To what extent do cell-penetrating peptides selectively cross the blood-brain barrier?

The blood-brain barrier protects the brain from toxic compounds. Its selective permeability is essential for the optimal function of the central nervous system. Some peptides can cross the blood-brain barrier. On the other hand, cell-penetrating peptides are able to overcome the cell membrane. During this research project, it was investigated whether these cell-penetrating peptides also can cross the blood-brain barrier. The chemical diversity of the already reported cell-penetrating peptides was investigated and a unified response for the extent of cellular uptake of peptides was introduced. Based on this study, a set of cell-penetrating peptides was rationally selected for further research. In order to more objectively compare the quantitative data on the blood-brain barrier influx of peptides, a classification system for blood-brain barrier influx was established. The purity of the selected synthetized cell-penetrating peptides was also investigated, which is essential for obtaining reliable research conclusions. Different chromatographic systems were compared for the analysis of the selected peptides. The investigated cell-penetrating peptides crossed the blood-brain barrier to a different extent. The influx varied from very low to very high and some peptides showed efflux out of the brain. There was no correlation observed between the blood-brain barrier transport kinetics and the extent of cellular uptake. During the aging process, the blood-brain barrier shows an increased permeability and, together with other age-related functional changes, should be taken into account during the development of medicines used by the elderly. Therefore, the current regulatory status of the development of geriatric medicines was investigated

Development of geriatric medicines: to GIP or not to GIP?

The elderly population represents a heterogeneous group of patients, which show co-morbidities and is often poly-medicated. Although geriatric patients form the main users of medicines, this group is underrepresented in clinical trials. Safe and efficacious use of drugs by this group is often not ensured. However, since these elderly population will numerically expand in the coming years, this patient group can no longer be neglected during the development and approval stage of medicines. We evaluated the current regulatory status for the geriatric medicines. Next, the views of the different stakeholders in this field will be analyzed. Finally, points of attention and suggestions concerning this regulatory discussion will be emphasized, like i.a. the approach of personalized medicine, the age and disease-related change in blood-brain barrier permeability and use of predictive models. Thus, by analogy with the paediatric investigation plan (PIP), coming to the conclusion whether to GIP or not to GIP

Exploring the chemical-functional space of cell-penetrating peptides

Cell penetrating peptides (CPPs) are an increasingly growing part of fundamental and applied peptide research. Using their capacity to cross cell barriers, they have already been successfully applied as carriers for problematic cargos like DNA, (si)RNA, proteins and other peptides, (poly/oligo) saccharides and small molecules. Several hundreds of CPPs, showing different properties and activities, are already reported in the literature. To clarify the different types of actions in cell-penetrating behaviour, a database of more than 200 peptides was build, covering the CPPs described over the last five years and for which quantitative data were available. Seen the wide range of techniques, cell lines, peptide concentrations and other operational parameters used to quantify the cellular uptake, a unified response for cellular uptake was firstly defined based upon a concentration corrected standardized response relative to the concentration corrected response of Penetratin, a well known and characterized CPP. In this way a “meta-analytical” comparison of the cellular uptake of different CPPs is established, which was currently hardly needed. Therefore a chemical space was developed using more than 3000 descriptors, calculated from the optimized 3D-structure of the CPPs. By combining these descriptors and the unified responses, clusters of peptides are obtained from which model CPPs can be rationally selected and QSPRs and mechanisms of action established

Exploring the Brainpeps database

Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened to new therapeutics for CNS diseases and pain management. Recently, we have constructed the Brainpeps database (brainpeps.ugent.be) to give an overview of the available BBB transport data of peptides, which are scattered in the literature [1]. One possible application of the Brainpeps database is the study of structure-property relationships (QSPRs). Before peptides can be used as drugs, their impurity profile needs to be examined as part of the International Conference on Harmonization (ICH) risk assessment of peptide drugs. Compared to small molecules, no in-silico predictive programs are available for toxicity screening of the different peptide impurities towards passing the BBB. To predict the BBB-behaviour of peptides as well as their impurities, we explored the Brainpeps database. During this presentation, the first results of the modelling experiments are presented. Our starting hypothesis is that the interactions of peptides at the blood-brain barrier are comparable with those of peptides in HPLC systems. Therefore, we determined the retention characteristics on different fused-core HPLC systems of a set of model peptides selected from the Brainpeps database and explored the relationship between the chromatographic characteristics and their BBB-influx properties [2]. In conclusion, using the Brainpeps database and experimental HPLC data, a first step towards in-silico profiling of peptides, including their impurities, at the blood-brain barrier level is taken. More chromatographic analyses of BBB peptides and harmonization on testing the BBB transport of peptides are future challenges to validate and unify this model. References [1] Van Dorpe S., Bronselaer A., Nielandt J., Stalmans S., Wynendaele E., Audenaert K., Van de Wiele C., Burvenich C., Peremans K., Hsuchou H., De Tré G., De Spiegeleer B. Brainpeps: the blood-brain barrier peptide database. Brain Struct Funct (2012), DOI: 10.1007/s00429-011-0375-0. [2] D’Hondt M., Van Dorpe S., Gevaert B., Wynendaele E., Stalmans S., Peremans K., Burvenich C., De Spiegeleer B. Fused-core RP-HPLC modelling of peptides. Journal of Pharmaceutical Analysis (2012), Accepted for publication

