Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

PurposeBasal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements.MethodsSelf-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection.ResultsWith depletion of phenol, insulin degludec and another fatty diacid–insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T3R3 conformation in presence of phenol and zinc, changing to T6 with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly.ConclusionsInsulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.

The interior of chlorosomes of green bacteria forms an unusual antenna system organized without proteins. The steady-spectra (absorption, circular dichroism, and linear dichroism) have been modeled using the Frenkel Hamiltonian for the large tubular aggregates of bacteriochlorophylls with geometries corresponding to those proposed for Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. For the Cf. aurantiacus aggregates we apply a structure used previously (V. I. Prokhorenko., D. B. Steensgaard, and A. R. Holzwarth, Biophys: J. 2000, 79:2105-2120), whereas for the Cb. tepidum aggregates a new extended model of double-tube aggregates, based on recently published solid-state nuclear magnetic resonance studies (B.-J. van Rossum, B. Y. van Duhl, D. B. Steensgaard, T. S. Balaban, A. R. Holzwarth, K. Schaffner, and H. J. M. de Groot, Biochemistry 2001, 40:1587-1595), is developed. We find that the circular dichroism spectra depend strongly on the aggregate length for both types of chlorosomes. Their shape changes from "type-II" (negative at short wavelengths to positive at long wavelengths) to the "mixed-type" (negative-positive-negative) in the nomenclature proposed in K. Griebenow, A. R. Holzwarth, F. van Mourik, and R. van Grondelle, Biochim: Biophys. Acta 1991, 1058:194-202, for an aggregate length of 30-40 bacteriochlorophyll molecules per stack. This "size effect" on the circular dichroism spectra is caused by appearance of macroscopic chirality due to circular distribution of the transition dipole moment of the monomers. We visualize these distributions, and also the corresponding Frenkel excitons, using a novel presentation technique. The observed size effects provide a key to explain many previously puzzling and seemingly contradictory experimental data in the literature on the circular and linear dichroism spectra of seemingly identical types of chlorosomes.

The energy transfer processes in isolated chlorosomes from green bacteria Chlorobium tepidum and Chloroflexus aurantiacus have been studied at low temperatures (1.27 K) by two-pulse photon echo and one-color transient absorption techniques with approximately 100 fs resolution. The decay of the coherence in both types of chlorosomes is characterized by four different dephasing times stretching from approximately 100 fs up to 300 ps. The fastest component reflects dephasing that is due to interaction of bacteriochlorophylls with the phonon bath, whereas the other components correspond to dephasing due to different energy transfer processes such as distribution of excitation along the rod-like aggregates, energy exchange between different rods in the chlorosome, and energy transfer to the base plate. As a basis for the interpretation of the excitation dephasing and energy transfer pathways, a superlattice-like structural model is proposed based on recent experimental data and computer modeling of the Bchl c aggregates (1994. Photosynth. Res. 41:225-233.) This model predicts a fine structure of the Q(y) absorption band that is fully supported by the present photon echo data.

Heteronuclear 2-D and 3-D magic-angle spinning NMR dipolar correlation spectroscopy was applied to determine solid-state (1)H shifts for aggregated bacteriochlorophyll c (BChl c) in uniformly (13)C-enriched light harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum. A complete assignment of 29 different observable resonances of the 61 protons of the aggregated BChl c in the intact chlorosomes is obtained. Aggregation shifts relative to monomeric BChl c in solution are detected for protons attached to rings I, II, and III/V and to their side chains. The 2(1)-H(3), 3(2)-H(3), and 3(1)-H resonances are shifted upfield by -2.2, -1, and -3.3 ppm, respectively, relative to monomeric BChl c in solution. Although the resonances are inhomogeneously broadened and reveal considerable global structural heterogeneity, the 5-CH and the 7-Me responses are doubled, which provides evidence for the existence of at least two relatively well-defined structurally different arrangements. Ab initio quantum chemical modeling studies were performed to refine a model for the self-assembled BChl c with two different types of BChl stacks. The BChl in the stacks can adopt either anti- or syn-configuration of the coordinative bond, where anti and syn designate the relative orientation of the Mg-OH bond relative to the direction of the 17-17(1) bond. The analogy between aggregation shifts for BChl c in the chlorosome and for self-assembled chlorophyll a/H(2)O is explored, and a bilayer model for the tubular supra-structure of sheets of BChl c is proposed, from a homology modeling approach.