Magnetic Resonance Signal Amplification by Reversible Exchange of Selective PyFALGEA Oligopeptide Ligands Towards Epidermal Growth Factor Receptors.

The biorelevant PyFALGEA oligopeptide ligand, which is selective towards the epidermal growth factor receptor (EGFR), has been successfully employed as a substrate in magnetic resonance signal amplification by reversible exchange (SABRE) experiments. It is demonstrated that PyFALGEA and the iridium catalyst IMes form a PyFALGEA:IMes molecular complex. The interaction between PyFALGEA:IMes and H2 results in a ternary SABRE complex. Selective 1D EXSY experiments reveal that this complex is labile, which is an essential condition for successful hyperpolarization by SABRE. Polarization transfer from parahydrogen to PyFALGEA is observed leading to significant enhancement of the 1 H NMR signals of PyFALGEA. Different iridium catalysts and peptides are inspected to discuss the influence of their molecular structures on the efficiency of hyperpolarization. It is observed that PyFALGEA oligopeptide hyperpolarization is more efficient when an iridium catalyst with a sterically less demanding NHC ligand system such as IMesBn is employed. Experiments with shorter analogues of PyFALGEA, that is, PyLGEA and PyEA, show that the bulky phenylalanine from the PyFALGEA oligopeptide causes steric hindrance in the SABRE complex, which hampers hyperpolarization with IMes. Finally, a single-scan 1 H NMR SABRE experiment of PyFALGEA with IMesBn revealed a unique pattern of NMR lines in the hydride region, which can be treated as a fingerprint of this important oligopeptide.

Triazolopeptides Inhibiting the Interaction between Neuropilin-1 and Vascular Endothelial Growth Factor-165.

Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 µM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 µM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure-activity relationships.

Ischemia/Reperfusion-Induced Translocation of PKCßII to Mitochondria as an Important Mediator of a Protective Signaling Mechanism in an Ischemia-Resistant Region of the Hippocampus.

Emerging reports indicate that activated PKC isoforms that translocate to the mitochondria are pro- or anti-apoptotic to mitochondrial function. Here, we concentrate on the role of PKCß translocated to mitochondria in relation to the fate of neurons following cerebral ischemia. As we have demonstrated previously ischemia/reperfusion injury (I/R) results in translocation of PKCß from cytoplasm to mitochondria, but only in ischemia-resistant regions of the hippocampus (CA2-4, DG), we hypothesize that this translocation may be a mediator of a protective signaling mechanism in this region. We have therefore sought to demonstrate a possible relationship between PKCßII translocation and ischemic resistance of CA2-4, DG. Here, we reveal that I/R injury induces a marked elevation of PKCßII protein levels, and consequent enzymatic activity, in CA2-4, DG in the mitochondrial fraction. Moreover, the administration of an isozyme-selective PKCßII inhibitor showed inhibition of I/R-induced translocation of PKCßII to the mitochondria and an increase in neuronal death following I/R injury in CA1 and CA2-4, DG in both an in vivo and an in vitro model of ischemia. The present results suggest that PKCßII translocated to mitochondria is involved in providing ischemic resistance of CA2-4, DG. However, the exact mechanisms by which PKCßII-mediated neuroprotection is achieved are in need of further elucidation.

Conformational latitude - activity relationship of KPPR tetrapeptide analogues toward their ability to inhibit binding of vascular endothelial growth factor 165 to neuropilin-1.

Neuropilin-1 has been found to be overexpressed in several kinds of malignant tumors, and it is postulated that its interaction with the vascular endothelial growth factor 165 leads to progression of tumor vascularization and growth. Several analogues (KxxR) with various conformational latitudes have been synthesized and found as inhibitors of NRP-1. Detailed insight provided by molecular dynamics simulation allowed forming a clear relationship between flexibility of xx part of the molecule and its inhibitory activity.

Beware of Cocktails: Chain-Length Bidispersity Triggers Explosive Self-Assembly of Poly-L-Glutamic Acid ß2-Fibrils.

Chain-length polydispersity is among the least understood factors governing the fibrillation propensity of homopolypeptides. For monodisperse poly-L-glutamic acid (PLGA), the tendency to form fibrils depends of the main-chain length. Long-chained PLGA, so-called (Glu)200, fibrillates more readily than short (Glu)5 fragments. Here we show that conversion of α-helical (Glu)200 into amyloid-like ß-fibrils is dramatically accelerated in the presence of intrinsically disordered (Glu)5. While separately self-assembled fibrils of (Glu)200 and (Glu)5 reveal distinct morphological and infrared characteristics, accelerated fibrillation in mixed (Glu)200 and (Glu)5 leads to aggregates similar to neat (Glu)200 fibrils, even in excess of (Glu)5. According to molecular dynamics simulations and circular dichroism measurements, local events of "misfolding transfer" from (Glu)5 to (Glu)200 may play a key role in the initial stages of conformational dynamics underlying the observed phenomenon. Our results highlight chain-length polydispersity as a potent, although so-far unrecognized factor profoundly affecting the fibrillation propensity of homopolypeptides.

NMR signal enhancement by effective SABRE labeling of oligopeptides.

Signal amplification by reversible exchange (SABRE) can enhance nuclear magnetic resonance signals by several orders of magnitude. However, until now this was limited to a small number of model target molecules. Here, a new convenient method for SABRE activation applicable to a variety of synthetic model oligopeptides is demonstrated. For the first time, a highly SABRE-active pyridine-based biocompatible molecular framework is incorporated into synthetic oligopeptides. The SABRE activity is preserved, demonstrating the importance of such earmarking. Finally, a crucial exchange process responsible for SABRE activity is identified and discussed.

