IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFβ.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFβ. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes.

CTLA4-Ig-Based Bifunctional Costimulation Inhibitor Blocks CD28 and ICOS Signaling to Prevent T Cell Priming and Effector Function.

CTLA4-Ig/abatacept dampens activation of naive T cells by blocking costimulation via CD28. It is an approved drug for rheumatoid arthritis but failed to deliver efficacy in a number of other autoimmune diseases. One explanation is that activated T cells rely less on CD28 signaling and use alternate coreceptors for effector function.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches.

Background: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases.

Small Molecule IL-36γ Antagonist as a Novel Therapeutic Approach for Plaque Psoriasis.

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate.

IL-36 receptor antagonistic antibodies inhibit inflammatory responses in preclinical models of psoriasiform dermatitis.

Psoriasis vulgaris (PV) results from activation of IL-23/Th17 immune pathway and is further amplified by cytokines/chemokines from skin cells. Among skin-derived pro-inflammatory cytokines, IL-36 family members are highly upregulated in PV patients and play a critical role in general pustular psoriasis. However, there is limited data showing crosstalk between the IL-23 and IL-36 pathways in PV.

Cell-Surface Receptor-Ligand Interaction Analysis with Homogeneous Time-Resolved FRET and Metabolic Glycan Engineering: Application to Transmembrane and GPI-Anchored Receptors.

Ligand-binding assays are the linchpin of drug discovery and medicinal chemistry. Cell-surface receptors and their ligands have traditionally been characterized by radioligand-binding assays, which have low temporal and spatial resolution and entail safety risks. Here, we report a powerful alternative (GlycoFRET), where terbium-labeled fluorescent reporters are irreversibly attached to receptors by metabolic glycan engineering.

Generation and characterization of ABBV642, a dual variable domain immunoglobulin molecule (DVD-Ig) that potently neutralizes VEGF and PDGF-BB and is designed for the treatment of exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis.

Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor.

Background: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds.

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode.

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.

Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study.

Background: Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2).

Development of an in vitro potency bioassay for therapeutic IL-13 antagonists: the A-549 cell bioassay.

Interleukin 13 is a key cytokine that mediates airway hyper-responsiveness and mucus over-production, and several anti-IL-13 therapeutic antibodies are currently in clinical development for the treatment of asthma. Conventional cell-based bioassays for evaluating neutralization potencies of IL-13 antagonists are semi-quantitative or with a low sensitivity. Here, we report the development of a highly sensitive bioassay to assess the potency of IL-13 antagonists using human lung epithelial A-549 cells that produce thymus and activation-regulated chemokine (TARC) in response to IL-13 stimulation.

An efficient method to express and refold a truncated human procaspase-9: a caspase with activity toward Glu-X bonds.

A truncated form of human procaspase-9 missing the first 111 amino acids, and a variety of mutants derived therefrom, have been expressed in Escherichia coli inclusion bodies. Upon refolding to active enzymes, Delta(1-111) procaspase-9 and mutants were recovered at purity greater than 95% and with a final yield of 20-35 mg/L cell culture. Our active procaspase-9 retains its pro-segment, while undergoing major auto processing at Asp315 and a minor (20%) cleavage at Glu306.

Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme.

Both germinal (gACE) and somatic (sACE) isozymes of angiotensin-converting enzyme (ACE) are type I ectoproteins whose enzymatically active ectodomains are cleaved and shed by a membrane-bound protease. Here, we report a role of protein tyrosine phosphorylation in regulating this process. Strong enhancements of ACE cleavage secretion was observed upon enhancing protein Tyr phosphorylation by treating gACE- or sACE-expressing cells with pervanadate, an inhibitor of protein Tyr phosphatases.