Synthesis of a spin-labeled anti-estrogen as a dynamic motion probe for the estrogen receptor ligand binding domain.

The preparation and characterization of a novel nitroxide spin probe based on a steroidal anti-estrogen is described. The probe 5 demonstrated very high binding affinity for both the alpha and beta isoforms of the estrogen receptor-ligand binding domain. EPR spectrometric studies demonstrate conformational constraints for the ligand, consistent with the nitroxyl moiety occupying a position just beyond the receptor-solvent interface.

TLR Stimulation Produces IFN-ß as the Primary Driver of IFN Signaling in Nonlymphoid Primary Human Cells.

Several human autoimmune diseases are characterized by increased expression of type 1 IFN-stimulated genes in both the peripheral blood and tissue. The contributions of different type I IFNs to this gene signature are uncertain as the type I IFN family consists of 13 alphas and one each of ß, ε, κ, and ω subtypes. We sought to investigate the contribution of various IFNs to IFN signaling in primary human cell types. We stimulated primary skin, muscle, kidney, and PBMCs from normal healthy human donors with various TLR ligands and measured the expression of type I IFN subtypes and activation of downstream signaling by quantitative PCR. We show that IFNB1 is the dominant type I IFN expressed upon TLR3 and TLR4 stimulation, and its expression profile is associated with subsequent MX1 transcription. Furthermore, using an IFN-ß-specific neutralizing Ab, we show that MX1 expression is inhibited in a dose-dependent manner, suggesting that IFN-ß is the primary driver of IFN-stimulated genes following TLR3 and TLR4 engagement. Stimulation with TLR7/8 and TLR9 ligands induced IFNB1 and IFNA subtypes and MX1 expression only in PBMCs and not in tissue resident cell types. Concordantly, IFN-ß neutralization had no effect on MX1 expression in PBMCs potentially because of the combination of IFNB1 and IFNA expression. Combined, these data highlight the potential role for IFN-ß in driving local inflammatory responses in clinically relevant human tissue types and opportunities to treat local inflammation by targeting IFN-ß.

Dynamic conformational responses of a human cannabinoid receptor-1 helix domain to its membrane environment.

The influence of membrane environment on human cannabinoid 1 (hCB(1)) receptor transmembrane helix (TMH) conformational dynamics was investigated by solid-state NMR and site-directed spin labeling/EPR with a synthetic peptide, hCB(1)(T377-E416), corresponding to the receptor's C-terminal component, i.e., TMH7 and its intracellular alpha-helical extension (H8) (TMH7/H8). Solid-state NMR experiments with mechanically aligned hCB(1)(T377-E416) specifically (2)H- or (15)N-labeled at Ala380 and reconstituted in membrane-mimetic dimyristoylphosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) bilayers demonstrate that the conformation of the TMH7/H8 peptide is more heterogeneous in the thinner DMPC bilayer than in the thicker POPC bilayer. As revealed by EPR studies on hCB(1)(T377-E416) spin-labeled at Cys382 and reconstituted into the phospholipid bilayers, the spin label partitions actively between hydrophobic and hydrophilic environments. In the DMPC bilayer, the hydrophobic component dominates, regardless of temperature. Mobility parameters (DeltaH(0)(-1)) are 0.3 and 0.73 G for the peptide in the DMPC or POPC bilayer environment, respectively. Interspin distances of doubly labeled hCB(1)(T377-E416) peptide reconstituted into a TFE/H(2)O mixture or a POPC or DMPC bilayer were estimated to be 10.6 +/- 0.5, 16.8 +/- 1, and 11.6 +/- 0.8 A, respectively. The extent of coupling (>or=50%) between spin labels located at i and i + 4 in a TFE/H(2)O mixture or a POPC bilayer is indicative of an alpha-helical TMH conformation, whereas the much lower coupling (14%) when the peptide is in a DMPC bilayer suggests a high degree of peptide conformational heterogeneity. These data demonstrate that hCB(1)(T377-E416) backbone dynamics as well as spin-label rotameric freedom are sensitive to and altered by the peptide's phospholipid bilayer environment, which exerts a dynamic influence on the conformation of a TMH critical to signal transmission by the hCB(1) receptor.

