Structural aspects of molecular recognition in the immune system. Part I: Acquired immunity (IUPAC Technical Report)

Humoral immunity allows the body to mount a defense against pathogens and foreign substances, and to respond with memory to subsequent exposures. The molecular participants may also recognize self-structures, leading to attack on the body and autoimmune disease. The main players in humoral immunity are antibody-producing B lymphocytes, and several classes of T lymphocytes. This review deals with the molecular details of recognition of antigens by soluble antibodies, and of substances presented to receptors on the surfaces of T cells (TCRs). The prototype antibody consists of a dimer of dimers, two heavy (H) chains and two light (L) chains, with antigen recognition capacity lying in variable "head” regions of an H-L pair. Most crystallographic studies are done with this substructure, called a Fab fragment, bound in a soluble antigen complex. Homologous to this arrangement, the prototype TCR consists of two chains (α and β) that complex not soluble antigen, but usually a short peptide or other small molecule presented by proteins of the major histocompatibility complex. In each case a general background on the historical development of understanding the molecular recognition interface is given, followed by a number of examples of crystal structures from the recent literature that have allowed us to refine our understanding of the complex recognition process. Variations on the prototypical structures are also considered. The spectrum of recognition strategies involves interplay of lock-and-key with flexibility, varying degrees of entropic and enthalpic contributions, surface shaping by entrapped water molecules, and combinations of stabilizing hydrogen bonding, electrostatic interactions, salt bridging, and van der Waals forces. Preeminent in the recent literature are details of antibody binding to influenza A and human immunodeficiency viral antigens. Both viral antigens and attempts to understand autoimmunity are prominent in the recent TCR literatur

Structural aspects of molecular recognition in the immune system. Part II: Pattern recognition receptors (IUPAC Technical Report)

The vertebrate immune system uses pattern recognition receptors (PRRs) to detect a large variety of molecular signatures (pathogen-associated molecular patterns, PAMPs) from a broad range of different invading pathogens. The PAMPs range in size from relatively small molecules, to others of intermediate size such as bacterial lipopolysaccharide, lipopeptides, and oligosaccharides, to macromolecules such as viral DNA, RNA, and pathogen-derived proteins such as flagellin. Underlying this functional diversity of PRRs is a surprisingly small number of structurally distinct protein folds that include leucine-rich repeats in Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the DExH box helicase domain in RIG-like receptors (RLRs), and C-type lectin domains (CTLDs) in the C-type lectins. Following PAMP recognition by the PRRs, downstream signaling pathways activate the innate immune system to respond to invading pathogenic organisms. The resulting stimulatory response is also vital for a balanced adaptive immune response to the pathogen, mediated by circulating antibodies and/or cytotoxic T cells. However, an aberrant stimulation of the innate immune system can also lead to excessive inflammatory and toxic stress responses. Exciting opportunities are now arising for the design of small synthetic molecules that bind to PRRs and influence downstream signaling pathways. Such molecules can be useful tools to modulate immune responses, for example, as adjuvants to stimulate adaptive immune responses to a vaccine, or as therapeutic agents to dampen aberrant immune responses, such as inflammation. The design of agonists or antagonists of PRRs can now benefit from a surge in knowledge of the 3D structures of PRRs, many in complexes with their natural ligands. This review article describes recent progress in structural studies of PRRs (TLRs, NLRs, CTLs, and RLRs), which is required for an understanding of how they specifically recognize structurally diverse "foreign” PAMPs amongst a background of other "self” molecules, sometimes closely related in structure, that are present in the human bod

Synthesis and antimicrobial activity against Pseudomonas aeruginosa of macrocyclic β-hairpin peptidomimetic antibiotics containing N-methylated amino acids

Antimicrobial resistance among Gram-negative bacteria is a growing problem, fueled by the paucity of new antibiotics that target these microorganisms. One novel family of macrocyclic β-hairpin-shaped peptidomimetics was recently shown to act specifically against Pseudomonas spp. by a novel mechanism of action, targeting the outer membrane protein LptD, which mediates lipopolysaccharide transport to the cell surface during outer membrane biogenesis. Here we explore the mode of binding of one of these β-hairpin peptidomimetics to LptD in Pseudomonas aeruginosa, by examining the effects on antimicrobial activity following N-methylation of individual peptide bonds. An N-methyl scan of the cyclic peptide revealed that residues on both sides of the β-hairpin structure at a non-hydrogen bonding position likely mediate hydrogen-bonding interactions with the target LptD. Structural analyses by NMR spectroscopy further reinforce the conclusion that the folded β-hairpin structure of the peptidomimetic is critical for binding to the target LptD. Finally, new NMe analogues with potent activity have been identified, which opens new avenues for optimization in this family of antimicrobial peptides

