Structural Insight into the Self-Assembly of a Pharmaceutically Optimized Insulin Analogue Obtained by Small-Angle X-ray Scattering.

B29Nε-lithocholyl-γ-l-ßGlu-desB30 human insulin [NN344] belongs to a group of insulins with fatty acid or sterol modifications. These insulin analogues have been found to form subcutaneous depots upon injection and hereby have a protracted release profile in vivo. In the present study, B29Nε-lithocholyl-γ-l-Glu-desB30 human insulin was investigated using in-solution small-angle X-ray scattering (SAXS) at chemical conditions designed to mimic three stages during treatment in vivo: in-vial/pen, postinjection, and longer times after injection. We found that the specific insulin analogue formed a mixture of mono- and dihexamers under in-vial/pen conditions of low salt and stabilizing phenol. At postinjection, conditions mimicking a subcutaneous depot, B29Nε-lithocholyl-γ-l-Glu-desB30 human insulin, formed very long straight soluble hexamer-based rods stacked along the Zn(II)-axis. The self-assembly was triggered by an increase in salt concentration when going from vial to physiological conditions. Mimicking longer times after injection and the additional removal of phenol caused the length of the rods to decrease significantly. Finally, we found that the self-assembly could be controlled by varying the amount of modification at the interaction interface by making mixed hexamers of B29Nε-lithocholyl-γ-l-Glu-desB30 and desB30 human insulin. This opens extra possibilities for controlling the release profile of very-long-acting insulins.

A de Novo-Designed Monomeric, Compact Three-Helix-Bundle Protein on a Carbohydrate Template.

De novo design and chemical synthesis of proteins and of other artificial structures that mimic them is a central strategy for understanding protein folding and for accessing proteins with new functions. We have previously described carbohydrates that act as templates for the assembly of artificial proteins, so-called carboproteins. The hypothesis is that the template preorganizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from the template could facilitate protein folding. Here we report the design and synthesis of three-helix-bundle carboproteins on deoxyhexopyranosides. The carboproteins were analyzed by CD, analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and NMR spectroscopy, and this revealed the formation of the first compact and folded monomeric carboprotein, distinctly different from a molten globule. En route to this carboprotein we observed a clear effect originating from the template on protein folding.

Self-assembly of designed coiled coil peptides studied by small-angle X-ray scattering and analytical ultracentrifugation.

α-Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix-containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small-angle X-ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self-assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self-assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC.

Microwave heating in solid-phase peptide synthesis.

The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins - mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(α)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form ß-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of ß-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).

Perfluoroalkyl chains direct novel self-assembly of insulin.

The self-assembly of biopharmaceutical peptides into multimeric, nanoscale objects, as well as their disassembly to monomers, is central for their mode of action. Here, we describe a bioorthogonal strategy, using a non-native recognition principle, for control of protein self-assembly based on intermolecular fluorous interactions and demonstrate it for the small protein insulin. Perfluorinated alkyl chains of varying length were attached to desB30 human insulin by acylation of the ε-amine of the side-chain of LysB29. The insulin analogues were formulated with Zn(II) and phenol to form hexamers. The self-segregation of fluorous groups directed the insulin hexamers to self-assemble. The structures of the systems were investigated by circular dichroism (CD) spectroscopy and synchrotron small-angle X-ray scattering. Also, the binding affinity to the insulin receptor was measured. Interestingly, varying the length of the perfluoroalkyl chain provided three different scenarios for self-assembly; the short chains hardly affected the native hexameric structure, the medium-length chains induced fractal-like structures with the insulin hexamer as the fundamental building block, while the longest chains lead to the formation of structures with local cylindrical geometry. This hierarchical self-assembly system, which combines Zn(II) mediated hexamer formation with fluorous interactions, is a promising tool to control the formation of high molecular weight complexes of insulin and potentially other proteins.

Automated 'X-Y' robot for peptide synthesis with microwave heating: application to difficult peptide sequences and protein domains.

Precise microwave heating has emerged as a valuable method to aid solid-phase peptide synthesis (SPPS). New methods and reliable protocols, as well as their embodiment in automated instruments, are required to fully use this potential. Here we describe a new automated robotic instrument for SPPS with microwave heating, report protocols for its reliable use and report the application to the synthesis of long sequences, including the beta-amyloid 1-42 peptide. The instrument is built around a valve-free robot originally developed for parallel peptide synthesis, where the robotic arm transports reagents instead of pumping reagents via valves. This is the first example of an 'X-Y' robotic microwave-assisted synthesizer developed for the assembly of long peptides. Although the instrument maintains its capability for parallel synthesis at room temperature, in this paper, we focus on sequential peptide synthesis with microwave heating. With this valve-free instrument and the protocols developed for its use, fast and efficient syntheses of long and difficult peptide sequences were achieved.

Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis.

A class of small molecules displaying comparable activities with peptide ligands BAM22 and corticostatin-14 at both the human and rhesus monkey MrgX1 and MrgX2 receptors, respectively, was discovered. A comparative study to compare solid-phase and solution-phase chemistries for the efficient synthesis of the active class, tetracyclic benzimidazoles, was undertaken. The solid-phase chemistry was found to be superior both for the synthesis of analogs and for the synthesis of gram quantities.

Identification and functional validation of PNAs that inhibit murine CD40 expression by redirection of splicing.

Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.

Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid.

Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages. On the basis of this peptide motif, the present study aimed at identifying the structural features, which define effective peptide carriers for cellular delivery of PNA. While the structure-activity relationship study revealed some clear correlations, only a few modifications actually led to an overall improvement as compared to the parent octa(l-lysine) conjugate. In a preliminary PK/tissue distribution study in healthy mice, the parent conjugate exhibited relatively broad tissue distribution and only modest elimination via excretion within the time frame of the study.

Microwave-assisted solid-phase peptide synthesis using the biotage Syro Wave™.

Fast and precise heating by microwave irradiation during solid-phase peptide synthesis (SPPS) can reduce reaction times as well as provide better purities and greater yields for the synthesis of difficult peptides. Microwave- assisted SPPS has proven to be a useful and reliable tool for the synthesis of peptides as well as small proteins. It is particularly well suited for sequences with a high propensity to form ß-sheet-type structures and for sterically difficult couplings. In this protocol, conditions and detailed procedures are described for performing microwave-assisted SPPS using the Syro Wave™. Here we describe the synthesis of two difficult peptide sequences: the first is derived from the C-terminus of the MuLV CTL epitope, the second is a de novo designed peptide with a C-terminal alkyne.

Synthesis and evaluation of optically pure dioxolanes as inhibitors of hepatitis C virus RNA replication.

A series of optically pure 1,3-dioxolane nucleoside mimics was synthesized by a synthetic route that allowed incorporation of a 5R-methyl substituent from commercially available starting materials. The pyrrolo[2,3-d]pyrimidine heterocycle was chosen as a substitute for the purine derivative. Coupling of the pyrrolo[2,3-d]pyrimidine and the dioxolane was performed under solid-liquid phase transfer conditions. The ability to inhibit HCV RNA replication was assessed in a cell based subgenomic replicon assay. None of the described compounds displayed significant anti-HCV activity.

Synthesis and biological evaluation of 5R- and 5S-methyl substituted D- and L-configuration 1,3-dioxolane nucleoside analogs.

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-methyldioxolane. Dioxolanes of alpha-D-, beta-D-, alpha-L-, and beta-L-configuration were prepared, that included 5-methyl derivatives of both 5R and 5S configuration. Molecular mechanics calculations indicate that the 5S and 5R diastereoisomeric 1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic replicon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-dioxolanes were found to be inactive.