Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

Receptor subtype discrimination using extensive shape complementary designed interfaces.

To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.

Topological control of cytokine receptor signaling induces differential effects in hematopoiesis.

Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems.

Chemically Modifying Viruses for Diverse Applications.

Long fascinating to biologists, viruses offer nanometer-scale benchtops for building molecular-scale devices and materials. Viruses tolerate a wide range of chemical modifications including reaction conditions, pH values, and temperatures. Recent examples of nongenetic manipulation of viral surfaces have extended viruses into applications ranging from biomedical imaging, drug delivery, tissue regeneration, and biosensors to materials for catalysis and energy generation. Chemical reactions on the phage surface include both covalent and noncovalent modifications, including some applied in conjunction with genetic modifications. Here, we survey viruses chemically augmented with capabilities limited only by imagination.

Biosensing with Virus Electrode Hybrids.

Virus electrodes address two major challenges associated with biosensing. First, the surface of the viruses can be readily tailored for specific, high affinity binding to targeted biomarkers. Second, the viruses are entrapped in a conducting polymer for electrical resistance-based, quantitative measurement of biomarker concentration. To further enhance device sensitivity, two different ligands can be attached to the virus surface, and increase the apparent affinity for the biomarker. In the example presented here, the two ligands bind to the analyte in a bidentate binding mode with a chelate-based avidity effect, and result in a 100 pM experimentally observed limit of detection for the cancer biomarker prostate-specific membrane antigen. The approach does not require enzymatic amplification, and allows reagent-free, real-time measurements. This article presents general protocols for the development of such biosensors with modified viruses for the enhanced detection of arbitrary target proteins.

Dual genetically encoded phage-displayed ligands.

M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display.

Sub-nanomolar detection of prostate-specific membrane antigen in synthetic urine by synergistic, dual-ligand phage.

The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnosis and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. This report describes a bioaffinity matrix of viruses integrated into PEDOT films for electrochemical sensing of prostate-specific membrane antigen (PSMA), a prostate cancer biomarker. High sensitivity to PSMA resulted from synergistic action by two different ligands to PSMA on the same phage particle. One ligand was genetically encoded, and the secondary recognition ligand was chemically synthesized to wrap around the phage. The dual ligands result in a bidentate binder with high-copy, dense ligand display for enhanced PSMA detection through a chelate-based avidity effect. Biosensing with virus-PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification.