Design and validation of bioorthogonal probes for cellular disease models

Well-validated chemical probes enable testing of biological hypotheses, investigation of target tractability and translatability to the clinical phase. Consequently, these important tool compounds play a key role in the drug discovery process. Moreover, their use for target validation may ultimately help to decrease the attrition rate encountered by new molecular entities in clinical trials. Despite the number of publications describing well-validated chemical probes, for several reasons, it remains a difficult challenge to identify and select the appropriate high quality molecule enabling the desired biological and pharmacological studies in the relevant disease model. Various reports describe sets of principles to be fulfilled by high quality chemical probes, which mainly rely on verifying that chemical probes are potent, engage their intended targets in the relevant cellular model, have sufficient exposure at the desired site of action and express functional pharmacological activities by a selective modulation of their targets. Classical chemical probes include LMW ligands, which inform on the functional consequences of interacting with a particular bio-logical target in a model system. To get information on other parameters (such as sub-cellular distribution of target or compound) different tools may need to be used, often requiring specific chemical functionalization in order to observe the molecule using the currently available technologies. Compared to classical chemical probes, bioorthogonal probes can also be small molecules that elicit a functional response but with the additional advantage of being able to undergo reaction with a variety of chemical reporters (e.g. fluorophores) in situ, in bio-logical model systems such as cells and animals. Therefore, this increases their versatility; for example their visualization using imaging technologies (bioluminescence and fluorescence imaging) can provide important insights on compound permeability, in-tracellular distribution, and potentially co-localization with its protein target in relevant cellular disease models. Hence, the use of a bespoke bioorthogonal probe may answer key biological questions, which are usually only addressed by using multiple classical probes. Herein, we describe the design and the synthesis of bioorthogonal probes to study the inhibition of the p53-Mdm2 protein-protein interaction. Furthermore, we report the development of a set of assays based on fluorescence and bioluminescence techniques enabling the validation of these molecules in a cellular osteosarcoma model. Our approach for chemical probe design and validation in cellular disease models may be applied in principle to a wide range of novel drug discovery projects to provide early mechanistic understanding.

Small Molecule Based Approaches to Inhibit Macrophage Migration Inhibitory Factor (MIF) Activities and Elucidate its Role in Health and Disease

Hajer Ouertatani-Sakouhi

Macrophage migration inhibitory factor (MIF) is a major mediator in innate immunity and inflammation and a potential therapeutic target in multiple inflammatory, infectious and autoimmune diseases including cancer. Current therapeutic strategies for targeting MIF focus on modulating its biological activities using anti-MIF neutralizing antibodies or developing inhibitors of its tautomerase activity. Although the identity of its natural substrate remains unknown, several small molecule inhibitors have been reported to be effective inhibitors of MIF tautomerase activity in vitro. All these inhibitors were identified by rational design and structure-activity studies based on the structure of the catalytic site and/or non physiologic substrates of MIF. The lack of suitable high-throughput screening (HTS) assays has hindered the screening of chemical libraries and discovery of more diverse inhibitors. Motivated by the goal of discovering diverse classes of MIF inhibitors that modulate MIF activity specific targeting of its biochemical, conformation and quaternary structure properties, both activity based endpoint and kinetic HTS assays were developed, optimized and performed on diverse set of libraries. Using the endpoint assay, out of 15440 compounds screened, twelve novel inhibitors of MIF's catalytic activity (∼ 0.1% hit rate) with IC50s in the range of 1.5 to 15.5 µM were identified and validated. The interaction site and mechanism of action of all these inhibitors were defined using structure activity studies and a battery of biochemical and biophysical methods, including mass spectrometry, light scattering, and NMR. The effect on MIF biological activities was also examined on MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Using the kinetic-based activity assay, we screened 80,000 small molecules and identified and validated thirteen novel inhibitors of MIF catalytic activity with inhibition constant (Ki,app) values ranging from 0.5 to 13 µM. According to the structure and potency some of these molecules could be of great therapeutic potential. The MIF inhibitors emerging from these studies could be divided into four classes: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1 which could be classified into 5 groups; 2) a novel class of catalytic site inhibitors, 3) a class of trimer destabilizing inhibitors. These findings demonstrate the potential of HTS assays as attractive means to identify novel classes of MIF inhibitors as lead drug candidates and/or chemical tools for elucidating the biochemical and structural bases underlying the multifunctionality of MIF's in health and disease. Having these molecules in hand, will advance our understanding of protein-protein interactions mechanism for MIF oligomerization and may help to elucidate the role of MIF enzymatic activity in health and disease.

