Design and Synthesis of Potent, Long-Acting Lipidated Relaxin-2 Analogs.

Peptide hormone relaxin-2, a member of the insulin family of peptides, plays a key role in hemodynamics and renal function and has shown preclinical efficacy in multiple disease models, including acute heart failure, fibrosis, preeclampsia, and corneal wound healing. Recently, serelaxin, a recombinant version of relaxin-2, has been studied in a large phase 3 clinical trial (RELAX-AHF-2) for acute decompensated heart failure patients with disappointing outcome. The poor in vivo half-life of relaxin-2 may have limited its therapeutic efficacy and long-term cardiovascular benefit. Herein, we have developed a semisynthetic methodology and generated potent, fatty acid-conjugated relaxin analogs with long-acting pharmacokinetic (PK) profile in rodents. The enhanced PK properties translated into improved and long-lasting pharmacodynamic effect in pubic ligament elongation (PLE) studies. The resultant novel relaxin analog, R9-13, represents the first long-acting relaxin-2 analog and could potentially improve the clinical efficacy and outcome for this important peptide hormone. This semisynthetic methodology could also be applied to other cysteine-rich peptides and proteins for half-life extension.

The Dependence of Carbohydrate-Aromatic Interaction Strengths on the Structure of the Carbohydrate.

Interactions between proteins and carbohydrates are ubiquitous in biology. Therefore, understanding the factors that determine their affinity and selectivity are correspondingly important. Herein, we have determined the relative strengths of intramolecular interactions between a series of monosaccharides and an aromatic ring close to the glycosylation site in an N-glycoprotein host. We employed the enhanced aromatic sequon, a structural motif found in the reverse turns of some N-glycoproteins, to facilitate face-to-face monosaccharide-aromatic interactions. A protein host was used because the dependence of the folding energetics on the identity of the monosaccharide can be accurately measured to assess the strength of the carbohydrate-aromatic interaction. Our data demonstrate that the carbohydrate-aromatic interaction strengths are moderately affected by changes in the stereochemistry and identity of the substituents on the pyranose rings of the sugars. Galactose seems to make the weakest and allose the strongest sugar-aromatic interactions, with glucose, N-acetylglucosamine (GlcNAc) and mannose in between. The NMR solution structures of several of the monosaccharide-containing N-glycoproteins were solved to further understand the origins of the similarities and differences between the monosaccharide-aromatic interaction energies. Peracetylation of the monosaccharides substantially increases the strength of the sugar-aromatic interaction in the context of our N-glycoprotein host. Finally, we discuss our results in light of recent literature regarding the contribution of electrostatics to CH-π interactions and speculate on what our observations imply about the absolute conservation of GlcNAc as the monosaccharide through which N-linked glycans are attached to glycoproteins in eukaryotes.

Sialoside Arrays: New Synthetic Strategies and Applications.

Sialic acid-containing carbohydrates, or sialosides, play critical roles in many biological events and in diseases, including viral and bacterial infections, the immune response, the progression of tumor cell metastasis, etc. Despite the importance, the limited access to complex sialosides had prevented extensive studies on the function and significance of sialic acid structural diversity. However, recent advances in synthetic sialoside chemistry, such as the novel chemoenzymatic or stereochemical approach, have produced homogeneous size- and structure-defined sialosides to create diverse sialosides for array application. The advantage of sialoside arrays is the multivalent display of arrayed sialosides which can serve to mimic cell surface display; thus, an array-based technique is well suited for investigations of the real sialoside-mediated interactions in nature. In brief, this chapter discusses the novel strategies for synthesizing sialosides with selected examples of applications to illustrate the potential of sialoside arrays and further forecast to the trend of using nanotechnology in sialoside arrays.

Iminosugar C-glycoside analogues of α-D-GlcNAc-1-phosphate: synthesis and bacterial transglycosylase inhibition.

We herein describe the first synthesis of iminosugar C-glycosides of α-D-GlcNAc-1-phosphate in 10 steps starting from unprotected D-GlcNAc. A diastereoselective intramolecular iodoamination-cyclization as the key step was employed to construct the central piperidine ring of the iminosugar and the C-glycosidic structure of α-D-GlcNAc. Finally, the iminosugar phosphonate and its elongated phosphate analogue were accessed. These phosphorus-containing iminosugars were coupled efficiently with lipophilic monophosphates to give lipid-linked pyrophosphate derivatives, which are lipid II mimetics endowed with potent inhibitory properties toward bacterial transglycosylases (TGase).

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets.

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Glycans on influenza hemagglutinin affect receptor binding and immune response.

Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.

Toward automated oligosaccharide synthesis.

Carbohydrates have been shown to play important roles in biological processes. The pace of development in carbohydrate research is, however, relatively slow due to the problems associated with the complexity of carbohydrate structures and the lack of general synthetic methods and tools available for the study of this class of biomolecules. Recent advances in synthesis have demonstrated that many of these problems can be circumvented. In this Review, we describe the methods developed to tackle the problems of carbohydrate-mediated biological processes, with particular focus on the issue related to the development of the automated synthesis of oligosaccharides. Further applications of carbohydrate microarrays and vaccines to human diseases are also highlighted.

Differential receptor binding affinities of influenza hemagglutinins on glycan arrays.

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.

Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode.

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 A resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB-dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB-ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L45 plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA-PriB-ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.