Exploration of quorum sensing peptides: the missing link between microbiome and cancer outcome?

The human microbiome was recently associated with diverse diseases. The explanation for this relationship is however not yet clarified. In this research, the interaction of quorum sensing peptides, produced by the bacteria, with human cancer cells was explored. First, the chemical characteristics of already known quorum sensing peptides (and analogues), their bacterial origin and quorum sensing related properties were summarized in the Quorumpeps database. The chemical diversity of these quorum sensing peptides was then analyzed, after which diverse model-peptides were selected for further research. After chemical synthesis of these peptides, the purity was investigated, next to the identity of the impurities. A thorough quality control is necessary to correctly interpret functional results. Eventually, the effect of the quorum sensing peptides on human cancer cell behaviour was investigated: we demonstrated that some quorum sensing peptides induced colon and breast cancer cell invasion and promoted angiogenesis; both processes can be linked to cancer metastasis. Some quorum sensing peptides were also found to pass the blood-brain barrier, indicating that these peptides can exert an effect on the brain tissue. Quorum sensing peptides can pass the intestinal barrier as well and thus reach the blood circulation; once they are present in the blood, they can interact with cells throughout the human body. The quorum sensing peptides were also found to remain sufficiently stable in human plasma. Moreover, the quorum sensing peptides demonstrated no haemolytic and no direct cell-killing effects. Finally, we reviewed the quality aspects of radiolabelled peptides as used not only in the biomedical research but also as diagnostics or therapeutics in the current shift towards personalized medicine

Characterisation of modified pharmaceutical proteins: the somatropin case

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin [1]. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is currently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. This yields 9 possible labelling sites for somatropin: 38, 41, 70, 115, 140, 145, 158, 168 and 172. As site-specific labelling is necessary to avoid active region interactions, characterisation of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and Staphylococcus aureus V-8 proteases. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact position of amino acid modifications

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

Faecal microbiota transplantation : a regulatory hurdle?

During faecal microbiota transplantation, stool from a healthy donor is transplanted to treat a variety of dysbiosis-associated gut diseases. Competent authorities are faced with the challenge to provide adequate regulation. Currently, regulatory harmonization is completely lacking and authorities apply non-existing to most stringent requirements. A regulatory approach for faecal microbiota transplantation could be inserting faecal microbiota transplantation in the gene-, cell-and tissue regulations, including the hospital exemption system in the European Advanced Therapy Medicinal Products regulation, providing a pragmatic and efficacy-risk balanced approach and granting all patients as a matter of principle access to this therapy

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