Home parenteral nutrition with an omega-3-fatty-acid-enriched MCT/LCT lipid emulsion in patients with chronic intestinal failure (the HOME study): study protocol for a randomized, controlled, multicenter, international clinical trial.

BACKGROUND: Home parenteral nutrition (HPN) is a life-preserving therapy for patients with chronic intestinal failure (CIF) indicated for patients who cannot achieve their nutritional requirements by enteral intake. Intravenously administered lipid emulsions (ILEs) are an essential component of HPN, providing energy and essential fatty acids, but can become a risk factor for intestinal-failure-associated liver disease (IFALD). In HPN patients, major effort is taken in the prevention of IFALD. Novel ILEs containing a proportion of omega-3 polyunsaturated fatty acids (n-3 PUFA) could be of benefit, but the data on the use of n-3 PUFA in HPN patients are still limited. METHODS/DESIGN: The HOME study is a prospective, randomized, controlled, double-blind, multicenter, international clinical trial conducted in European hospitals that treat HPN patients. A total of 160 patients (80 per group) will be randomly assigned to receive the n-3 PUFA-enriched medium/long-chain triglyceride (MCT/LCT) ILE (Lipidem/Lipoplus® 200 mg/ml, B. Braun Melsungen AG) or the MCT/LCT ILE (Lipofundin® MCT/LCT/Medialipide® 20%, B. Braun Melsungen AG) for a projected period of 8 weeks. The primary endpoint is the combined change of liver function parameters (total bilirubin, aspartate transaminase and alanine transaminase) from baseline to final visit. Secondary objectives are the further evaluation of the safety and tolerability as well as the efficacy of the ILEs. DISCUSSION: Currently, there are only very few randomized controlled trials (RCTs) investigating the use of ILEs in HPN, and there are very few data at all on the use of n-3 PUFAs. The working hypothesis is that n-3 PUFA-enriched ILE is safe and well-tolerated especially with regard to liver function in patients requiring HPN. The expected outcome is to provide reliable data to support this thesis thanks to a considerable number of CIF patients, consequently to broaden the present evidence on the use of ILEs in HPN. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03282955. Registered on 14 September 2017.

A general strategy for antibody library screening via conversion of transient target binding into permanent reporter deposition.

We report here a generally applicable method for the selective covalent attachment of a reporter molecule to a replicating entity that allows one to obtain specific binders from a single round of library screening. We show that selective biotinylation of phage particles displaying a binder to any given target can be achieved by application of a coupled enzyme reaction on the surface of the target-binding phage particles that includes a peroxidase, an oxidase and a catalase. Due to the covalent linkage of biotin together with the tight and stable interaction of biotin with streptavidin, very stringent wash conditions for removal of nonspecific binders can be applied. The method termed (3)CARD (triple catalytic reporter deposition) was successfully applied to single-round screening of a phage display library of camelid single-domain antibodies against three different target proteins.

Protein production in Yarrowia lipolytica via fusion to the secreted lipase Lip2p.

We established a strategy for protein production and purification via expression in Yarrowia lipolytica as Lip2p fusion protein. To evaluate the expression system a cysteine-rich miniprotein, an antibody fragment and an enzyme showing galactose oxidase activity were chosen. These proteins have varying disulfide bond content, size, and structural complexity. Endogenous lipase Lip2p was used as a fusion partner to direct the fused proteins to the extracellular medium. A linker sequence was introduced at the junction of Lip2p and the respective fused protein that contains a hexahistidine tag followed by a TEV protease cleavage site. This allows for a specific and simple purification via IMAC for capturing the secreted proteins from the supernatant followed by a second IMAC for removing all contaminants after proteolytic release of the protein of interest. Up to 174 mg/L fusion protein was obtained using shake flask cultivation. Functionality of each of the purified proteins was confirmed by individual assays. Expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for an online monitoring of accumulation of secreted fusion proteins in the medium by exploiting the intrinsic lipase activity of the fusion.

Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1.

Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.

