We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. . NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family.

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. In vitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. In vivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated. The introduction of i to i + 4 glutamic acid-lysine lactam constraints in c[Glu (18)-Lys (22)][Gly (8)]GLP-1(7-37)-NH2 (6), c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-NH2 (10), and c[Glu (23)-Lys (27)][Gly (8)]GLP-1(7-37)-NH2 (11) resulted in potent functional activity and receptor affinities comparable to native GLP-1. Selected GLP-1 peptides were chemoselectively PEGylated in order to prolong their in vivo activity. PEGylated peptides [Gly (8),Aib (22)]GLP-1(7-37)-Cys ((PEG))-Ala-NH2 (23) and c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-Cys ((PEG))-Ser-Gly-NH2 (24) retained picomolar functional potency and avid receptor binding properties. Importantly, PEGylated GLP-1 peptide 23 exhibited sustained in vivo efficacy with respect to blood glucose reduction and decreased body weight for several days in nonhuman primates.

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Conotoxins are cysteine-rich peptides from the venom of the predatory marine snail of the genus Conus. These toxins are classified according to their primary structure and biological activity and include the ␣-conotoxin class, which possess a two-loop framework containing two disulfide bonds and are specific inhibitors of nicotinic acetylcholine receptors (nAChRs).1 Native neuronal nAChRs are composed from a number of distinct subunits (␣2-␣7 and ␣9; ␤2-␤4), which combine to form functional receptors showing a range of pharmacological properties. PnIA and PnIB 2 are 16-residue peptides isolated from the venom of the molluscivorous Conus pennaceus that differ by two amino acids at positions 10 and 11 (see Table I). PnIA and PnIB were originally reported to block ACh-evoked responses in Aplysia neurons (1) and, more recently, to exhibit activity in bovine adrenal chromaffin cells, but not at the mammalian neuromuscular junction (2).The x-ray crystal structures of both PnIA (6) and PnIB (7) are similar, comprising an ␣-helix between residues 5 and 12, a 3 10 helical turn at the N terminus, and consecutive ␤-turns at the C terminus. In both structures, the side chains of residues 10 and 11 are exposed on the surface of the molecules and hence mutation of these residues would not be expected to produce significant changes in the global fold. Data obtained from 1 H NMR experiments in the current study confirm this is indeed the case. The high degree of surface exposure of these residues and their lack of structural perturbation means that changes in activity among these peptides can be correlated directly to different residue side chains having different binding interactions at different nAChR subtypes.Preliminary studies have shown that PnIA and PnIB differentially inhibit the nicotine-induced catecholamine release from bovine chromaffin cells (2). Differences in potency must be due to the residues at positions 10 and 11 of these conotoxins. Position 10 is of particular interest as this position typically contains different hydrophobic residues in other neuronal nAChR-selective conotoxins, EpI (3), MII (4), and ImI (5). To further elucidate the role of the residues at positions 10 and 11 in conferring nAChR subtype selectivity, the modified toxins [A10L]PnIA and [N11S]PnIA (see Table I) were synthesized. The aim of this study was to determine the selectivity of both the native and modified ␣-conotoxins PnIA and PnIB for the different nAChR subtypes, which constitute the whole-cell ACh-induced current in mammalian peripheral neurons, and to provide information as to the relative contribution of these nAChR subunits to the whole-cell response. These studies reveal significant differences in the selectivity and potency of PnIA and PnIB that arise through a key mutation at position 10 of these ␣-conotoxins. EXPERIMENTAL PROCEDURES Materials for Conotoxin Synthesis-N-Boc-L-amino