Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. ...Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min), 5.63 μl/(g × min) and 6.02 μl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.
Bacteria communicate with each other by the use of signaling molecules, a process called 'quorum sensing'. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced ...by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (Kin = 20.87 μl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (Kin = 2.68 μl/(g×min)) and very low (Kin = 0.18 μl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.
Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid ...DNA and (si)RNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP)-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA) and a quantitative structure-property relationship study (QSPR), using chemo-molecular descriptors and our newly defined CP-response, learned that besides typical well-known properties of CPPs, i.e. positive charge and amphipathicity, the shape, structure complexity and the 3D-pattern of constituting atoms influence the cellular uptake capacity of peptides.
Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of ...these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy of different clinical diseases.
The neuromedin peptides are peripherally and centrally produced, but until now, it is generally believed that they only function as locally acting compounds without any quantitative knowledge about ...their blood-brain barrier (BBB) passage. Here, we characterize the transport kinetics of four neuromedins (NMU, NMN, NMB and NT) across the BBB, as well as their metabolization profile, and evaluate if they can act as endocrine hormones.
Using the in vivo mouse model, multiple time regression (MTR), capillary depletion (CD) and brain efflux studies were performed. Data was fitted using linear (NMU, NT and NMB) or biphasic modeling (NMU and NMN). Three of the four investigated peptides, i.e. NMU, NT and NMN, showed a significant influx into the brain with unidirectional influx rate constants of 1.31 and 0.75 μL/(g × min) for NMU and NT respectively and initial influx constants (K1) of 72.14 and 7.55 μL/(g × min) and net influx constants (K) of 1.28 and 1.36 × 10−16 μL/(g×min) for NMU and NMN respectively. The influx of NMB was negligible. Only NMN and NT showed a significant efflux out of the brain with an efflux constant (kout) of 0.042 min−1 and 0.053 min−1 respectively.
Our results indicate that locally produced neuromedin peptides and/or fragments can be transported through the whole body, including passing the BBB, and taken up by different organs/tissues, supporting the idea that the neuromedins could have a much bigger role in the regulation of biological processes than currently assumed.
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•The peptides NMU, NT and NMN show a clear influx from the blood to the brain.•The peptides that enter the brain capillaries continue to the brain parenchyma.•NMN and NT show an efflux from the brain to the blood.•The stability of all peptides was evaluated and the metabolites identified.
During fundamental research, it is recommended to evaluate the test compound identity and purity in order to obtain reliable study outcomes. For peptides, quality control (QC) analyses are routinely ...performed using reversed-phase liquid chromatography coupled to an ultraviolet (UV) detector system. These traditional QC methods, using a C18 column and a linear gradient with formic acid (FA) as acidic modifier in the mobile phase, might not result in optimal chromatographic performance for basic peptides due to their cationic nature and hence may lead to erroneous results. Therefore, the influence of the used chromatographic system on the final QC results of basic peptides was evaluated using five cationic cell-penetrating peptides and five C18-chromatographic systems, differing in the column particle size (high performance liquid chromatography (HPLC) versus ultra-high performance liquid chromatography (UHPLC)), the acidic modifier (FA versus trifluoroacetic acid (TFA)), and the column temperature (30 °C versus 60 °C). Our results indicate that a UHPLC system with the C18 column thermostated at 30 °C and a mobile phase containing TFA, provides the most suitable routine QC analysis method for cationic peptides, outperforming in sensitivity and resolution compared to the other systems. We also demonstrate the use of a single quad mass spectrometry (MS) detector system during QC analysis of (cationic) peptides, allowing identification of the peptide and its impurities, as well as the evaluation of the peak purity.
Infections by antibiotic-resistant bacteria are becoming a great risk for human health, leading to an urgent need for new efficient antibacterial therapies. The short, proline-rich antimicrobial ...peptides from insects gained a lot of interest as a potential antibacterial treatment, having a low toxicity profile and being mainly active against Gram-negative bacteria. To know whether these antimicrobial peptides can be used for the treatment of cerebral infections, the blood-brain barrier transport characteristics of these peptides were investigated. This study describes the results of the in vivo blood-brain barrier experiments in mice, as well as the in vitro metabolic stability in mouse plasma and brain of apidaecin Api137, oncocin, drosocin and drosocin Pro5Hyp. The four investigated peptides showed a significant influx into the brain with a K(in) ranging between 0.37 and 0.86 µL/g x min and brain distribution volumes of 19.6 to 25.8 µL/g. Only for drosocin, a significant efflux was determined, with a k(out) of 0.22 min(-1). After entering the brain, oncocin was for approximately 80% trapped in the endothelial cells, while the other peptides reached the brain parenchyma for about 70%. All peptides were stable in plasma and brain during the experiments, with estimated metabolic half-lives ranging between 47 min and 637 min. We conclude that the investigated short, proline-rich antimicrobial peptides show an influx into the brain, which make them a promising antibacterial treatment of cerebral infections.
