The Effects of Sequence Variation on Genome-wide NRF2 Binding--New Target Genes and Regulatory SNPs.

Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide binding sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126-3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity.

Development of molecules stimulating the activity of KLK3 - an update.

Kallikrein-related peptidase-3 (KLK3, known also as prostate-specific antigen, PSA) is highly expressed in the prostate. KLK3 possess antiangiogenic activity, which we have found to be related to its proteolytic activity. Thus, it may be possible to slow down the growth of prostatic tumors by enhancing this activity. We have developed peptides that enhance the proteolytic activity of KLK3. As these peptides are degraded in circulation and rapidly excreted, we have started to modify them and have succeeded in creating bioactive and more stable pseudopeptides. We have also identified small molecules stimulating the activity of KLK3, especially in synergy with peptides.

Smart Porous Silicon Nanoparticles with Polymeric Coatings for Sequential Combination Therapy.

In spite of the advances in drug delivery, the preparation of smart nanocomposites capable of precisely controlled release of multiple drugs for sequential combination therapy is still challenging. Here, a novel drug delivery nanocomposite was prepared by coating porous silicon (PSi) nanoparticles with poly(beta-amino ester) (PAE) and Pluronic F-127, respectively. Two anticancer drugs, doxorubicin (DOX) and paclitaxel (PTX), were separately loaded into the core of PSi and the shell of F127. The nanocomposite displayed enhanced colloidal stability and good cytocompatibility. Moreover, a spatiotemporal drug release was achieved for sequential combination therapy by precisely controlling the release kinetics of the two tested drugs. The release of PTX and DOX occurred in a time-staggered manner; PTX was released much faster and earlier than DOX at pH 7.0. The grafted PAE on the external surface of PSi acted as a pH-responsive nanovalve for the site-specific release of DOX. In vitro cytotoxicity tests demonstrated that the DOX and PTX coloaded nanoparticles exhibited a better synergistic effect than the free drugs in inducing cellular apoptosis. Therefore, the present study demonstrates a promising strategy to enhance the efficiency of combination cancer therapies by precisely controlling the release kinetics of different drugs.

Electrostatic interaction on loading of therapeutic peptide GLP-1 into porous silicon nanoparticles.

Porous silicon (PSi) nanoparticles' tunable properties are facilitating their use at highly challenging medical tasks such as peptide delivery. Because of many different mechanisms that are affecting the interaction between the peptide and the particle, the drug incorporation into the mesoporous delivery system is not straightforward. We have studied the adsorption and loading of incretin hormone glucagon like peptide 1 (GLP-1) on PSi nanoparticles. The results show that the highest loading degree can be achieved in pH values near the isoelectric point of peptide, and the phenomenon is independent of the surface's zeta potential. In order to study the interaction between the peptide and the nanoparticle, we studied the adsorption with lower concentrations and noticed that also non-Coulombic forces have a big role in adsorption of GLP-1. Adsorption is effective and pH-independent especially on low peptide concentrations and onto more hydrophobic nanoparticles. Reversibility of adsorption was studied as a function of buffer pH. When the loading is compared to the total mass of the formulation, the loading degree is 29%, and during desorption experiments 25% is released in 4 h and can be considered as a reversible loading degree. Thus, the peptides adsorbed first seem to create irreversibly adsorbed layer that facilitates reversible adsorption of following peptides.

Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 µM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides.

The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

Novel systemically active galanin receptor 2 ligands in depression-like behavior.

Neuropeptide galanin and its three G-protein coupled receptors, galanin receptor type 1-galanin receptor type 3 (GalR1-GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression-like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide-based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant-like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant-like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.

Peptides binding to prostate-specific antigen enhance its antiangiogenic activity.

