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.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl 1 ]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES {N-nonanoyl, des-Ser 1 [L-thioproline 2 , L-cyclohexylglycine 3 ]-RANTES(2-68)}, which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1 H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel sheet, where the last two strands form a hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.Obesity and associated disorders such as diabetes are now at epidemic levels in the United States and other developed countries (1, 2). An understanding at the molecular level of the normal processes governing energy homeostasis and weight regulation is therefore essential for developing a comprehensive understanding of these disorders and producing effective pharmaceutical treatments. Recent studies have demonstrated the key role played by brain melanocortin receptors (MCRs) in the regulation of energy homeostasis (3-6). Although MCRs are found in various tissues, these studies indicate that the specific receptors MC3R and MC4R are directly implicated in energy balance. All MCRs are G-protein-coupled receptors (GPCRs) that up-regulate the production of cAMP in the presence of small endogenous peptide agonists (7). These agonists are derived in vivo from the single pro-opiomelanocortin peptide (POMC) that is cleaved post-translationally to form the melanocortinstimulating hormones (MSHs) and the adrenocorticotropin hormone (ACTH). MCR function is also modulated by potent endogenous antagonists known as the agouti protein and the agouti-related protein (AGRP) that indeed may exert even greater control over MCR signaling than the peptide agonists (8-11). Relative to the small peptide agonists, these antagonists are larger in size and possess a five-dis...