Identification of VEGFR2 as the Histatin-1 receptor in endothelial cells.

Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.

5-HT2 Receptor Subfamily and the Halogen Bond Promise.

The binding of C-4-halogenated 1-(4-X-2,5-dimethoxyphenyl)-2-aminopropane (DOX) serotonin agonist psychedelics at all three 5-HT2 receptor subtypes is up to two orders of magnitude stronger for X = Cl, Br, or I (but not F) than when C-4 bears a hydrogen atom and more than expected from their hydrophobicities. Our docking and molecular dynamics simulations agree with the fact that increasing the polarizability of halogens results in halogen-oxygen distances to specific backbone C═O groups, and C-X···O angles, in ranges expected for halogen bonds (XBs), which could contribute to the high affinities observed. Good linear correlations are found for each receptor type, indicating that the binding pocket-ligand affinity is enhanced as the XB interaction becomes stronger (i.e., I ≈ Br > Cl > F). It is also striking to note how the linear equations unveil that the receptor's response on the strength of the XB interaction is quite similar among 5-HT2A and 5-HT2C, whereas the 5-HT2B's sensitivity is less. The calculated dipole polarizabilities in the binding pocket of the receptors reflect the experimental affinity values, indicating that less-polarizable and harder binding sites are more prone to XB formation.

Major Histocompatibility Complex Class I-Related Chain A (MICA) Allelic Variants Associate With Susceptibility and Prognosis of Gastric Cancer.

Gastric cancer (GC) is the fifth most prevalent type of cancer worldwide. Gastric tumor cells express MICA protein, a ligand to NKG2D receptor that triggers natural killer (NK) cells effector functions for early tumor elimination. MICA gene is highly polymorphic, thus originating alleles that encode protein variants with a controversial role in cancer. The main goal of this work was to study MICA gene polymorphisms and their relationship with the susceptibility and prognosis of GC. Fifty patients with GC and 50 healthy volunteers were included in this study. MICA alleles were identified using Sanger sequencing methods. The analysis of MICA gene sequence revealed 13 MICA sequences and 5 MICA-short tandem repeats (STR) alleles in the studied cohorts We identified MICA*002 (*A9) as the most frequent allele in both, patients and controls, followed by MICA*008 allele (*A5.1). MICA*009/049 allele was significantly associated with increased risk of GC (OR: 5.11 [95% CI: 1.39-18.74], p = 0.014). The analysis of MICA-STR alleles revealed a higher frequency of MICA*A5 in healthy individuals than GC patients (OR = 0.34 [95% CI: 0.12-0.98], p = 0.046). Survival analysis after gastrectomy showed that patients with MICA*002/002 or MICA*002/004 alleles had significantly higher survival rates than those patients bearing MICA*002/008 (p = 0.014) or MICA*002/009 (MICA*002/049) alleles (p = 0.040). The presence of threonine in the position MICA-181 (MICA*009/049 allele) was more frequent in GC patients than controls (p = 0.023). Molecular analysis of MICA-181 showed that the presence of threonine provides greater mobility to the protein than arginine in the same position (MICA*004), which could explain, at least in part, some immune evasion mechanisms developed by the tumor. In conclusion, our findings suggest that the study of MICA alleles is crucial to search for new therapeutic approaches and may be useful for the evaluation of risk and prognosis of GC and personalized therapy.

Dibenzofuranylethylamines as 5-HT2A/2C Receptor Agonists.

The human 5-HT2 receptor subtypes have high sequence identity in their orthosteric ligand-binding domain, and many agonists are poorly selective between the 5-HT2A and 5-HT2C subtypes. Nevertheless, their activation is associated with different pharmacological outcomes. We synthesized five phenethylamine analogs in which the benzene ring is replaced by a bulky dibenzo[b,d]furan moiety and found a couple with >70-fold 5-HT2C selectivity. Molecular docking studies of the most potent compound (5) at both receptor subtypes revealed the likely structural basis of its selectivity. Although in both cases, some crucial interactions are conserved, the change of the Ala2225.46 residue in the 5-HT2C receptor to the larger Ser2425.46 in the 5-HT2A subtype, which is the only structural difference between the orthosteric binding pockets of both receptors, weakens a π-π stacking interaction between the dibenzofuran moiety and the important Phe6.52 residue and breaks a hydrogen bond between the dibenzofuran oxygen and Ser5.43, explaining the selectivity of compound 5 for the 5-HT2C receptor. We believe that this effect of the residue at position 5.46 merits further exploration in the search for selective 5-HT2C receptor agonists that are of considerable interest in the treatment of schizophrenia and substance abuse.