BACKGROUND: Proteolytically active prostate-specific antigen (PSA or kallikrein-related peptidase 3, KLK3) has been shown to exert antiangiogenic properties. High levels of PSA in prostatic tumors may thus slow down cancer progression by inhibiting angiogenesis. We hypothesize that factors stimulating the activity of PSA could be used to reduce prostate tumor growth. Using phage display, we have developed peptides C4 and B2 that stimulate the enzymatic activity of PSA. Our aim was to study whether these peptides enhance the antiangiogenic activity of PSA. METHODS: We used an in vitro angiogenesis assay where human umbilical vein endothelial cells (HUVECs) form tubular networks when they are grown on Matrigel. Proteolytically active PSA and peptides that stimulate the activity of PSA were added to the cells. Endothelial cell tube formation was quantified and expressed as an angiogenesis index. RESULTS: PSA reduced the angiogenesis index to ∼50% of controls both in serum-containing and serum-free medium. The addition of peptide C4 or B2 together with PSA caused a significant further decrease in angiogenesis index to ∼70% of that caused by PSA alone. A similar decrease in angiogenesis index was observed when PSA concentration was increased 2.4-fold of that used with peptides. CONCLUSIONS: The inhibitory effect of PSA on tube formation can be enhanced by the addition of peptides that stimulate the activity of PSA. This supports our hypothesis that stimulation of PSA activity can be used to reduce angiogenesis and thereby inhibit prostate tumor growth.

[Nanoscale tailoring of the surface properties of biomaterials and drug carriers]. / Nanoteknologia biomateriaalien ja lääkkeiden kantaja-aineiden pintojen räätälöinnissä.

Functionalities of biomaterials and drug delivery systems are improved by tailoring their surface properties using modern nanotechnology. Orthopedic implants and invasive electrodes are examples of implantable biomaterials. Biological interactions of orthopedic implants can be optimized by the synergetic effect of surface micro- and nanotexturing with a chemical composition of coating. Further, mechanical flexibility and electrochemical characteristics of invasive electrodes are improved by using micro- and nanotechnology. In nano-size drug delivery systems, surface properties of nanocarriers strongly affect their safety and efficacy. Mesoporous silicon nanoparticles are example of nanocarriers those properties can be tailored for drug delivery applications.

A robust approach to make inorganic nanovectors biotraceable.

Nuclear medicine imaging plays an important role in nanomedicine. However, it is still challenging to develop a versatile platform to make the nonviral nanovectors used in cancer therapy biotraceable. In the present study, a robust approach to radiolabel inorganic nanovectors for SPECT and PET imaging was developed. The approach was based on the bisphosphonates (BP) conjugated on the nanovector, mesoporous silicon (PSi) nanoparticles. BP served as an efficient chelator for various radionuclides. For both of the 99mTc and 68Ga radionuclides utilized, the radiochemical purity and radiochemical yield were ∼99% and ∼90%, respectively. Because of the short decay time of the radionuclides, an easy, fast and effective PEGylation method was developed to improve the residence time in systemic circulation. Both PEG-99mTc-BP-PSi and PEG-68Ga-BP-PSi NPs, where PEGylation was performed after the labeling, had excellent colloidal and radiochemical stability in vitro. The plain particles without PEGylation accumulated fast in the reticuloendothelial system organs upon intravenous administration, while PEGylation prolonged the residence time of the particles in systemic circulation. Overall, the developed approach proved to be applicable for labeling nonviral nanovectors with various radionuclides easily and robustly. Considering the nature of mesoporous nanoparticles, the approach does not hamper the addition of other functionalities on the vector, nor its capability to carry high payloads.

Allergen-specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy.

Although allergen-specific CD4(+) T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401-restricted responses of peripheral blood-derived memory (CD4(+)CD45RO(+)) and naïve (CD4(+)CD45RA(+)) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2(127-142)-specific memory T cells in the peripheral blood-derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2(127-142)-specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2(127-142)-specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long-term Bos d 2(127-142)-specific T-cell lines generated from both memory and naïve T-cell pools from individuals with allergy proliferated more strongly, produced more IL-4 and IL-10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T-cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen-specific T-cell repertoires differ between individuals with or without allergy.

Biogenic nanoporous silicon carrier improves the efficacy of buparvaquone against resistant visceral leishmaniasis.

