Identification and X-ray Co-crystal Structure of a Small-Molecule Activator of LFA-1-ICAM-1 Binding

The integrin Leucocyte function associated antigen 1 (LFA-1) is a heterodimeric immune receptor ubiquitously expressed on all leucocytes. Its interaction with Intercellular adhesion molecule 1 (ICAM-1) provides a critical recognition event between T-cells and antigen presenting cells in the immune systems efforts to pull off an early stage cell mediated immune response.[1–3] The LFA-1/ICAM-1 axis has thus been explored as a target interaction for drug discovery.[4–7] Furthermore, the structural changes of LFA-1 upon activation and interaction with ICAM-1 also make the LFA-1/ICAM-1 interaction an interesting example of protein-protein interaction (PPI) inhibition by small molecule inhibitors.[8,9] While protein-protein interaction inhibition by small molecules is considered to be the ultimate art in drug design, even fewer examples of true agonists of PPIs have been reported.[10–12] As for LFA-1, such activators would have interesting applications in rare hereditary genetic disorders called Leucocyte adhesion deficiency (LAD) or as potential enhancers of tumour immunotherapy.[13,14] Although, one such activator has been described recently, closer biological investigation has shown that it ultimately worked as an inhibitor on a cellular level by locking the LFA-1/ICAM-1 interaction when reversibility was needed for detachment of immune cells from endothelial surfaces and tissue infiltration.[15] Herein we describe the identification and structural biology of IBE-667, an ICAM-1 binding enhancer for LFA-1 from on-bead screening of tagged one-bead one-compound combinatorial libraries by confocal nanoscanning and bead picking (CONA).[16] Cellular assays demonstrate the activity of IBE-667 in promoting the binding of LFA-1 on activated immune cells to ICAM-1. X-ray structure based analysis did not only allow us to explain the molecular features of IBE-667 binding to LFA-1 but also offers an explanation for its mode of action

Hintergrundtext Medizinforschung am PSI

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Caractérisation de cristaux de complexes protéines-ligands

L’étude des protéines et des interactions de celles-ci sont un enjeu important dans la recherche de traitement pour différentes maladies. Dans le contexte de mon stage, je me suis intéressée à certaines protéines impliquées dans des cancers ainsi que dans des réactions inflammatoires. Afin de trouver pour ces protéines des ligands potentiels, je vais utiliser différentes techniques telles que : la cristallisation combinée à la diffraction aux rayons X, la thermofluorimétrie, ou encore la titration calorimétrique isotherme. Les ligands obtenus pourront être par la suite optimisés afin d’avoir entre autre une meilleure affinité pour la protéine d’intérêt. La cristallographie à rayons X est une des méthodes les plus utilisées pour obtenir une structure 3D à haute résolution de complexes protéine-ligand. La structure de ces complexes est primordiale dans le développement de nouveaux médicaments afin de déterminer le type d’interaction mis en place. Les ligands montrant une interaction vont être modifiés de façon à ce que celle-ci soit la plus forte possible. Car en cas de forte interaction entre la protéine et le ligand, la cinétique d’action de la protéine est modifié. La protéine ne remplit donc plus son rôle

Caractérisation de cristaux de complexes protéines-ligands

L’étude des protéines et des interactions de celles-ci sont un enjeu important dans la recherche de traitement pour différentes maladies. Dans le contexte de mon stage, je vais m’intéresser à certaines protéines impliquées dans des cancers ainsi que dans des réactions inflammatoires. Enfin de trouver des ligands potentiels pour ces protéines, je vais utiliser différentes techniques tels que : l’utilisation de la cristallisation aux rayons-X, la thermo-fluorimétrie, ou encore la calorimétrie isotherme à titration. Les ligands obtenus pourront être par la suite optimisé afin d’avoir une meilleure affinité pour la protéine d’intérêt

Structure of human cyclophilin A in complex with the novel immunosuppressant sanglifehrin A at 1.6 A resolution.

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 A resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA.SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA.SFA complexes is the molecular species mediating the immunosuppressive effect

Improved lymphocyte function-associated antigen-1 (LFA-1) inhibition by statin derivatives: molecular basis determined by x-ray analysis and monitoring of LFA-1 conformational changes in vitro and ex vivo.

The integrin lymphocyte function-associated antigen-1 (LFA-1) (alphaLbeta2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering drug lovastatin, which binds to an allosteric site of the alphaL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases

Behavioral dynamics of conversation, (mis)communication and coordination in noisy environments

Abstract During conversations people coordinate simultaneous channels of verbal and nonverbal information to hear and be heard. But the presence of background noise levels such as those found in cafes and restaurants can be a barrier to conversational success. Here, we used speech and motion-tracking to reveal the reciprocal processes people use to communicate in noisy environments. Conversations between twenty-two pairs of typical-hearing adults were elicited under different conditions of background noise, while standing or sitting around a table. With the onset of background noise, pairs rapidly adjusted their interpersonal distance and speech level, with the degree of initial change dependent on noise level and talker configuration. Following this transient phase, pairs settled into a sustaining phase in which reciprocal speech and movement-based coordination processes synergistically maintained effective communication, again with the magnitude of stability of these coordination processes covarying with noise level and talker configuration. Finally, as communication breakdowns increased at high noise levels, pairs exhibited resetting behaviors to help restore communication—decreasing interpersonal distance and/or increasing speech levels in response to communication breakdowns. Approximately 78 dB SPL defined a threshold where behavioral processes were no longer sufficient for maintaining effective conversation and communication breakdowns rapidly increased

Crystallization and preliminary X-ray crystallographic study of interleukin-8

AbstractInterleukin-8 (neutrophil-activating factor; NAP-1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three-dimensional structural study at a resolution higher than 2.4 Å. The crystals belong to the space group P3121 or P3221 and have unit cell dimensions a = b = 40.9 Å, c = 90.3 Å

The Central Valine Concept Provides an Entry in a New Class of Non Peptide Inhibitors of the P53-MDM2 Interaction

Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein-protein interaction , we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53-MDM2 interaction