Molecular-scale force measurement in a coiled-coil peptide dimer by electron spin resonance.

A new method for measuring forces between small protein domains based on double electron-electron resonance (DEER) spectroscopy is demonstrated using a model peptide derived from the alpha-helical coiled-coil leucine zipper of yeast transcriptional activator GCN4. The equilibrium distribution of distances between two nitroxide spin labels rigidly attached to the helices of the dimer was determined by DEER and yielded a closing force of 100 +/- 10 pN between monomers, in excellent agreement with theoretical predictions.

Co-inhibitory T cell receptor KLRG1: human cancer expression and efficacy of neutralization in murine cancer models.

BACKGROUND: KLRG1 is a lymphocyte co-inhibitory, or immune checkpoint, receptor expressed predominantly on late-differentiated effector and effector memory CD8+ T and NK cells. Targeting of KLRG1 neutralization in murine cancer models has not previously been reported. METHODS: We studied KLRG1 expression in human blood and tumor samples from available genomic datasets. Anti-KLRG1 neutralizing antibody was studied in the murine 4T1 breast cancer as monotherapy, and in the MC38 colon cancer and B16F10 melanoma models as combination therapy with anti-PD-1 antibody. RESULTS: In human blood and tumor samples, KLRG1 expression is aligned with cytotoxic T and NK cell differentiation, and upregulated in human tumor samples after a variety of therapies, potentially contributing to adaptive resistance. In in vivo murine models, anti-KLRG1 antibody monotherapy in the 4T1 breast cancer model reduced lung metastases (decreased lung weights p=0.04; decreased nodule count p=0.002), while anti-KLRG1 + anti-PD-1 combination therapy in the MC38 colon cancer and B16F10 melanoma models produced synergistic benefit greater than anti-PD-1 alone for tumor volume (MC38 p=0.01; B16F10 p=0.007) and survival (MC38 p=0.02; B16F10 p=0.002). CONCLUSIONS: These studies provide the first evidence that inhibition of the KLRG1 pathway enhances immune control of cancer in murine models, and provide target validation for KLRG1 targeting of human cancer. The mechanism of efficacy of KLRG1 blockade in murine models remains to be determined.

Designed BH3 peptides with high affinity and specificity for targeting Mcl-1 in cells.

Mcl-1 is overexpressed in many cancers and can confer resistance to cell-death signaling in refractory disease. Molecules that specifically inhibit Mcl-1 hold potential for diagnosing and disrupting Mcl-1-dependent cell survival. We selected three peptides from a yeast-surface display library that showed moderate specificity and affinity for binding to Mcl-1 over Bfl-1, Bcl-xL, Bcl-2, and Bcl-w. Specificity for Mcl-1 was improved by introducing threonine at peptide position 2e. The most specific peptide, MS1, bound Mcl-1 with 40-fold or greater specificity over four other human Bcl-2 paralogs. In BH3 profiling assays, MS1 caused depolarization in several human Mcl-1-dependent cell lines with EC50 values of ∼3 µM, contrasted with EC50 values of >100 µM for Bcl-2-, Bcl-xL-, or Bfl-1-dependent cell lines. MS1 is at least 30-fold more potent in this assay than the previously used Mcl-1 targeting reagent NoxaA BH3. These peptides can be used to detect Mcl-1 dependency in cells and provide leads for developing Mcl-1 targeting therapeutics.

Mcl-1-Bim complexes accommodate surprising point mutations via minor structural changes.

Mcl-1 is an antiapoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the alpha-helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl-2 family function. We report the crystal structure of human Mcl-1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl-1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine-to-alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix alpha3 accommodating an isoleucine-to-tyrosine mutation at this same position. We surveyed the variation in available Mcl-1 and Bcl-x(L) structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3-only proteins with Mcl-1. With the antiapoptotic Bcl-2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl-1.