Visceral leishmaniasis is a vector-borne protozoan infection that is fatal if untreated. There is no vaccination against the disease, and the current chemotherapeutic agents are ineffective due to increased resistance and severe side effects. Buparvaquone is a potential drug against the leishmaniases, but it is highly hydrophobic resulting in poor bioavailability and low therapeutic efficacy. Herein, we loaded the drug into silicon nanoparticles produced from barley husk, which is an agricultural residue and widely available. The buparvaquone-loaded nanoparticles were several times more selective to kill the intracellular parasites being non-toxic to macrophages compared to the pure buparvaquone and other conventionally used anti-leishmanial agents. Furthermore, the in vivo results revealed that the intraperitoneally injected buparvaquone-loaded nanoparticles suppressed the parasite burden close to 100%. By contrast, pure buparvaquone suppressed the burden only by 50% with corresponding doses. As the conclusion, the biogenic silicon nanoparticles are promising carriers to significantly improve the therapeutic efficacy and selectivity of buparvaquone against resistant visceral leishmaniasis opening a new avenue for low-cost treatment against this neglected tropical disease threatening especially the poor people in developing nations.

Identification of IGFBP-3 fragments generated by KLK2 and prevention of fragmentation by KLK2-inhibiting peptides.

Kallikrein-related peptidase 2 (KLK2) degrades insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in vitro. IGFBP-3 forms complexes with IGFs, preventing them from binding to their receptors and stimulating cell proliferation and survival. IGF-independent actions have also been described for IGFBP-3. The degradation of IGFBP-3 by KLK2 or other proteases in the prostate may promote the growth of prostate cancer. We studied IGFBP-3 degradation by immunoblotting and two specific immunoassays, one recognizing only native non-fragmented IGFBP-3 and the other one recognizing both intact and proteolytically cleaved IGFBP-3. Peptides were used to inhibit the enzyme activity of KLK2 and cleavage sites in IGFBP-3 were identified by mass spectrometry. KLK2 proteolyzed IGFBP-3 into several small fragments, mostly after Arg residues, in keeping with the trypsin-like activity of KLK2. The fragmentation could be inhibited by KLK2-inhibiting peptides in a dose-dependent fashion. As degradation of IGFBP-3 could lead to a more aggressive cancer phenotype, inhibition of KLK2 activity might be useful for treatment of prostate cancer and other diseases associated with increased KLK2 activity.

Identification of novel peptide inhibitors for human trypsins.

Human trypsin isoenzymes share extensive sequence similarity, but certain differences in their activity and susceptibility to inhibitors have been observed. Using phage display technology, we identified seven different peptides that bind to and inhibit the activity of trypsin-3, a minor trypsin isoform expressed in pancreas and brain. All of the peptides contain at least two of the amino acids tryptophan, alanine and arginine, whereas proline was found closer to the N-terminus in all but one peptide. All peptides contain two or more cysteines, suggesting a cyclic structure. However, we were able to make synthetic linear variants of these peptides without losing bioactivity. Alanine replacement experiments for one of the peptides suggest that the IPXXWFR motif is important for activity. By molecular modeling the same amino acids were found to interact with trypsin-3. The peptides also inhibit trypsin-1, but only weakly, if at all, trypsin-2 and -C. As trypsin is a highly active enzyme which can activate protease-activated receptors and enzymes that participate in proteolytic cascades involved in tumor invasion and metastasis, these peptides might be useful lead molecules for the development of drugs for diseases associated with increased trypsin activity.

Mimetics of the disulfide bridge between the N- and C-terminal cysteines of the KLK3-stimulating peptide B-2.

Human prostate produces kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen), which is widely used as a prostate cancer marker. Proteolytically active KLK3 has been shown to inhibit angiogenesis and its expression decreases in poorly differentiated tumors. Thus, it may be possible to control prostate cancer growth with agents that stimulate the proteolytic activity of KLK3. We have earlier developed synthetic peptides, which bind specifically to KLK3 and promote its proteolytic activity. These peptides are cyclic, all containing a disulfide bridge between the N- and C-terminal cysteines. To increase the in vivo stability of the KLK3-stimulating peptide B-2, we made differently cyclized analogues by replacing both terminal cysteines and the disulfide bridge between them. A replacement consisting of gamma-amino butyric acid and aspartic acid, where the amino group from the former was linked to the main chain carboxyl group of the latter, was found to be, at high concentrations, more active than the B-2 peptide. Furthermore, as compared to the parent peptide, this analog had an improved stability in plasma and against the enzymatic degradation by KLK3. In addition, the series of analogues also provided valuable information of the structure-activity relationships of the B-2 peptide.

