Blockade of activin type II receptors with a dual anti-ActRIIA/IIB antibody is critical to promote maximal skeletal muscle hypertrophy.

The TGF-ß family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc "trap," to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit.

Ligandability Assessment of IL-1ß by Integrated Hit Identification Approaches.

Human interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1ß binders that interfere with IL-1ß signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1ß, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1ß as a target residue suitable for the development of covalent, low-molecular-weight IL-1ß antagonists.

Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1ß.

Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.

Atrial natriuretic peptide, a regulator of nuclear factor-kappaB activation in vivo.

Natriuretic peptides (NPs) comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. Along this line, atrial NP (ANP) (international non-proprietary name: carperitide, HANP) is an approved drug for the treatment of acute heart failure. In recent years, evidence has been given that the NP system possesses a far broader biological spectrum than the regulation of blood pressure and volume homeostasis. In fact, a substantial amount of in vitro work indicates that ANP affects important inflammatory processes and signaling pathways. Quite surprisingly, however, no information exists on the in vivo antiinflammatory potential and signaling of ANP. We show here that pretreatment of lipopolysaccharide (Salmonella abortus equi, 2.5 mg/kg)-challenged mice with ANP (5 microg/kg iv, 15 min) rapidly inhibits nuclear factor-kappaB activation via inhibition of phosphorylation and degradation of the IkappaB-alpha protein. ANP also reduces Akt activation upon lipopolysaccharide injection. In ANP-pretreated mice, the increase of TNF-alpha serum concentration is markedly prevented; most importantly, the survival of these animals improved. These findings demonstrate both in vitro and in vivo an antiinflammatory profile of ANP that deserves to be further investigated in a therapeutic perspective.

Orthorhombic sphere packings. II. Bivariant lattice complexes.

All homogeneous sphere packings and all interpenetrating layers of spheres were derived that refer to the 22 orthorhombic bivariant lattice complexes. In total, sphere packings of 90 different types have been found. Only for 47 of these types is the maximal inherent symmetry of their sphere packings orthorhombic. Some examples demonstrate the usefulness of sphere packings for the comparison and description of crystal structures.

Hexagonal and trigonal sphere packings. IV. Trivariant lattice complexes of trigonal space groups.

The 13 trivariant lattice complexes with trigonal symmetry are compatible with 218 types of homogeneous sphere packings, 7 types of interpenetrating sphere packings and one type of interpenetrating layers of spheres. Altogether, the lattice complexes with trigonal characteristic space group (with 0, 1, 2 or 3 degrees of freedom) give rise to 225 types of sphere packing. 110 of these types are compatible exclusively with one of the 13 trivariant lattice complexes, 31 in addition with some of the invariant, univariant or bivariant lattice complexes, whereas 6 types occur solely in such a lattice complex. 65 types encompass special sphere packings that can also be generated with hexagonal symmetry [Sowa, Koch & Fischer (2003). Acta Cryst. A59, 317-326; Sowa & Koch (2004). Acta Cryst. A60, 158-166; Sowa & Koch (2005). Acta Cryst. A61, 331-342]; cubic inherent symmetry occurs for certain sphere packings [Fischer (2004). Acta Cryst. A60, 246-249] belonging to 13 types. The maximal inherent symmetry is trigonal for 147 of the 225 types. The sphere packings of 61 types can be subdivided into connected layer-like subunits, those of 86 types into connected rod-like subunits. Such subunits may be used to construct some kind of ;descriptive symbols' that reflect certain properties of the sphere packings. Interpenetrating sphere packings with cubic inherent symmetry belong to one of the 7 types. All interpenetrating sphere layers that belong to the only type occurring in the trigonal crystal system show hexagonal inherent symmetry. Some examples depict crystal structures that can be described by means of sphere packings.

Orthorhombic sphere packings. I. Invariant and univariant lattice complexes.

All homogeneous sphere packings and all interpenetrating sphere packings were derived that refer to the 6 invariant and the 11 univariant lattice complexes belonging to the orthorhombic crystal system. In total, sphere packings of 38 types have been found. Only for 17 types is the maximal inherent symmetry of their sphere packings orthorhombic. By means of a number of examples, the applicability of sphere packings for the comparison and description of simple crystal structures is demonstrated.

Interpenetration of homogeneous sphere packings and of two-periodic layers of spheres.

All systems of interpenetrating sphere packings that occur with highest symmetry in the cubic, hexagonal or tetragonal crystal family are tabulated. Homogeneous sphere packings belonging to 49 different types may be intertwined to systems of interpenetrating sphere packings belonging to 74 types. For all compatible lattice complexes, the coordinate and lattice parameters are given. The corresponding patterns of interpenetration are analysed. For the interpenetration of two, three, four, five and eight sphere packings, eleven, three, five, one and two different patterns, respectively, are distinguished. In addition, four types of interpenetrating layers of spheres were found. Each such sphere configuration splits into two or three subsets of parallel sphere layers with an angle of 90 degrees or of 120 degrees , respectively, between the directions of the normals of the layers. A single sphere layer corresponds either to a honeycomb net or to a net built up from quadrangles and octagons.

Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway.

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.