Adverse effects of dipeptidyl peptidases 8 and 9 inhibition in rodents revisited.

AIM: To evaluate the association between inhibition of dipeptidyl peptidase (DPP)-8 and/or DPP-9 organ toxicities and mortality in rodents. RESEARCH DESIGN AND METHODS: The relative selectivity of the DPP-4 inhibitor, vildagliptin, was determined by comparing its K(I) (concentration of compound yielding 50% inhibition of the enzyme) values for inhibition of recombinant human DPP-4, DPP-8 and DPP-9 assessed in vitro. In experiments performed in vivo, vildagliptin was administered by gavage for 13 weeks, at doses up to 1500 mg/kg/day in CD-1 mice and at doses up to 900 mg/kg/day in Wistar rats. Plasma concentrations of vildagliptin were assessed at week 12, and toxicities previously ascribed to inhibition of DPP-8 and/or DPP-9 were assessed at week 13. RESULTS: The K(I) values for vildagliptin-induced inhibition of DPP-4, DPP-8 and DPP-9 were 3, 810 and 95 nM respectively. The mean plasma concentration 24 h after dose after 12-week daily dosing with 1500 mg/kg/day in mice was 2279 nM. The mean plasma drug level 24 h after dose after 12-week daily dosing with 900 mg/kg/day in rats was 5729 nM. These high doses maintained plasma drug levels well above the K(I) values for DPP-8 and DPP-9 throughout a 24-h period. At these high doses, the toxicities of a selective DPP-8/DPP-9 inhibitor that were reported previously (100% mortality in mice, alopecia, thrombocytopenia, reticulocytopenia, enlarged lymph nodes, splenomegaly and 20% mortality in rats) were not observed. CONCLUSIONS: Inhibition of DPP-8 and DPP-9 per se does not lead to organ toxicities and mortality in rodents. Thus, a mechanism other than DPP-8/DPP-9 inhibition likely underlies the toxicity previously reported to be associated with a selective DPP-8/DPP-9 inhibitor.

The lifetime of insulin hexamers.

The kinetic stability of insulin hexamers containing two metal ions was investigated by means of hybridization experiments. Insulin was covalently labeled at the N(epsilon)-amino group of Lys(B29) by a fluorescence donor and acceptor group, respectively. The labels neither affect the tertiary structure nor interfere with self-association. Equimolar solutions of pure donor and acceptor insulin hexamers were mixed, and the hybridization was monitored by fluorescence resonance energy transfer. With the total insulin concentration remaining constant and the association/dissociation equilibria unperturbed, the subunit interchange between hexamers is an entropy-driven relaxation process that ends at statistical distribution of the labels over 16 types of hexamers differing by their composition. The analytical description of the interchange kinetics on the basis of a plausible model has yielded the first experimental values for the lifetime of the hexamers. The lifetime is reciprocal to the product of the concentration of the exchanged species and the interchange rate constant: tau = 1/(c. k). Measured for different concentrations, temperatures, metal ions, and ligand-dependent conformational states, the lifetime was found to cover a range from minutes for T(6) to days for R(6) hexamers. The approach can be used under an unlimited variety of conditions. The information it provides is of obvious relevance for the handling, storage, and pharmacokinetic properties of insulin preparations.

Characterization and binding specificity of the monomeric STAT3-SH2 domain.

Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction. STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains. The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus. Here we describe the expression, purification, and refolding of the STAT3-SH2 domain. Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy. The STAT3-SH2 domain undergoes a conformational change upon dimerization. Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides. The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130.

Analysis of protein self-association at constant concentration by fluorescence-energy transfer.

Fluorescence-resonance-energy transfer from subunits labelled with a fluorescence donor group to subunits labelled with a fluorescence acceptor group can be used for quantitative analysis of protein self-association. The present approach evaluates fluorescence measurements on mixtures of equimolar solutions of donor-labelled and acceptor-labelled protein composed by systematic variation of the volume ratio. Its attractive feature is that it allows the determination of equilibrium constants at fixed total concentration. Problems encountered by most other methods, which require the equilibria to be followed to high dilution, are avoided. Conditions to be fulfilled are that a reactive site is available on the protein for specific introduction of the labels and that labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Forster distance of the donor/acceptor pair complies with its separation. While dimerisation constants can be determined exclusively by fluorescence measurements, the analysis of more complex cases of self-association depends on additional independent information. This communication reports on an application of the approach to the association/dissociation equilibrium between insulin monomers and dimers. Labelling of insulin at the epsilon-amino group of LysB29 does not disturb the conformation nor does it affect dimerisation. 2-Aminobenzoyl and 3-nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most other fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Forster distance of 29 A matches their 32-A separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor-insulin and acceptor-insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K12, of 0.72x10(5) M(-1). Its agreement with values obtained with other methods demonstrates that the present approach is a reliable alternative.