Amyloidogenic Properties of Short α-L-Glutamic Acid Oligomers.

Poly-L-glutamic acid (PLGA) forms amyloid-like ß2-fibrils with the main spectral component of vibrational amide I' band unusually shifted below 1600 cm(-1). This distinct infrared feature has been attributed to the presence of bifurcated hydrogen bonds coupling C═O and N-D (N-H) groups of the main chains to glutamate side chains. Here, we investigate how decreasing the chain length of PLGA affects its capacity to form ß2-fibrils. A series of acidified aqueous solutions of synthetic (l-Glu)n peptides (n ≈ 200, 10, 6, 5, 4, and 3) were incubated at high temperature. We observed that n = 4 is the critical chain length for which formation of aggregates with the ß2-like infrared features is still observed under such conditions. Interestingly, according to atomic force microscopy (AFM), the self-assembly of (L-Glu)n chains varying vastly in length produces fibrils with rather uniform diameters of approximately 4-6 nm. Kinetic experiments on (L-Glu)5 and (L-Glu)200 peptides indicate that the fibrillation is significantly accelerated not only in the presence of homologous seeds but also upon cross-seeding, suggesting thereby a common self-assembly theme for (L-Glu)n chains of various lengths. Our results are discussed in the context of mechanisms of amyloidogenic fibrillation of homopolypeptides.

Chirality and Rigidity in Triazole-Modified Peptidomimetics Interacting with Neuropilin-1.

The interaction of Neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has been shown to promote angiogenesis under physiological and pathological conditions. Angiogenesis around tumors is a major factor allowing for their growth and spread. Disrupting NRP-1/VEGF complex formation is thus a promising pathway for the development of new anticancer pharmaceuticals. A large body of work has been produced in the last two decades detailing the development of inhibitors of NRP-1/VEGF complex formation. Among those were peptide A7R and its smaller derivatives KXXR and K(Har)XXR. It has been previously reported that replacement of the XX backbone with triazole residues has a positive effect on the proteolytic stability of inhibitors. It has also been reported that a higher dihedral angle range restriction of the XX backbone has a positive effect on the activity of inhibitors. In this work, we have designed new triazole derivatives of K(Har)XXR inhibitors with substitution allowing for higher range restriction of the XX backbone. The obtained peptidomimetics have greater activity than their less restricted counterparts. One of the newly obtained structures has greater affinity than the reference peptide A7R.

Preparation of RNAs with non-canonical 5' ends using novel di- and trinucleotide reagents for co-transcriptional capping.

Many eukaryotic and some bacterial RNAs are modified at the 5' end by the addition of cap structures. In addition to the classic 7-methylguanosine 5' cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to in vitro transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations. To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5'-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5' capping.

Branched pentapeptides as potent inhibitors of the vascular endothelial growth factor 165 binding to Neuropilin-1: Design, synthesis and biological activity.

The demonstrated involvement of VEGF165/NRP-1 complex in pathological angiogenesis has catalyzed interest in blocking this interaction to combat angiogenesis dependent diseases. It was shown before that Lys-Pro-Pro-Arg is a fairly strong inhibitor of the VEGF165/NRP-1 interaction. Our current findings suggest that the side chain elongation of the Lys1 by branching it with additional homoarginine (Har) residue, to obtain Lys(Har)-Pro-Pro-Arg, allows more effective inhibition. Moreover, increasing the flexibility of the middle part of molecule, in particular with simultaneous introduction of additional interacting elements at the second or third position, produced compounds up to 30-fold more active (IC50 = 0.2 µM) than the heptapeptide ATWLPPR (A7R) which is one of the first peptide known as an effective antagonist of the VEGF165 binding to NRP-1 and in vivo decreases breast cancer angiogenesis and growth. Herein, we present also the structure-activity study of Lys(Har)-Pro-Pro-Arg, discussing the design, synthesis, inhibitory activity, proteolytic stability and molecular modeling of the prepared derivatives. For two of the most active analogs the high proteolytic stability was also observed. These studies provide the next step for elucidating the optimal structure of the small peptidic inhibitors of VEGF165/NRP-1 interaction that could serve as research tools or be prospective drug candidates.

Effects of terminal capping on the fibrillation of short (L-Glu)n peptides.

Several homopolypeptides including poly-l-glutamic acid (PLGA) form amyloid-like fibrils under favorable physicochemical conditions. We have shown recently that even short uncapped (Glu)n peptides (for n>3) form fibrillar ß-aggregates which cross-seed with amyloid fibrils obtained from high molecular weight fractions of PLGA. Here we investigate effects of N-terminal acetylation and C-terminal amidation on the amyloidogenic tendencies of (Glu)n peptides containing 3, 4, and 5 residues. Our results based primarily on time-lapse FT-IR spectroscopy and AFM microscopy indicate that selective modifications of C-termini (and, to a lesser degree, of N-termini) decrease capacity of tetra- and pentapeptides to form fibrils. On the other hand, peptides modified at both ends appear to form fibrils as fast as unmodified analogues. In fact, the double terminal modification enables fibrillation of (Glu)3 which is not fibrillogenic in the unmodified state. The AFM data suggests that the double capping results in the aggregates becoming more tape-like or acquiring noticeable tendencies to bend. According to seeding and cross-seeding experiments, there is a high degree of promiscuity between modified and unmodified peptides. Possible mechanisms explaining how amyloidogenic propensities of (Glu)n peptides are affected by terminal modifications have been discussed.

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1.

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence. Here, we present a structure-activity relationship study of the systematic optimization of amino acid residues in positions 1-3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP-1.