on June 22, 2023
  • Research

Published on June 7, 2023 Updated on June 7, 2023

PhD defense of Camille Lozada


Camille Lozada, PhD student at BioCIS, will defend her thesis on Thursday, June the 22nd at 2.30 pm in MIR amphitheatre, Neuville Campus

Camille Lozada will defend her PhD thesis untitled :
"Alteration of the membranotropic properties of cell-penetrating and fusion peptides by means of conformationally constrained tryptophan derivatives"

carried out under the direction of Prof. Nadège Lubin-Germain (CYU) et du Prof. Steven Ballet (VUB)
On Thursday, June the 22ndat 2.30 pm in MIR amphitheatre, Neuville campus
Membranotropic peptides are generally short sequences able to interact with membranes. Among these bioactive peptides, two categories of peptides were studied: cell-penetrating peptides (CPPs) and viral fusion peptides (FPs). CPPs are commonly cationic and have been well-studied for their capacity to cross cellular membranes. Therefore, they have been much utilized to improve the cellular uptake of polar therapeutic agents. On the contrary, FPs are short hydrophobic sequences found in viral proteins. They are essential for enveloped viruses because of their capacity to insert into the host cellular membrane, to subsequently allow the viral membrane to merge during the infection process. We developed a vectorization method using FPs of hepatitis C virus (HCV) as anchors into biological membranes. However, preliminary results showed a moderate membranotropic activity, probably due to their lack of structuration, a crucial feature for membranotropic behavior. Therefore, it was suggested to introduce constrained amino acids to induce or facilitate peptide structuration. Two cyclic Trp surrogates were chosen as local constraints and synthesized as suitable building blocks to be incorporated in unstructured membranotropic peptides: a tetrahydro-β-carboline derivative Tcc, and an indoloazepinone scaffold Aia. These surrogates present constrained dihedral angles and limited side chain orientations which were hypothesized to influence the peptide’s structure and/or membranotropic activity. To study the influence of the introduction of these local constraints on membrane activity and structuration, we selected four reference peptides: one CPP (RW9) and three viral peptides (HCV7, Flav, and C8). These peptides were synthesized by SPPS and Trp residues were replaced by either Tcc or Aia building blocks, varying the number of modifications and their positions, eventually resulting in a series of 34 peptides. Their membranotropic activities were studied by monitoring Trp fluorescence in the presence of liposomes, allowing to calculate partition coefficients (Kp). Results showed that improvement of the activity by introducing Tcc or Aia was sequence dependent and influenced by the number of these modifications. The most potent peptides were found to be [Aia4]RW9 (H-RRW-Aia-Arg-RWRR-NH2) and [Tcc1,4]C8 (Ac-Tcc-ED-TccVGWI-NH2) which showed Kp values 3-fold higher than their native counterparts. Further studies by molecular dynamics (MD) simulations were in correlation with the trend experimentally observed. Further structural studies were performed by circular dichroism (CD). Finally, tagged versions of the peptides with fluorescein (FITC) were added to culture medium of CHO-K1 cells for permeation studies using confocal microscopy. Future studies will demonstrate the capacity of the modified sequences to transport various types of bioactive cargoes into cells.