Dynamic Combinatorial Optimization of In Vitro and In Vivo Heparin Antidotes.

Heparin-like macromolecules are widely used in clinics as anticoagulant, antiviral, and anticancer drugs. However, the search of heparin antidotes based on small synthetic molecules to control blood coagulation still remains a challenging task due to the physicochemical properties of this anionic polysaccharide. Here, we use a dynamic combinatorial chemistry approach to optimize heparin binders with submicromolar affinity. The recognition of heparin by the most amplified members of the dynamic library has been studied with different experimental (SPR, fluorescence, NMR) and theoretical approaches, rendering a detailed interaction model. The enzymatic assays with selected library members confirm the correlation between the dynamic covalent screening and the in vitro heparin inhibition. Moreover, both ex vivo and in vivo blood coagulation assays with mice show that the optimized molecules are potent antidotes with potential use as heparin reversal drugs. Overall, these results underscore the power of dynamic combinatorial chemistry targeting complex and elusive biopolymers.

Hybrid Cyclobutane/Proline-Containing Peptidomimetics: The Conformational Constraint Influences Their Cell-Penetration Ability.

A new family of hybrid ß,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-ß-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane ß-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties.

Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches.

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1 flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.

Pseudo-Wang Handle for the Preparation of Fully Protected Peptides. Synthesis of Liraglutide by Fragment Condensation.

A handle for the protection of the C-terminus of peptides after cleaving with low concentration of trifluoroacetic acid (2-4%) is reported. The handle prevents polymerization reactions in the convergent condensation of peptidic fragments. Moreover, it is traceless, being removed during the final deprotection step of the peptide synthesis. This cheap and convenient handle is easily attached to the solid support, causing no disturbance to peptide elongation and thus proving to be useful in the convergent synthesis of long peptides.

Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

Plasmablasts During Acute Dengue Infection Represent a Small Subset of a Broader Virus-specific Memory B Cell Pool.

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.

Application of Gene Expression Trajectories Initiated from ErbB Receptor Activation Highlights the Dynamics of Divergent Promoter Usage.

Understanding how cells use complex transcriptional programs to alter their fate in response to specific stimuli is an important question in biology. For the MCF-7 human breast cancer cell line, we applied gene expression trajectory models to identify the genes involved in driving cell fate transitions. We modified trajectory models to account for the scenario where cells were exposed to different stimuli, in this case epidermal growth factor and heregulin, to arrive at different cell fates, i.e. proliferation and differentiation respectively. Using genome-wide CAGE time series data collected from the FANTOM5 consortium, we identified the sets of promoters that were involved in the transition of MCF-7 cells to their specific fates versus those with expression changes that were generic to both stimuli. Of the 1,552 promoters identified, 1,091 had stimulus-specific expression while 461 promoters had generic expression profiles over the time course surveyed. Many of these stimulus-specific promoters mapped to key regulators of the ERK (extracellular signal-regulated kinases) signaling pathway such as FHL2 (four and a half LIM domains 2). We observed that in general, generic promoters peaked in their expression early on in the time course, while stimulus-specific promoters tended to show activation of their expression at a later stage. The genes that mapped to stimulus-specific promoters were enriched for pathways that control focal adhesion, p53 signaling and MAPK signaling while generic promoters were enriched for cell death, transcription and the cell cycle. We identified 162 genes that were controlled by an alternative promoter during the time course where a subset of 37 genes had separate promoters that were classified as stimulus-specific and generic. The results of our study highlighted the degree of complexity involved in regulating a cell fate transition where multiple promoters mapping to the same gene can demonstrate quite divergent expression profiles.

Searching for new cell-penetrating agents: hybrid cyclobutane-proline γ,γ-peptides.

Two generations of hybrid γ,γ-peptides containing cyclobutane amino acids and cis-γ-amino-L-proline joined in alternation have been synthesized and their capacity to cross the eukaryotic cell membrane has been evaluated. The first generation consists of di-, tetra- and hexapeptides, and their properties have been analyzed as well as the influence of peptide length and chirality of the cyclobutane residues. Results have shown that the absolute configuration of the cyclobutane amino acid does not have a relevant influence. The second generation consists of hybrid γ,γ-hexapeptides with a common backbone and distinct side chains introduced with different linkage types through the α-amino group (N(α)) of the proline monomers. These peptides have been shown to be non-toxic towards HeLa cells and to internalize them effectively, the best results being obtained for the peptides with a spacer of five carbons between the N(α) atom and the guanidinium group. The introduction of cyclobutane residues inside the sequence affords a good balance between charge and hydrophobicity, reducing the number of positive charges. This results in lower toxicity and similar cell-uptake properties when compared to previously described peptide agents.

Efficient γ-amino-proline-derived cell penetrating peptide-superparamagnetic iron oxide nanoparticle conjugates via aniline-catalyzed oxime chemistry as bimodal imaging nanoagents.

Aniline-catalyzed oxime chemistry was employed to conjugate a γ-amino-proline-derived cell penetrating peptide to superparamagnetic iron oxide nanoparticles (SPIONs). Internalization of the novel nanoconjugate into HeLa cells was found to be remarkably higher compared to the analogous TAT-SPION conjugate.

Synthesis and structural study of highly constrained hybrid cyclobutane-proline γ,γ-peptides.

Two diastereomeric series of hybrid γ,γ-peptides derived from conveniently protected derivatives of (1R,2S)- and (1S,2R)-3-amino-2,2-dimethylcyclobutane-1-carboxylic acid and cis-4-amino-L: -proline joined in alternation have efficiently been prepared through convergent synthesis. High-resolution NMR experiments show that these compounds present defined conformations in solution affording very compact structures as the result of intra and inter residue hydrogen-bonded ring formation. (R,S)-cyclobutane containing peptides adopt more twisted conformations than (S,R) diastereomers. In addition, all these γ-peptides have high tendency to aggregation providing vesicles of nanometric size, which were stable when allowed to stand for several days, as verified by transmission electron microscopy.