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Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Peptides derived from the heptad repeat 2 (HR2) region of the HIV fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, enfuvirtide, is used for the treatment of therapy-experienced AIDS patients. The mechanism of action of these peptides is binding to a critical intermediate along the virus–cell fusion pathway, and accordingly, increasing the affinity for the intermediate yields more potent inhibitors. We took a different approach, namely to increase the potency of the HR2 peptide inhibitor C34 by targeting it to the cell compartment where fusion occurs, and we show here that a simple, yet powerful way to accomplish this is attachment of a cholesterol group. C34 derivatized with cholesterol (C34-Chol) shows dramatically increased antiviral potency on a panel of primary isolates, with IC 90 values 15- to 300-fold lower than enfuvirtide and the second-generation inhibitor T1249, making C34-Chol the most potent HIV fusion inhibitor to date. Consistent with its anticipated mechanism of action, the antiviral activity of C34-Chol is unusually persistent: washing target cells after incubation with C34-Chol, but before triggering fusion, increases IC 50 only 7-fold, relative to a 400-fold increase observed for C34. Moreover, derivatization with cholesterol extends the half-life of the peptide in vivo. In the mouse, s.c. administration of 3.5 mg/kg C34-Chol yields a plasma concentration 24 h after injection >300-fold higher than the measured IC 90 values. Because the fusion machinery targeted by C34-Chol is similar in several other enveloped viruses, we believe that these findings may be of general utility.

Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001) Proc. Natl. Acad. Sci. USA 98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17) 3 , has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17) 3 ( i ) inhibits fusion in a cell-cell fusion assay (IC 50 = 260 pM); ( ii )is the most potent fusion inhibitor described to date (IC 50 = 40-380 pM) in a single-cycle infectivity assay against HIV HXB2 , HIV NL4-3 , and HIV MN-1 ; ( iii ) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and ( iv ) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17) 3 was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17) 3 an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.

The synthesis of a high affinity mannose receptor ligand, appropriately functionalized for chemoselective ligation with an antigen or DNA-binding moieties is described. By a combination of solid- and solution-phase chemistry a versatile synthesis of the target structure was accomplished. Examples of subsequent ligation reactions are described. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

Infection by Hepatitis C Virus (HCV) leads to a slowly progressing disease that over two decades can lead to liver cirrhosis or liver cancer. Currently, one of the most promising approaches to anti-HCV therapy is the development of inhibitors of the NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors of NS3/4A have been described, all taking advantage of binding to the S subsite of the enzyme. Inspection of the S' subsite of NS3/4A shows binding pockets which might be exploited for inhibitor binding, but due to the fact that ground-state binding to the S' subsite is not used by the substrate, this does not represent a suitable starting point. We have now optimized S'-binding in the context of noncleavable decapeptides spanning P6-P4'. Binding was sequentially increased by introduction of the previously optimized P-region [Ingallinella et al. (1998) Biochemistry 37, 8906-8914], change of the P4' residue, and combinatorial optimization of positions P2'-P3'. The overall process led to an increase in binding of more than 3 orders of magnitude, with the best decapeptide showing IC(50)200 pM. The binding mode of the decapeptides described in the present work shares features with the binding mode of the natural substrates, together with novel interactions within the S' subsite. Therefore, these peptides may represent an entry point for a novel class of NS3 inhibitors.

We report the synthesis and the characterization of N-(4-aminobutyl)-N- ethyl-isoluminol (ABEI) macrocyclic lactone, an activated ester with an unusual macrocyclic structure, and its use for efficient ABEI conjugation to proteins. Compared to the equivalent reagent normally used for chemiluminescence protein labeling, the ABEI macrocyclic lactone displays improved chemical properties, including stability and reactivity. We show the simple synthesis and the use of ABEI macrocyclic lactone for efficient chemiluminescence labeling of monoclonal antibody mixtures currently used in clinical immunodiagnostic assays for the detection of the HIV p24 antigen in patients. © 2013 Elsevier Ltd. All rights reserved.

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

Much effort for a therapy against hepatitis C virus (HCV) is devoted to the search of inhibitors of the virally-encoded protease NS3, which is required for maturation of HCV polyprotein [1]. In order to cleave its substrates NS3 must form a complex with the 54residue viral cofactor protein NS4A, whose activity is mimicked by a synthetic peptide (Pep4A) corresponding to amino acids 21-34 [1]. Binding of Pep4A was shown to induce important tertiary structure changes in the enzyme [2]. We have recently developed substrate-derived hexapeptide inhibitors of NS3 with affinities in the low nanomolar range [3]. Structure-activity relationships, molecular modelling, site-directed mutagenesis and most recently NMR have been used to gain knowledge about the salient features of inhibitor binding [3-5]. In the present work we have further studied the interaction between NS3, NS4A and the inhibitors, and found that major conformational changes take place in the enzyme upon binary and ternary complex formation.

Hairpin polyamides are high-affinity, sequence selective DNA binders. The use of a safety-catch linker for the solid phase synthesis of hairpin polyamides allows for easy preparation of derivatives ready for chemoselective ligation with unprotected peptides. Examples of ligations reported include thioether bond formation and thioester-mediated amide bond formation ('Native Chemical Ligation').

One of the most promising approaches to anti-HCV therapy is the development of inhibitors of the viral NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors have been described [1], all taking advantage of binding to the S site of the enzyme. By studying the interaction of these inhibitors with NS3 [2], we showed that binding occurs according to an induced-fit mechanism. In the absence of a cofactor, multiple binding modes are apparent, while in the presence of a cofactor all P-inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by CD spectroscopy. Inhibitor complex formation is associated with stabilization of the enzyme, as highlighted by limited proteolysis experiments.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.

The interactions of peptide inhibitors, obtained by the optimization of Nterminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel b-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole. # 1999 Academic Press

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkuhler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.