Do cell-penetrating peptides cross the blood-brain barrier

Cell-penetrating peptides (CPPs) are a chemically diverse group of peptides, able to cross cell membranes. In literature, several studies are available describing a successful coupling of a brain-impermeable compound to a CPP to transport the molecule across the blood-brain barrier (BBB). Quantitative kinetic BBB transport studies of cell-penetrating peptides are however not yet available. To characterize quantitatively the BBB behavior of CPPs, the in vivo BBB transport and the in vitro metabolic stability of a diverse set of CPPs was determined. The chemical-functional space of the CPPs was first explored using chemo-molecular descriptors and a newly defined cell-penetrating response [1]. Then, five chemically and functionally diverse CPPs were selected representing the CPP space. Their BBB influx and efflux was quantified, as well as their distribution to the brain capillaries and parenchyma. Furthermore, the in vitro metabolic stability of these CPPs in mouse serum and brain was determined to ensure the reliability of our BBB transport conclusions. Finally, preliminary results of the in-brain region distribution will be presented. Our results indicate that the investigated CPPs show divergent BBB transport kinetics, which may be explained by their structural difference and their mechanism of cellular influx

How the exploration of the chemical space of cell-penetrating peptides helps to understand their functionality

Cell-penetrating peptides (CPPs) gained in recent years a lot of interest in the applied biomedical research field because of their ability to cross cellular barriers without causing significant lethal membrane damage. They have already been successfully applied to bring cell-impermeable cargoes into the cell interior. However, to date, only limited applications have reached the clinical phase of drug development. Currently, harmonization is needed in the cellular uptake studies of CPPs in order to efficiently draw reliable conclusions on their uptake mechanisms and structural properties. Therefore, we explored the chemical space of representative CPPs using chemo-molecular descriptors, which numerically express their chemical properties. Together with a newly defined cell-penetrating (CP) response for cellular influx, calculated using available quantitative cellular uptake data, the chemo-molecular descriptors were used to clarify which structural characteristics of the CPPs influence to what extent their cell-penetrating properties.[1] Our study indicated that CPPs are a chemically and CP-functionally diverse group of peptides.[1] Moreover, the newly defined CP-response and subgroups of CPPs help to select representative model peptides for further investigating their functionalities like e.g. uptake mechanisms and behaviour at different membrane interfaces, like the blood-brain barrier.[2] Another application of the exploration of the chemical space of the CPPs, is to investigate their chemical and functional relationship with the antimicrobial peptides, which are historically investigated as a separate peptide group.[3] References 1. S. Stalmans et al. Plos One. (2013), 8, e71752. 2. S. Stalmans et al. (submitted, 2014) 3. S. Stalmans et al. Protein Peptide Lett (2014), 21, 399-406

A computational high-throughput screening approach of iNKT-agonists: a novel tool to find optimized iNKT cell ligands

Depending on the environment and the activating glycolipids, iNKT cells are known to induce T-helper 1 and/or T-helper 2 cytokines. This highly versatile nature makes these innate-like cells very interesting targets for immunomodulation. As many pathologies as well as physiological ageing are associated with altered immune responses, iNKT cells could play a role in new therapies. Many analogs of the glycolipid alpha-galactosylceramide (a-GalCer) are known to activate iNKT-cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and/or Th2 cytokines. The design of iNKT cell ligands with selective Th1 and Th2 properties requires refined structural insights. Therefore, the chemical space of 333 currently known iNKT activators, including several newly tested analogs, was visualized by more than 3000 chemical descriptors which were calculated for each individual analog. The immunological responses consisted of four different cytokines in five different test-systems. With these two information-sets, structure-activity models were developed using a system biology computational approach. We present highly sensitive and specific predictive models that can be further exploited as high-throughput instruments to in-silico screen potential glycolipids, thereby reducing the attrition rate

Quorumpeps database : chemical space, microbial origin and functionality of quorum sensing peptides

Quorum-sensing (QS) peptides are biologically attractive molecules, with a wide diversity of structures and prone to modifications altering or presenting new functionalities. Therefore, the Quorumpeps database (http://quorumpeps.ugent.be) is developed to give a structured overview of the QS oligopeptides, describing their microbial origin (species), functionality (method, result and receptor), peptide links and chemical characteristics (3D-structure-derived physicochemical properties). The chemical diversity observed within this group of QS signalling molecules can be used to develop new synthetic bio-active compounds

An in silico approach for modelling T-helper polarizing iNKT cell agonists

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