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Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent 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.

CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro . D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a “hot spot” for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.

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.

Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic “match” between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA 0 precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA 0 vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA 0 , the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA 0 conjugate is not as efficacious as for influenza B virus.

The causative agent of a recent outbreak of an atypical pneumonia, known as severe acute respiratory syndrome (SARS), has been identified as a coronavirus (CoV) not belonging to any of the previously identified groups. Fusion of coronaviruses with the host cell is mediated by the envelope spike protein. Two regions within the spike protein of SARS-CoV have been identified, showing a high degree of sequence conservation with the other CoV, which are characterized by the presence of heptad repeats (HR1 and HR2). By using synthetic and recombinant peptides corresponding to the HR1 and HR2 regions, we were able to characterize the fusion-active complex formed by this novel CoV by CD, native PAGE, proteolysis protection analysis, and size-exclusion chromatography. HR1 and HR2 of SARS-CoV associate into an antiparallel six-helix bundle, with structural features typical of the other known class I fusion proteins. We have also mapped the specific boundaries of the region, within the longer HR1 domain, making contact with the shorter HR2 domain. Notably, the inner HR1 coiled coil is a stable α-helical domain even in the absence of interaction with the HR2 region. Inhibitors binding to HR regions of fusion proteins have been shown to be efficacious against many viruses, notably HIV. Our results may help in the design of anti-SARS therapeutics.

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I β turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.

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.

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon-like peptide 1 (GLP-1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose-dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37-amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP-1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment-associated nausea than GLP-1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half-life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP-IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP-IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258-2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use.

A binding assay suitable for the identification of active site-directed inhibitors of the hepatitis C virus serine protease NS3 was developed. A C-terminal extension of 13 residues that is specifically recognized by the Escherichia coli biotin holoenzyme synthetase (Bir A) was fused to a truncated NS3 protease domain, allowing the efficient production of in vivo biotinylated protease. This enzyme was purified and shown to have the same properties as its wild-type counterpart concerning substrate binding and turnover, interaction with a cofactor peptide, and inhibition by three different classes of inhibitors. Immobilization of the biotinylated protease, using streptavidin-coated scintillation proximity beads, allowed detection, by scintillation counting, of its interaction with a tritiated active site ligand spanning the whole substrate binding site of the protease from P6 to P4′. Immobilization did not measurably affect accessibility to either the active site or the cofactor binding site of the protease as judged by the unchanged affinities for a cofactor peptide and for two active site binders. Using the displacement of the radioligand as readout, we were able to set up a rapid, robust, and fully automated assay, suitable for the selective identification of novel active site ligands of the NS3 protease.

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

The complete primary structure of human erythrocyte acylpeptide hydrolase has been determined by using a combination of different mass spectrometric procedures and sequencing techniques. These data allowed us to correct the incomplete nucleotide sequence of the DNF15S2 locus on the short arm of human chromosome 3 at region 21, coding for the enzyme. The protein consists of 732 amino acid residues and is acetylated at the N-terminus. Alkylation experiments on the native enzyme demonstrated that all 17 cysteine residues present in the polypeptide chain are in reduced form. Multiple sequence alignment did not reveal striking similarity with proteases of known tertiary structure with the exception of members of the serine oligopeptidase family. Limited proteolysis experiments generated a C-terminal portion, containing all the catalytic triad elements responsible for proteolytic activity, and an N-terminal domain of unknown function, both still strongly associated in a completely active nicked form. The site of tryptic hydrolysis was identified as Arg193. The secondary structural organization of the protease domain of the enzyme is consistent with the alpha/beta hydrolase fold.

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.