An increasing number of studies demonstrate the ability of peptides to cross the blood-brain barrier (BBB), opening perspectives for a new class of therapeutics for central nervous system diseases. ...However, information on the BBB transport of peptides suffer from a wide variety in used methods and experimental set-up. Therefore, it is currently difficult, if not impossible, to classify peptides according to their BBB influx characteristics. To allow direct comparison of BBB influx results of peptides, we introduce a classification method and unified response for BBB influx transport of peptides. First, the results of BBB influx response types (i.e. Kin (MTR), Kin (Perfusion), Pin vitro and Pin vivo), which quantitatively express brain influx, were classified into five classes of BBB influx magnitude based on the distribution of these results for the individual response types. Then, these classes were converted to a BBBin-response, representing a scaled value ranging from zero (no influx) to ten (high influx), independent from the BBB influx response type from which it was derived. This unified response can immediately be applied for new BBB influx results of peptides and represents a ballpark figure for BBB influx and allows direct comparison and ranking of peptides independent of the response type.
Chronic pain is an undertreated epidemic affecting quality of life in at least 20% of the global population, and CNS-related side effects, tolerance, and addiction are common features of current ...medications. α-Conotoxin Vc1.1 potently elicits prolonged analgesia in preclinical chronic constriction injury and chronic visceral hypersensitivity models of neuropathic pain. A backbone-cyclized variant, cVc1.1, exhibits superior in vitro stability and is orally active, but its in vivo half-life and disposition, both critical in informing drug candidate progression, remain unexplored. Here, we investigate the pharmacological influence of the peptidic bridge differentiating linear and cyclic Vc1.1 in various preclinical PK/PD rodent models. While previous in vitro studies had indicated cyclization conferred increased stability for cVc1.1, in vivo the peptides exhibited similar half-lives and oral bioavailabilities. The ratios of free drug exposure metrics (Cmax × fu,p and AUC0-inf × fu,p) following oral dosing vs. their respective in vitro IC50s at the GABAB receptor were comparable for Vc1.1 and cVc1.1, indicating similar drug efficiency indexes. MALDI imaging, radiolabel, and LC-MS biodistribution studies of cVc1.1 in rodents demonstrated that the intact cyclopeptide and several metabolites persist in the GI tract for at least 4 h, long after the plasma levels of the intact peptide had fallen below the target IC50. Biodistribution analyses of IV administered 125I-labelled cVc1.1 revealed accumulation primarily in the kidneys consistent with renal elimination, and combined with insignificant uptake in brain, suggested a low likelihood of CNS-related side effects.
•cVc1.1 and Vc1.1 exhibit low oral bioavailability and similar half-lives in rat PK.•MALDI imaging demonstrates cVc1.1 in the GI tract for >4 h post oral dosing.•cVc1.1 exhibits negligible penetration into the brain.•Drug efficiency indices of cVc1.1 and Vc1.1 are similar to marketed peptide drugs.•Conotoxins are potent neuroactive peptides with potential for the treatment of pain.
Many analogues of the glycolipid alpha-galactosylceramide (α-GalCer) are known to activate iNKT cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of ...Th1 and Th2 cytokines. Because of iNKT cell involvement and associated Th1/Th2 cytokine changes in a broad spectrum of human diseases, the design of iNKT cell ligands with selective Th1 and Th2 properties has been the subject of extensive research. This search for novel iNKT cell ligands requires refined structural insights. Here we will visualize the chemical space of 333 currently known iNKT cell activators, including several newly tested analogues, by more than 3000 chemical descriptors which were calculated for each individual analogue. To evaluate the immunological responses we analyzed five different cytokines in five different test-systems. We linked the chemical space to the immunological space using a system biology computational approach resulting in highly sensitive and specific predictive models. Moreover, these models correspond with the current insights of iNKT cell activation by α-GalCer analogues, explaining the Th1 and Th2 biased responses, downstream of iNKT cell activation. We anticipate that such models will be of great value for the future design of iNKT cell agonists.
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