Accelerated Nucleation of Hydroxyapatite Using an Engineered Hydrophobin Fusion Protein.

Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.

Hydrophobin can prevent secondary protein adsorption on hydrophobic substrates without exchange.

By combining several surface analytical tools, we show that an adsorbed layer of the protein H*Protein B prevents the adsorption of secondary proteins bovine serum albumin, casein, or collagen at low-salinity conditions and at pH 8. H*Protein B is an industrially producible fusion protein of the hydrophobin family, known for its high interfacial activity. While applications of hydrophobin have been reported to facilitate adhesion of proteins under different pH conditions, careful analysis by quartz-crystal microbalance and ellipsometry prove that no additional adsorption can be found on top of the H*Protein B layer in this study. Surface analysis by X-ray photoelectron spectroscopy and secondary ion mass spectrometry proves that the hydrophobin layer stays intact even after hours of exposure to solutions of the secondary proteins and that no exchange of proteins can be detected.

Recombinantly produced hydrophobins from fungal analogues as highly surface-active performance proteins.

Hydrophobins are available from natural resources only in milligram amounts. BASF succeeded in a recombinant production process, up-scaled to pilot plant production in kilogram scale. Strain and protein optimization by modulation of gene expression and generation of fusion proteins finally leads to two class I hydrophobins called H*Protein A and H*Protein B. By analytical ultracentrifugation, we confirm that the self-association of H*Proteins in solution is governed by their sequence, because oligomerization is induced by the same mechanisms (pH > 6, temperature >> 5 degrees C, concentration > 0.2 mg/ml) as for the well-known native hydrophobins SC3 and HFB II. Additionally, we established the triggering of structure formation by bridging with divalent ions and the stabilization of dimers and tetramers by monovalent ions or surfactants. This interplay with surfactants can be exploited synergistically: The capacity for emulsification of a 300 ppm standard surfactant solution is boosted from 0 to 100% by the addition of a mere 1 ppm of our new hydrophobins, with H*Protein A and H*Protein B having specific application profiles. This astonishing performance is rationalized by the finding that the same minute admixtures enhance significantly the interfacial elastic modulus, thus stabilizing interfaces against coalescence and phase separation.

Design and synthesis of novel potent and selective integrin alphanubeta3 antagonists--novel synthetic routes to isoquinolinone, benzoxazinone, and quinazolinone acetates.

An unexpected ring contraction of benzazepinone based alpha(nu)beta(3) antagonists led to the design of quinolinone-type derivatives. Novel and efficient synthetic routes to isoquinolinone, benzoxazinone, and quinazolinone acetates were established. Nanomolar alpha(nu)beta(3) antagonists based on these new scaffolds were prepared. Moreover, benzoxazinones 15a and 15b exhibited high microsomal stability and good permeability.

Adsorption and rheological behavior of an amphiphilic protein at oil/water interfaces.

Hydrophobins are highly surface active proteins which self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. We investigate hydrophobin self-assembly at oil/water interfaces to deepen the understanding of protein behavior in order to improve our biomimetic synthesis. Therefore, we carried out pendant drop measurements of hydrophobin stabilized oil/water systems determining the time-dependent IFT and the dilatational rheology with additional adaptation to the Serrien protein model. We show that the class I hydrophobin H(∗)Protein B adsorbs at an oil/water interface where it forms a densely-packed interfacial protein layer, which dissipates energy during droplet oscillation. Furthermore, the interfacial protein layer exhibits shear thinning behavior.

On the incorporation of functionalities into hydroxyapatite capsules.

Nature is capable of building materials with tailor-made properties under ambient conditions for specific applications. We apply some of the basic building principles of biomineralization in this paper: we stabilize an oil/water emulsion with the protein hydrophobin and mineralize this emulsion, resulting in hollow mineral capsules. The use of an emulsion as a liquid template enables precise size control over the final capsules. We mimic nature by using complexing agents and surfactants as additives to alter the properties of the growing mineral. We also modify the mineral itself by addition of different cations. Furthermore, we show the inclusion of silver into the capsules. This should add antibacterial properties to the capsules and shows exemplarily that catalytically active metals can be included. While the manual process needs numerous working steps and long waiting times, we ease the whole process by automation and use phosphatases to shorten synthesis time. Our experiments show the flexibility and adaptability of our system, making it an ideal platform for various possible applications such as drug transport and especially as microreactors.

Use of laccase as a novel, versatile reporter system in filamentous fungi.

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

Activity of the endothelin system in kidney allograft recipients is not associated with progression of chronic graft dysfunction.

Endothelin (ET) A receptor antagonists have been shown to be beneficial in rat models of chronic kidney allograft dysfunction. We investigated urinary and plasma ET-1 (uET-1, pET-1) and BigET-1 (uBigET-1, pBigET-1) concentrations, and plasma soluble ET-converting enzyme (ECE) concentration in 310 adult Caucasian kidney allograft recipients with graft survival of more than 2 years from the outpatients department of our clinic. All patients were on cyclosporine A- or FK506-based immunosuppression protocols. From all available measurements since transplantation, we calculated the slope of serum creatinine(-1)/year (slopeCrea) as a parameter for progression of chronic graft dysfunction, as well as the mean of serum creatinine (meanCrea) from most recent year before measurements as a parameter for actual graft function. The slope of urinary protein excretion/year (slopeProt) and mean of urinary protein concentration (meanProt) from most recent year was calculated analogue. uET-1 and uBigET-1 were adjusted for protein excretion by calculating uET-1/meanProt and uBigET-1/meanProt. Blood and urine probes for measurements were always drawn immediately before morning dosage of immunosuppressants. There was no significant correlation of any measured component of the ET system with slopeCrea or slopeProt. MeanCrea (mg/dl) was significantly correlated with pBigET-1 (fmol/ml) and pET-1 (fmol/ml) (pBigET-1: r=0.179, P=0.001; pET-1: r=0.161, P=0.009). The other measured components of the ET systems were not significant correlated with meanCrea. In conclusion, the actual graft function is associated with elevated pET-1 and BigET-1 concentrations as it is well known from other forms of impaired kidney function. However, the actual concentration of ET-1, soluble ECE, and BigET-1 in urine and plasma in our study is not associated with parameters for progression of chronic graft dysfunction.