Combinatorial tuning of peptidic drug candidates: high-affinity matriptase inhibitors through incremental structure-guided optimization.

Herein we report a convenient strategy for the development of novel, highly-potent peptidic inhibitors of the trypsin-like serine protease matriptase based on the monocyclic variant of the sunflower trypsin inihibitor-1 (SFTI-1[1,14]). We screened SFTI-1[1,14] variants possessing incremental modifications of the parent peptide for beneficial binding properties. This compound library comprising 6 peptides and 16 triazole-containing peptidomimetics was established via structure-guided rational design and synthesized using a divergent strategy employing "copper-click" chemistry. The most favorable amino acid substitutions were combined in one framework yielding potent SFTI-1-derived matriptase inhibitor-1 (SDMI-1) and the truncated dodecapeptide variant (SDMI-2) with single-digit nanomolar inhibition constants. In silico studies indicated that the improved matriptase affinity compared to the parent peptide is caused by the successful establishment of additional favorable proton donor-acceptor interactions between basic inhibitor side chains and acidic residues on the surface of the target enzyme. SDMI-1 and 2 are potent inhibitors of the pharmaceutically relevant protease matriptase at a near physiological pH and, thus, may find applications in therapy or diagnostics.

Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III).

In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.

Oxidative folding of peptides with cystine-knot architectures: kinetic studies and optimization of folding conditions.

Bioactive peptides often contain several disulfide bonds that provide the main contribution to conformational rigidity and structural, thermal, or biological stability. Among them, cystine-knot peptides-commonly named "knottins"-make up a subclass with several thousand natural members. Hence, they are considered promising frameworks for peptide-based pharmaceuticals. Although cystine-knot peptides are available through chemical and recombinant synthetic routes, oxidative folding to afford the bioactive isomers still remains a crucial step. We therefore investigated the oxidative folding of ten protease-inhibiting peptides from two knottin families, as well as that of an HIV entry inhibitor and of aprotinin, under two conventional sets of folding conditions and by a newly developed procedure. Kinetic studies identified folding conditions that resulted in correctly folded miniproteins with high rates of conversion even for highly hydrophobic and aggregation-prone peptides in concentrated solutions.

Between two worlds: a comparative study on in vitro and in silico inhibition of trypsin and matriptase by redox-stable SFTI-1 variants at near physiological pH.

A comparative study on in vitro and in silico inhibition of trypsin and matriptase by derivatives of the sunflower trypsin inhibitor-1 at near physiological pH is reported. Besides wild-type bicyclic SFTI-1, monocyclic variants possessing native cystine as well as redox-stable triazolyl side-chain macrocyclization motifs were studied for the first time in matriptase inhibition assays. Interestingly, monocyclic SFTI-1[1,14] demonstrated higher potency against this pharmacologically relevant protease compared to its bicyclic counterpart. Structural analysis of binding/inhibition of investigated SFTI-1 derivatives was performed using a combination of molecular dynamics simulations and docking experiments. In silico data were in good accordance with in vitro results, indicating the importance of the terminal inhibitor regions for the affinity towards matriptase. Presented work gives new perspectives for the optimization of the SFTI-1 framework towards in vivo applications.

From pico to nano: biofunctionalization of cube-octameric silsesquioxanes by peptides and miniproteins.

Polyhedral silsesquioxanes are considered valuable conjugation scaffolds. Nevertheless, only a few examples of silsesquioxane-assembled peptide oligomers have been reported to date. We developed a new bioorthogonal cube-octameric silsesquioxane (COSS) scaffold bearing eight aminooxy coupling sites allowing for the conjugation of diverse peptides via oxime ligation. We found that the coupling efficacy depends on the ligand in view of steric hindrance and electrostatic repulsion. For the first time scaffold-based conjugation of cystine-knot miniproteins having a backbone of about thirty amino acids was successfully accomplished without loss of bioactivity. Atomic force microscopy (AFM) provided further knowledge on the size of COSS verifying them as picoscaffolds growing upon bioconjugation to nano-dimension.