Immunotherapeutic potential of the immunodominant T-cell epitope of lipocalin allergen Bos d 2 and its analogues.

Lipocalin allergens, which contain most of the important animal-derived respiratory sensitizers, induce T helper type 2 (Th2) deviation, but the reasons for this are not clear. To explore the prospects for peptide-based allergen immunotherapy and to elucidate the characteristics of the immunodominant epitope of Bos d 2, BALB/c mice were immunized with a peptide containing the epitope, peptides containing its analogues, peptides from the corresponding regions of other lipocalin proteins, and peptides with a homologous sequence. We observed that murine spleen cells recognized the immunodominant epitope of Bos d 2, p127-142, in almost the same way as human Bos d 2-specific T cells did. Enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) analyses showed that p127-142 and a corresponding peptide from horse Equ c 1 induced a Th2-deviated cellular response, whereas a homologous bacterial peptide from Spiroplasma citri induced a Th0-type response. Interestingly, the spleen cell response to the bacterial peptide and p127-142 was cross-reactive, that is, able to induce reciprocally the proliferation and cytokine production of primed spleen cells in vitro. More importantly, the peptides were able to skew the phenotype of T cells primed with the other peptide. Our results suggest that modified peptides can be useful in allergen immunotherapy.

The SPECT imaging shows the accumulation of neural progenitor cells into internal organs after systemic administration in middle cerebral artery occlusion rats.

The regenerative potential of stem cells from various sources has been under intense investigation in the experimental models of cerebral ischemia. To end up with a restorative therapeutic treatment, it is crucial to get the cell transplants to the site of injury. Here, we evaluated the feasibility of small animal SPECT/CT in assessing the definite accumulation of (111)In-oxine-labeled human embryonic stem (ES) cell-derived neural progenitors and rat hippocampal progenitors after intravenous or intra-arterial administration (femoral vein vs. common carotid artery) in middle cerebral artery occlusion (MCAO) and sham-operated rats. Cell detection was carried out immediately and 24h after the infusion using a SPECT/CT device. The results showed that after intravenous injections both cell types accumulated primarily into internal organs, instead of brain. In contrast, after intra-arterial injection, a weak signal was detected in the ischemic hemisphere. Additional studies showed that the detection sensitivity of SPECT/CT device was approximately 1000 (111)In-oxine-labeled cells and labeling did not affect the cell viability. In conclusion, a small animal SPECT is powerful technique to study the whole body biodistribution of cell-based therapies. Our data showed that intravenous administration is not an optimal route to deliver neural progenitor cell-containing transplants into the brain after MCAO in rats.

Peptide-mediated interference with baculovirus transduction.

Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells.

Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.

Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella.

The aim of this study was to clarify the growth rates of facultatively anaerobic Salmonella enterica serovar Enteritidis strain in aerobic and anaerobic conditions and the expression of type 1 fimbriae in relation to the growth phases. The cultivation was carried out in a Portable Microbe Enrichment Unit (PMEU) where in same conditions one can grow the cells in parallel by modifying, e.g. aerobiosis only. The results obtained show that although the anaerobic metabolism is generally believed to be a slower producer of biomass or metabolites, in these circumstances S. enterica serovar Enteritidis strain gave comparable growth rates in anaerobiosis with nitrogenation as in aerobic cultures with constant aeration. Fimbrial antigens were produced in the beginning of logarithmic phase of the growth cycle both in the aerobic and anaerobic conditions. The fimbria remained in the presence of oxygen. This capability is possibly used for the intrusion of oxygen containing tissues of host body by the invading pathogens. In conclusion S. enterica serovar Enteritidis strain suspensions grow equally well in constant nitrogenation and aeration, and fimbria were produced in both conditions, during the early logarithmic phase but they prevailed in the presence of aeration.