by adaptation and modulated to assure bacteria protection, evade immune surveillance, or reduce host immune responses. Deep structural understanding of TLRs signaling is essential for the modulation of the innate immune system in sepsis control and inflammation, during bacterial infection. To advance this knowledge, we have employed computational techniques to characterize the TLR4 molecular recognition of atypical LPSs from different opportunistic members of α2-Proteobacteria, including Brucella melitensis, Ochrobactrum anthropi, and Ochrobactrum intermedium, with diverse immunostimulatory activities. We contribute to unraveling the role of uncommon lipid A chemical features such as bearing very long-chain fatty acid chains, whose presence has been rarely reported, on modulating the proper heterodimerization of the TLR4 receptor complex. Moreover, we further evaluated the influence of the different oligosaccharide cores, including sugar composition and net charge, on TLR4 activation. Our studies contribute to elucidating, from the molecular and biological perspectives, the impact of the α2-Proteobacteria LPS cores and the chemical structure of the atypical lipid A for immune system evasion in opportunistic bacteria.This work was financially supported by the Spanish Ministry for Science and Innovation (grants CTQ2017-88353-R, and PID2020-113588RB-I00 to S.M.S.; grant PRE2018-086249 to A.M.R., and SAF2016-75988 to M.F.), Instituto de Salud Carlos III (RD16/0027/0006), European Commission – NextGenerationEU (Regulation EU 2020/2094) through CSIC's Global Health Platform (PTI Salud Global), Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM) and from Laboratorios Esteve to M.F. and institutional grants from “Fundación Ramón Areces” and “Banco de Santander” to CBMSO. Endless support from S.H.J. is sincerely acknowledged.Peer reviewe

Lipid‑A‑dependent and cholesterol‑dependent dynamics properties of liposomes from gram‑negative bacteria in ESKAPE

15 p.-9 fig.AntiMicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of lipid A, modulating the physico-chemical properties of the membrane and permeability to antibiotics. Liposomes are used as models of bacterial membrane infective vesicles. In this work, coarse-grained molecular dynamics simulations were used to model liposomes from ESKAPE Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa). We captured the role of lipid A, cardiolipin and cholesterol on liposome morphology and physico-chemical properties. Additionally, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf, were used to unveil their implications on membrane disruption. This study opens a promising starting point to understand molecular keys of bacterial membranes and to promote the discovery of new antimicrobials to overcome AMR.This work was financially supported by the Spanish Ministry for Science and Innovation (grants CTQ2017-88353-R, and PID2020-113588RB-I00; grant PRE2018-086249 for A.M.R.) and RES-BSC QSB-2020-2-0017.Peer reviewe

Understanding the antibacterial resistance: computational explorations in bacterial membranes

14 p.-8 fig.-1 graph. abst.Antimicrobial resistance (AMR) represents a major threat to global public health in the 21st century, dramatically increasing the pandemic expectations in the coming years. The ongoing need to develop new antimicrobial treatments that are effective against multi-drug-resistant pathogens has led the research community to investigate innovative strategies to tackle AMR. The bacterial cell envelope has been identified as one of the key molecular players responsible for antibiotic resistance, attracting considerable interest as a potential target for novel antimicrobials effective against AMR, to be used alone or in combination with other drugs. However, the multicomponent complexity of bacterial membranes provides a heterogeneous morphology, which is typically difficult to study at the molecular level by experimental techniques, in spite of the significant development of fast and efficient experimental protocols. In recent years, computational modeling, in particular, molecular dynamics simulations, has proven to be an effective tool to reveal key aspects in the architecture and membrane organization of bacterial cell walls. Here, after a general overview about bacterial membranes, AMR mechanisms, and experimental approaches to study AMR, we review the state-of-the-art computational approaches to investigate bacterial AMR envelopes, including their limitations and challenges ahead. Representative examples illustrate how these techniques improve our understanding of bacterial membrane resistance mechanisms, hopefully leading to the development of novel antimicrobial drugs escaping from bacterial resistance strategies.This work was financially supported by the Spanish Ministry for Science and Innovation (Grant Nos. CTQ2017-88353-R and PRE2018-086249 to A.M.R) and RES-BSC QSB-2020-2-0017. FSE, PON Ricerca e Innovazione 2014-2020, Azione I.1 “Dottorati Innovativi con caratterizzazione Industriale” is acknowledged for funding the Ph.D. grant to R.E.F. S.H.J. is gratefully acknowledged for his relentless support.Peer reviewe

Full-atom model of the agonist LPS-bound toll-like receptor 4 dimer in a membrane environment

21 p.-14 fig. In Memoriam Prof. Kilian Muñiz (R.I.P.). Excellent person, great heart.The innate immunity TLR4/MD-2 system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram-negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We here combine ab-initio calculations, molecular docking, all-atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies reveal functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular TIR domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD-2/LPS) 2 architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators.This work was financially supported by the Spanish Ministry for Science and Innovation (grants CTQ2014-57141-R, CTQ2017-88353-R, and PID2020-113588RB-I00; grants BES-2012-053653 for L.P.R., BES-2015-071588 for J.G.C. and PRE2018-086249 for A.M.R.), and the European Commission Marie Sklodowska-Curie actions (H2020-MSCA-ITN 642157 “TOLLerant”).Peer reviewe