Virtual screening for inhibitors of the human TSLP:TSLPR interaction

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathophysiology of various allergy disorders that are mediated by type 2 helper T cell (Th2) responses, such as asthma and atopic dermatitis. TSLP forms a ternary complex with the TSLP receptor (TSLPR) and the interleukin-7-receptor subunit alpha (IL-7Ra), thereby activating a signaling cascade that culminates in the release of pro-inflammatory mediators. In this study, we conducted an in silico characterization of the TSLP: TSLPR complex to investigate the drugability of this complex. Two commercially available fragment libraries were screened computationally for possible inhibitors and a selection of fragments was subsequently tested in vitro. The screening setup consisted of two orthogonal assays measuring TSLP binding to TSLPR: a BLI-based assay and a biochemical assay based on a TSLP: alkaline phosphatase fusion protein. Four fragments pertaining to diverse chemical classes were identified to reduce TSLP: TSLPR complex formation to less than 75% in millimolar concentrations. We have used unbiased molecular dynamics simulations to develop a Markov state model that characterized the binding pathway of the most interesting compound. This work provides a proof-ofprinciple for use of fragments in the inhibition of TSLP: TSLPR complexation

Entropic Tension in Crowded Membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function \emph{in vivo}. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than $2\;k_BT$ in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.Comment: 20 pages (inclduing supporting information), 4 figures, to appear in PLoS Comp. Bio

Chemical Biology is.....

Chemical Biology is a relatively new field, and as such is not yet simply or succinctly defined. It includes such a wide range of fundamental problems that this commentary could only include just a few snapshots of potential areas of interest. Overarching themes and selected recent successes and ideas in chemical biology are described to illustrate broadly the scope of the field, but should not be taken as exhaustive. The Chemical Biology Section of Chemistry Central Journal is pleased to receive manuscripts describing research into all and any aspects of the subject

Phage Display in the Quest for New Selective Recognition Elements for Biosensors

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies." The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.This study was supported by the Ministry of Economy and Competitiveness (Ministerio de Ciencia, Innovación y Universidades RTI2018-096410-B-C21). R.P. acknowledges UCM for a predoctoral grant and R.B. the PI17CIII/00045 grant from the AES-ISCIII program.S

Ligand-Receptor Interactions

The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the interest of biologists to the kinetic and mechanical properties of cell membrane receptors. The aim of this review is to give a description of these advances that benefitted from a largely multidisciplinar approach

Selective Pressure to Increase Charge in Immunodominant Epitopes of the H3 Hemagglutinin Influenza Protein

The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein–protein interactions, including antibody–antigen binding and ligand–receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R2 > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies

Separating Lentiviral Vector Injection and Induction of Gene Expression in Time, Does Not Prevent an Immune Response to rtTA in Rats

BACKGROUND: Lentiviral gene transfer can provide long-term expression of therapeutic genes such as erythropoietin. Because overexpression of erythropoietin can be toxic, regulated expression is needed. Doxycycline inducible vectors can regulate expression of therapeutic transgenes efficiently. However, because they express an immunogenic transactivator (rtTA), their utility for gene therapy is limited. In addition to immunogenic proteins that are expressed from inducible vectors, injection of the vector itself is likely to elicit an immune response because viral capsid proteins will induce "danger signals" that trigger an innate response and recruit inflammatory cells. METHODOLOGY AND PRINCIPAL FINDINGS: We have developed an autoregulatory lentiviral vector in which basal expression of rtTA is very low. This enabled us to temporally separate the injection of virus and the expression of the therapeutic gene and rtTA. Wistar rats were injected with an autoregulatory rat erythropoietin expression vector. Two or six weeks after injection, erythropoietin expression was induced by doxycycline. This resulted in an increase of the hematocrit, irrespective of the timing of the induction. However, most rats only responded once to doxycycline administration. Antibodies against rtTA were detected in the early and late induction groups. CONCLUSIONS: Our results suggest that, even when viral vector capsid proteins have disappeared, expression of foreign proteins in muscle will lead to an immune respons

Integrating water exclusion theory into βcontacts to predict binding free energy changes and binding hot spots

10.1186/1471-2105-15-57BMC Bioinformatics151-BBMI

PANIC-ATTAC: A Mouse Model for Inducible and Reversible β-Cell Ablation

OBJECTIVE—Islet transplantations have been performed clinically, but their practical applications are limited. An extensive effort has been made toward the identification of pancreatic β-cell stem cells that has yielded many insights to date, yet targeted reconstitution of β-cell mass remains elusive. Here, we present a mouse model for inducible and reversible ablation of pancreatic β-cells named the PANIC-ATTAC (pancreatic islet β-cell apoptosis through targeted activation of caspase 8) mouse

PuLSE:Quality control and quantification of peptide sequences explored by phage display libraries

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use