Pascal Kessler

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8⁺ T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.

The renin-angiotensin system (RAS) is one of the main regulatory systems of cardiovascular homeostasis. It is mainly composed of angiotensin-converting enzyme (ACE) and angiotensin II receptors AT1 and AT2. ACE and AT1 are targets of choice for the treatment of hypertension, whereas the AT2 receptor is still not exploited due to the lack of knowledge of its physiological properties. Peptide toxins from venoms display multiple biological functions associated with varied chemical and structural properties. If Brazilian viper toxins have been described to inhibit ACE, no animal toxin is known to act on AT1/AT2 receptors. We screened a library of toxins on angiotensin II receptors with a radioligand competition binding assay. Functional characterization of the selected toxin was conducted by measuring second messenger production, G-protein activation and β-arrestin 2 recruitment using bioluminescence resonance energy transfer (BRET) based biosensors. We identified one original toxin, A-CTX-cMila, which is a 7-residues cyclic peptide from Conus miliaris with no homology sequence with known angiotensin peptides nor identified toxins, displaying a 100-fold selectivity for AT1 over AT2. This toxin shows a competitive antagonism mode of action on AT1, blocking Gαq, Gαi3, GαoA, β-arrestin 2 pathways and ERK_1/2 activation. These results describe the first animal toxin active on angiotensin II receptors.

A robust, click‐chemistry‐inspired procedure for radiolabeling of cyclic ureas was developed. This protocol, suitable for all carbon isotopes (¹¹C, ¹³C, ¹⁴C), is based on the direct functionalization of carbon dioxide: the universal building block for carbon radiolabeling. The strategy is operationally simple and reproducible in different radiochemistry centers, exhibits remarkably wide substrate scope with short reaction times, and demonstrates superior reactivity as compared to previously reported systems. With this procedure, a variety of pharmaceuticals and an unprotected peptide were labeled with high radiochemical efficiency.

A robust, click‐chemistry‐inspired procedure for radiolabeling of cyclic ureas was developed. This protocol, suitable for all carbon isotopes (¹¹C, ¹³C, ¹⁴C), is based on the direct functionalization of carbon dioxide: the universal building block for carbon radiolabeling. The strategy is operationally simple and reproducible in different radiochemistry centers, exhibits remarkably wide substrate scope with short reaction times, and demonstrates superior reactivity as compared to previously reported systems. With this procedure, a variety of pharmaceuticals and an unprotected peptide were labeled with high radiochemical efficiency.

The HIV-1 envelope gp120 glycoprotein is made up of three autonomously folding domains that adopt different conformations in response to ligand binding. During HIV entry, the CD4 receptor engages a ∼14 Å deep cavity that penetrates the hydrophobic core of gp120 at the intersection of these three domains. Mutagenesis studies have demonstrated the importance of residues lining this cavity for virus entry. Here we use a set of CD4-mimetic miniproteins with different inserts into the Phe 43 cavity, to probe the structure of the cavity and decipher its role in ligand binding and neutralization. Through virological, biochemical and structural analyses we show that optimal filling of the Phe 43 cavity with a flexible ligand can lead to high affinity binding and broad and potent neutralization. CD4-mimetic miniprotein M48-U1, with a cyclohexylmethoxyphenyl insertion into the Phe 43 cavity showed best neutralization properties. M48-U1 bound YU2 gp120 with 25 pM affinity and neutralized all circulating HIV-1 isolates, except clade A/E viruses. The crystal structure of M48-U1 bound toYU2 gp120 was solved at 1.49 Å resolution. This was compared to the 2.1 Å resolution structure of a novel CD4-mimetic miniprotein, M48-U7 that inserts a flexible 5-phenoxypentan-1-ol moiety into the Phe 43 cavity. Structural analyses reveal residual ligand flexibility in the bound state. The M48-U1/U7-bound Phe 43 cavity resembled the cavity conformation in unliganded gp120 coree. These results provide an understanding of the structure-function relationship of Phe 43 cavity filling and define a mechanism for the broad and potent neutralization by the CD4-mimetic miniprotein M48-U1.

Background: Binding of the viral envelope protein (Env), and particularly of its gp120 subunit, to the cellular CD4 receptor is the first essential step of the HIV-1 entry process. The CD4 binding site (CD4bs) of gp120, and especially a recessed cavity occupied by the CD4 Phe43 residue, are known to be highly conserved among the different circulating subtypes and therefore constitute particularly interesting targets for vaccine and drug design. The miniCD4 proteins are a promising class of CD4bs inhibitors. Studying virus evolution under pressure of CD4bs inhibitors could provide insight on the gp120-CD4 interaction and viral entry. Results: The present study reports on the resistance induction of two subtype B HIV-1 against the most active miniCD4, M48U1, and its ancestor, M48, and how these mutated positions affect CD4bs recognition, entry efficiency, and sensitivity to other CD4bs inhibitors. Resistance against M48U1 was always associated with S375R/N substitution in both BaL and SF162; M48 resistance was associated with D474N substitution in SF162 and with H105Y substitution in BaL. In addition, some other mutations at position V255 and G471 were of importance for SF162 resistant viruses. Except for 474, all of these mutated positions are conserved, and introducing them into an SF162 Env expressing infectious molecular clone (pBRNL4.3 SF162) resulted in decreased entry efficiency. Furthermore, resistant mutants showed at least some cross-resistance towards other CD4bs inhibitors, the V3 monoclonal antibody 447-52D and some even against the monoclonal antibody 17b, of which the epitope overlaps the co-receptor binding site. Conclusions: The mutations H105Y, V255M, S375R/N, G471R/E, and D474N are found to be involved in resistance towards M48 and M48U1. All mutated positions are part of, or in close proximity to, the CD4bs; most are highly conserved, and all have an impact on the entry efficiency, suggesting their importance for optimal virus infectivity.

We developed a novel radioactive short bifunctional photoprobe, which could be coupled through a cleavable bond to an engineered cysteinyl residue on an analogue of a nicotinic acetylcholine receptor-specific α-neurotoxin. This cysteine was put on the tip of loop II in place of Arg33, a major residue for the interaction with the receptor. To facilitate the purification of the nAChR labeled subunits, we tagged the ligand with a desthiobiotin moiety. After irradiation of the photosensitive toxin−nAChR complex, gel electrophoresis showed that most of the radioactivity was attached to the α subunit (59%), followed by the γ subunit (28%), with the δ subunit (13%) being less labeled. On a preparative scale, the labeled subunits were purified on streptavidin beads before separation on SDS-PAGE. “In-gel” CNBr cleavage of the labeled α subunit followed by Edman degradation of the purified peptides showed that αTyr190 and αTyr198 were the most labeled residues, with a less important labeling on αCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.