Early molecular insights into thanatin analogues binding to A. baumannii LptA

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 μg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE- optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro

Structural aspects of molecular recognition in the immune system. Part II: Pattern recognition receptors

The vertebrate immune system uses pattern recognition receptors (PRRs) to detect a large variety of molecular signatures (pathogen-associated molecular patterns, PAMPs) from a broad range of different invading pathogens. The PAMPs range in size from relatively small molecules, to others of intermediate size such as bacterial lipopolysaccharide, lipopeptides, and oligosaccharides, to macromolecules such as viral DNA, RNA, and pathogen-derived proteins such as flagellin. Underlying this functional diversity of PRRs is a surprisingly small number of structurally distinct protein folds that include leucine-rich repeats in Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the DExH box helicase domain in RIG-like receptors (RLRs), and C-type lectin domains (CTLDs) in the C-type lectins. Following PAMP recognition by the PRRs, downstream signaling pathways activate the innate immune system to respond to invading pathogenic organisms. The resulting stimulatory response is also vital for a balanced adaptive immune response to the pathogen, mediated by circulating antibodies and/or cytotoxic T cells. However, an aberrant stimulation of the innate immune system can also lead to excessive inflammatory and toxic stress responses. Exciting opportunities are now arising for the design of small synthetic molecules that bind to PRRs and influence downstream signaling pathways. Such molecules can be useful tools to modulate immune responses, for example, as adjuvants to stimulate adaptive immune responses to a vaccine, or as therapeutic agents to dampen aberrant immune responses, such as inflammation. The design of agonists or antagonists of PRRs can now benefit from a surge in knowledge of the 3D structures of PRRs, many in complexes with their natural ligands. This review article describes recent progress in structural studies of PRRs (TLRs, NLRs, CTLs, and RLRs), which is required for an understanding of how they specifically recognize structurally diverse "foreign" PAMPs amongst a background of other "self" molecules, sometimes closely related in structure, that are present in the human body

Peptides in BioNMR Research

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ?-TIA to the human G-protein coupled ?1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics

Molecular Biological Comparison of Dental Pulp- and Apical Papilla-Derived Stem Cells

Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth

Purpose: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. Methods: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. Results: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. Conclusions: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors. Keywords: Bioactive small molecule compound; Cell invasion; FGFR; FRS2; Protein–protein interaction interference; Thermal proteome profilin

Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge

Structural aspects of molecular recognition in the immune system. Part I: Acquired immunity (IUPAC Technical Report)

Humoral immunity allows the body to mount a defense against pathogens and foreign substances, and to respond with memory to subsequent exposures. The molecular participants may also recognize self-structures, leading to attack on the body and autoimmune disease. The main players in humoral immunity are antibody-producing B lymphocytes, and several classes of T lymphocytes. This review deals with the molecular details of recognition of antigens by soluble antibodies, and of substances presented to receptors on the surfaces of T cells (TCRs). The prototype antibody consists of a dimer of dimers, two heavy (H) chains and two light (L) chains, with antigen recognition capacity lying in variable “head” regions of an H-L pair. Most crystallographic studies are done with this substructure, called a Fab fragment, bound in a soluble antigen complex. Homologous to this arrangement, the prototype TCR consists of two chains (α and β) that complex not soluble antigen, but usually a short peptide or other small molecule presented by proteins of the major histocompatibility complex. In each case a general background on the historical development of understanding the molecular recognition interface is given, followed by a number of examples of crystal structures from the recent literature that have allowed us to refine our understanding of the complex recognition process. Variations on the prototypical structures are also considered. The spectrum of recognition strategies involves interplay of lock-and-key with flexibility, varying degrees of entropic and enthalpic contributions, surface shaping by entrapped water molecules, and combinations of stabilizing hydrogen bonding, electrostatic interactions, salt bridging, and van der Waals forces. Preeminent in the recent literature are details of antibody binding to influenza A and human immunodeficiency viral antigens. Both viral antigens and attempts to understand autoimmunity are prominent in the recent TCR literature