Objective Structurally-improved GIP analogs were developed to determine precisely whether GIP receptor (GIPR) agonism or antagonism lowers body weight in obese mice. Methods A series of peptide-based GIP analogs, including structurally diverse agonists and a long-acting antagonist, were generated and characterized in vitro using functional assays in cell systems overexpressing human and mouse derived receptors. These analogs were characterized in vivo in DIO mice following acute dosing for effects on glycemic control, and following chronic dosing for effects on body weight and food intake. Pair-feeding studies and indirect calorimetry were used to survey the mechanism for body weight lowering. Congenital Gipr−/− and Glp1r−/− DIO mice were used to investigate the selectivity of the agonists and to ascribe the pharmacology to effects mediated by the GIPR. Results Non-acylated, Aib2 substituted analogs derived from human GIP sequence showed full in vitro potency at human GIPR and subtly reduced in vitro potency at mouse GIPR without cross-reactivity at GLP-1R. These GIPR agonists lowered acute blood glucose in wild-type and Glp1r−/− mice, and this effect was absent in Gipr−/− mice, which confirmed selectivity towards GIPR. Chronic treatment of DIO mice resulted in modest yet consistent, dose-dependent decreased body weight across many studies with diverse analogs. The mechanism for body weight lowering is due to reductions in food intake, not energy expenditure, as suggested by pair-feeding studies and indirect calorimetry assessment. The weight lowering effect was preserved in DIO Glp-1r−/− mice and absent in DIO Gipr −/− mice. The body weight lowering efficacy of GIPR agonists was enhanced with analogs that exhibit higher mouse GIPR potency, with increased frequency of administration, and with fatty-acylated peptides of extended duration of action. Additionally, a fatty-acylated, N-terminally truncated GIP analog was shown to have high in vitro antagonism potency for human and mouse GIPR without cross-reactive activity at mouse GLP-1R or mouse glucagon receptor (GcgR). This acylated antagonist sufficiently inhibited the acute effects of GIP to improve glucose tolerance in DIO mice. Chronic treatment of DIO mice with high doses of this acylated GIPR antagonist did not result in body weight change. Further, co-treatment of this acylated GIPR antagonist with liraglutide, an acylated GLP-1R agonist, to DIO mice did not result in increased body weight lowering relative to liraglutide-treated mice. Enhanced body weight lowering in DIO mice was evident however following co-treatment of long-acting selective individual agonists for GLP-1R and GIPR, consis...

A series of dipeptides AX and XA (X ) G, K, L, S, and V) were investigated by polarized visible Raman and FTIR-spectroscopy to examine the conformational determinants of the amide III band. A spectral decomposition combined with density functional calculations revealed that the amide III band has a multicomponent structure in that three different modes contribute to amide III vibrations. One of them (amide III 2 ) dominates the Raman spectra particularly of the cationic species. Its normal mode displays an in-phase combination of NH and C R1 H in plane bending vibrations, which makes it sensitive to changes of the dihedral angle ψ. Indeed, our Raman data show that amide III 2 varies with ψ but remains practically unaffected by variation of φ in the region between -95°and -75°, which is sampled by the investigated AX peptides. Our data support the Lord hypothesis that amide III depends solely on ψ (Lord, R. Appl. Spectrosc. 1977, 31, 187) but specifies to which of the amide III modes this statement applies. Our data further reveal that all amide III modes can interact with side chains vibrations. For some residues this causes a mode delocalization which yields a reduction of the Raman cross section. Amide S, which is a structure sensitive band resonance enhanced with UV-excitation, disappears for ψ-values outside of the β-sheet region due to changes of the normal mode compositions of several modes between 1300 and 1420 cm -1 . This explains its absence in the UV-Raman spectra of R-helical structures. Our data suggest that all AX peptides exhibit ψ angles around 150°.

ObjectiveTo signal, FGF19 and FGF21 require co-receptor βKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists.MethodsWe employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice.ResultsC-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice.ConclusionsWe report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.

The hormonally active form of vitamin D3, 1,25(OH)2D3 (calcitriol), exerts actions through VDR receptor, which acts as a transcriptional factor. Calcitriol is an immunomodulator that affects various immune cells, and several studies link it to many autoimmune diseases. BsmI polymorphism affects the level of VDR gene transcription, transcript stability, and posttranscriptional modifications. It seems to be related to the systemic lupus erythematosus (SLE). Our study examined the characteristics of VDR gene BsmI polymorphism in Polish SLE patients and their relationship with clinical manifestations of the disease. We genotyped 62 patients with SLE and 100 healthy controls using the real-time PCR. There were no differences observed in the frequency of BsmI genotypes in SLE patients and in the control group. There was no significant correlation between BsmI genotypes and clinical symptoms of SLE, but the AA genotype correlates with higher levels of antinuclear antibodies (ANA) in this group (r = 0.438; P = 0.002). A larger study examining BsmI and other VDR gene polymorphisms is needed. It may allow explaining differences in the clinical picture of the disease and choosing a personalized therapy.

Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.