Interaction of the endothelin system and calcineurin inhibitors after kidney transplantation.

Plasma endothelin (ET)-1 concentrations have been shown to be elevated in patients receiving calcineurin-inhibitors (CI). We investigated urinary and plasma ET-1 (uET-1, pET-1), BigET-1 (uBigET-1, pBigET-1) concentrations, and plasma soluble endothelin-converting enzyme (ECE) concentrations in 381 adult caucasian kidney allograft recipients with graft survival of more than 2 years from the outpatients department of our clinic. Blood and urine probes were always drawn immediately before morning dosage of immunosuppressants. Mean of urinary protein excretion (meanProt) and mean of serum creatinine (meanCrea) were calculated from all available measurements in the most recent year. uET-1 and uBigET-1 were adjusted for urinary protein excretion by calculating uET-1/meanProt and uBigET-1/meanProt. Patients (n=310) were on a cyclosporine A or FK506 (CI-group) based immunosuppression protocol, and 71 patients were immunosuppressed without use of CI (nonCI group). Time since transplantation was similar in both groups (mean+/-S.D.; CI-group: 7.55+/-2.50; nonCI-group: 7.74+/-3.06 years, P=0.240) as well as meanCrea (mean+/-S.D.; CI-group: 1.97+/-1.34; nonCI-group: 1.77+/-1.29 mg/dl, P=0.326). pET-1 was significantly higher in the CI-group, compared with nonCI (mean+/-S.D.; 0.87+/-1.4 versus 0.56+/-0.76 fmol/ml, P=0.011). pBigET-1 was similar (mean+/-S.D.; 0.85+/-1.41 versus 0.70+/-1.21 fmol/ml, P=0.33). ECE concentrations were higher in the CI group (mean+/-S.D.; 14.30+/-18.02 versus 9.23+/-7.42 ng/ml, P=0.001). uET-1/meanProt and uBigET-1/meanProt concentration were similar in the CI-group compared with the nonCI-group (mean+/-S.D.; uET-1/meanProt: 15+/-24 versus 21+/-40 pmol/g, P=0.139; uBigET-1/meanProt: 34+/-55 versus 19+/-23pmol/g, P=0.248). pET-1 elevation in patients receiving CI might be more likely to be due to elevated conversion of pBig-ET-1 by more ECE, and not to higher concentrations of pBigET-1.

Synthesis of highly potent and selective hetaryl ureas as integrin alpha(V)beta3-receptor antagonists.

Solid-phase synthesis and SAR of integrin alpha(V)beta3-receptor antagonists containing a urea moiety as non-basic guanidine mimetic are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta3 in the nanomolar range and high selectivity versus related integrins like alpha(IIb)beta3. For selected examples efficacy in functional cellular assays is demonstrated.

Design and synthesis of 1,5- and 2,5-substituted tetrahydrobenzazepinones as novel potent and selective integrin alphaVbeta3 antagonists.

The design and synthesis of novel integrin alpha(V)beta(3) antagonists based on a 1,5- or 2,5-substituted tetrahydrobenzaezpinone core is described. In vitro activity of respective compounds was determined via alpha(V)beta(3) binding assay, and selected derivatives were submitted to further characterization in functional cellular assays. SAR was obtained by modification of the benzazepinone core, variation of the spacer linking guanidine moiety and core, and modification of the guanidine mimetic. These efforts led to the identification of novel alpha(V)beta(3) inhibitors displaying potency in the subnanomolar range, selectivity versus alpha(IIb)beta(3) and functional efficacy in relevant cellular assays. A method for the preparation of enantiomerically pure derivatives was developed, and respective enantiomers evaluated in vitro. Compounds 31 and 37 were assessed for metabolic stability, resorption in the Caco-2 assay and pharmacokinetics.

Highly potent and selective alphaVbeta3-receptor antagonists: solid-phase synthesis and SAR of 1-substituted 4-amino-1H-pyrimidin-2-ones.

Solid-phase synthesis and SAR of alpha(V)beta(3)-receptor antagonists based on a N(1)-substituted 4-amino-1H-pyrimidin-2-one scaffold are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta(3) in the nano- to subnanomolar range and high selectivity versus related integrins like alpha(IIb)beta(3). For selected examples efficacy in functional cellular assays was demonstrated.

Synthesis and SAR of N-substituted dibenzazepinone derivatives as novel potent and selective alpha(V)beta(3) antagonists.

Synthesis and SARs of new integrin alpha(V)beta(3) antagonists based on an N-substituted dibenzazepinone scaffold are described. Variation of spacer and guanidine mimetic led to potent compounds exhibiting an IC(50) towards alpha(V)beta(3) in the nanomolar range, high selectivity versus integrin alpha(IIb)beta(3) and efficacy in functional cellular assays.