The signal transducer gp130--bacterial expression, refolding and properties of the carboxy-terminal domain of the cytokine-binding module.

Gp130 is the signal transducing receptor subunit of the so-called interleukin-6-type cytokines. This transmembrane protein is a member of the cytokine-receptor superfamily predicted to consist of six fibronectin-type-III-like domains in its extracellular part. The second and the third domain constitute the so-called cytokine-binding module. Domain 2 is characterized by a set of four conserved Cys residues, domain 3 by a conserved WSXWS motif. As a first approach to a more detailed characterization of the cytokine-binding domains of human gp130, we have expressed in Escherichia coli two forms of domain 3 differing in length. Both proteins were purified and refolded in a single step applying size-exclusion chromatography. According to the rotational correlation times deduced from fluorescence anisotropy decay, they do not form aggregates. CD and fluorescence spectroscopy were used to study thermal unfolding and denaturation by guanidinium hydrochloride. It was shown that N- and C-terminal extension by residues of the adjacent hinge regions substantially increase the thermal stability of the domain, which is conceivable from a molecular model. These results are the basis for further structural investigation by NMR spectroscopy.

Recombinant human O6-alkylguanine-DNA alkyltransferase induces conformational change in bound DNA.

Circular dichroism, and steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT bound to ds-DNA. Contrary to fluorescence, analysis of the far-UV CD spectra indicated a conformational change of AGT upon binding to DNA: its alpha-helical content is increased by approximately 12%. Analysis of near-UV CD spectra revealed that DNA was also affected, probably being separated into single strands locally.

Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 --> Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy.

Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.

Structure and rotational dynamics of fluorescently labeled insulin in aqueous solution and at the amphiphile-water interface of reversed micelles.

Insulin is a proteohormone with amphipathic three-dimensional structure and the ligand of a receptor, which itself spans the plasma membrane of glucose-metabolizing cells. In this study, the possible impact of amphiphiles on structural and dynamic properties of the hormone was investigated in reversed micelles mimicking the amphipathic nature of biological membranes. To make insulin susceptible to fluorescence measurements, two derivatives labeled with 2-aminobenzoic acid (Abz), N epsilon B29-Abz-insulin and [AbzB1]insulin, were prepared. First, the Abz-labeled insulins were shown by CD spectroscopy to exhibit conformational properties and self-association as well as the T-->R transition similar to the native hormone. By means of time-resolved fluorescence measurements, not only metal-ion induced hexamerization was observable in aqueous solution: The T-->R allosteric transition of the hexamer was shown to be accompanied by a diminution of its hydrodynamic radius. Second, structure and rotational dynamics of the labeled insulins were investigated in reversed micelles. In sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles, the main-chain conformation is similar to that in aqueous solution according to CD spectroscopy in the far-UV, whereas the weak circular dichroism in the near-UV is indicative of reduced aromatic contacts as well as of the absence of quaternary structure, and the CD spectra show the same shape as found for proteins in an intermediate state of folding referred to as the "molten globule". Fluorescence anisotropy decay measurements of N epsilon B29-Abz-insulin in reversed micelles of AOT, cetyltrimethylammonium bromide, and alpha-L-1,2-dioctanoylphosphatidylcholine showed that the internal mobility of the solubilizate is reduced compared to that in aqueous solution and that the rotational mobility of the labeled insulin decreases with decreasing micellar size. With respect to the immobilization, insulin interacts in a stronger way with the anionic than with the cationic or zwitterionic amphiphile; an integration into the amphiphile monolayer, however, could be ruled out in all cases. In conclusion, the results reveal an evident influence of amphiphiles on the structure and rotational dynamics of insulin. Further investigations should be focused on this finding also with regard to the possible importance of lipid-insulin interactions in vivo.