Design, synthesis, and biological activity of isophthalic acid derivatives targeted to the C1 domain of protein kinase C

Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([H-3]PDBu) from PKC alpha and delta at K-i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time. that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([H-3]PDBu) from PKC alpha and delta at K-i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time. that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([H-3]PDBu) from PKC alpha and delta at K-i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time. that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.Peer reviewe

New Structural Variants of Aeruginosin Produced by the Toxic Bloom Forming Cyanobacterium Nodularia spumigena

Peer reviewe

Addressing the Biochemical Foundations of a Glucose-Based "Trojan Horse"-Strategy to Boron Neutron Capture Therapy: From Chemical Synthesis to In Vitro Assessment

Boron neutron capture therapy (BNCT) for cancer is on the rise worldwide due to recent developments of in-hospital neutron accelerators which are expected to revolutionize patient treatments. There is an urgent need for improved boron delivery agents, and herein we have focused on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to BNCT could be based. By combining synthesis and molecular modeling with affinity and cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional boron delivery capacity in the in vitro assessment thereby pointing toward the significant potential embedded in this approach

The N-glycome of human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.</p> <p>Results</p> <p>The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Le^xand H type 2 antennae in sialylated complex-type N-glycans.</p> <p>Conclusion</p> <p>The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.</p

Identification and structural characterization of LytU, a unique peptidoglycan endopeptidase from the lysostaphin family

We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications.Peer reviewe

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728–1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nM). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.peerReviewe

Protein-oligosaccharide interaction mechanisms in chitinases engineered towards neolectins

Protein-carbohydrate interactions are essential in many biomolecular recognition events, such as inflammation, cell-cell recognition and adhesion, immunochemistry and human blood group type determination. A great deal of interest has thus arisen in isolation of medically important oligosaccharides. In this study our aim is to obtain atomic level information of the binding and to gain a deeper understanding of the factors determining the interactions between an oligosaccharide and a protein by using extended molecular dynamic (MD) simulations with explicit water. In addition to a conventional force field, a novel soft-core potential 1,2 originally developed for a priori modelling of surface loops of proteins without additional restraints was used here to the whole binding site of the modelled protein. The study concentrates on fungal 42kDa chitinase from Trichoderma harzianum 3, a naturally chitin degrading enzyme, containing an extended binding site providing a number of strong and specific interactions with up to 6-7 sugar units. Since these interactions come from a limited number of loops, the structure of chitinase (-barrel fold) provides an excellent platform for directed evolution studies. By mutating first the functional amino acid(s) and then altering the substrate specificity by locally directed saturation mutagenesis the functionality of a protein can be changed from a degrading enzyme to a specific binder. While experimentally determined three-dimensional structures were not available for the enzyme of interest, structural models were constructed based on the known structures of homologues. Experimentally determined sugar-protein complex structures of related chitinases were used in the initial simulations to evaluate the suitability of the force field parameters and simulation procedures. Classical MD (Gromacs) with a conventional force field and with a soft-core potential 1,2 is used to explore the conformational space of the chitinase loops and to study the functional behavior of the N-acetylglucosamine oligosaccharides and their derivates. Trajectories obtained from the simulations are used in analyzing the binding, especially the hydrogen bonding and hydrophobic interactions occurring via N/O-acetyl or O-methyl groups. The results from modeling are compared with the experimental data (mutagenesis, mass spectroscopy and nuclear magnetic resonance). Computational studies with the experimental work aim at development of neolectins, i.e. proteins selectively binding to given oligosaccharide structures, achieved by deactivating and engineering fungal chitinases towards the desired specificity and affinity. 1. Tappura, K.; Lahtela-Kakkonen, M; Teleman, O. J. Comput. Chem. 2000, 21(5), 388-97. 2. Tappura, K. Proteins 2001, 44(3), 167-79. 3. Boer, H.; Munck, N.; Natunen, J.; Wohlfahrt, G.; Söderlund, H.; Renkonen, O.; Koivula, A. (submitted 2004)