EPFL2010

Optimalisation de la photothérapie dynamique et de la photodétection de cancers précoces par spectroscopie résolue en temps de luminophores endogènes et exogènes

Pascal Uehlinger

The aim of this thesis is to optimize and obtain fundamental information on i) the photodetection (PD) of early stage cancers in the tracheo-bronchial tree by spectroscopic imaging of the tissue autofluorescence, ii) photodynamic therapy (PDT) applied in dermatology based on the use of protoporphyrin IX (PPIX) precursors. These two areas of photomedecine have been the subject of investigation at the air and soil pollution laboratory (LPAS) for the last 10 years. PD and PDT are areas of research that evolve in parallel, both having similar objectives: i.e. improving the patients' chances of survival by the early detection and treatment of cancer. Three main studies are presented, each in relation to a particular type of chromophore : Endogenous fluorophores generate the autofluorescence of biological tissue; some of these fluorophores can be synthesized endogenously in excess of their normal concentration following the administration of a biochemical precursor; finally, exogenous luminophores are artificially synthesized and administered to the organism where they can be used for detection. The introduction of this thesis presents several aspects of cancer as well as the theoretical background necessary for this study. At first we address time-resolved autofluorescence spectroscopy of bronchial tissue and the mechanisms at the origin of the contrasts existing between normal tissue and early stage tumours. Several hypotheses explain the reduction in intensity and spectral changes of the autofluorescence observed between early epidermal carcinoma and normal bronchial mucosa : 1) the reduction in the concentration of fluorophores in the malignant tissue, 2) the quenching of the fluorescence due to a change in the physico-chemical environment of the tumour, 3) an architectural effect such as a thickening of the malignant epithelium due to an abnormal cellular proliferation and 4) an increase in the concentration of light absorbers such as haemoglobin. In the course of our measurements no statistically significant difference in the fluorescence lifetime was observed between carcinoma in situ, moderate dysplasia and normal tissue, which leads us to conclude that hypotheses 1, 3 and/or 4 may well be at the origin of the differences in fluorescence intensity. The fluorescence lifetime decays are consistent, identifiable by a triple-exponential decay in the green and red part of the spectrum. This seems to indicate that one or two dominant fluorophores are involved which possibly, given the measured lifetimes, could be collagen and/or elastine. The second part of this thesis concerns the modulation of the production of PPIX (an endogenously induced fluorophore) in the skin, following the administration of 5-aminolevulinic acid or one of its esterified derivatives. These investigations have been carried out on the epidermis of human volunteers as well as on the epidermal equivalent (Epidex™). The final goal of these studies was on the one hand to increase the production of PPIX well below the skin surface to improve certain dermatological treatments such as the PDT of nodular basal cell carcinomas (BCC), and, on the other hand, the ability to use the results obtained with the Epidex™ model to produce PPIX specifically in hair follicles, which permits the development of a new method of hair removal by PDT. The tests using the Epidex™ model demonstrated that it is possible, by adding Desferal® (an iron chelator) or (L+) ascorbic acid iron (II) salt, to increase respectively decrease the production of PPIX as well as to modulate its elimination. These results open new perspectives such as the specific use of zinc-protoporphyrin (produced by skin cells following the dermatological use of Desferal®), and the identification of a specific precursor of PPIX for hair follicles. This precursor is an excellent "candidate" in the field of research for the development of a new method of hair removal by PDT. In the third part of this thesis we describe a preliminary study of optical sensors for the intravascular partial pressure of oxygen via the measurement of phosphorescence lifetime quenching. The phosphorescence lifetime of these species is directly related to the partial pressure of the oxygen dissolved (pO2). These oxygen sensors allow us, for instance, to measure the reduction of the pO2 during PDT, without phototoxicity. Palladium and ruthenium complexes were among the most promising compounds, and certain types of nanoparticules were used as vectors. Another interesting lead consists in the covalent modification of these "luminophores-molecules": in this context, the experiments conducted in collaboration with the "Laboratoire de photonique et interfaces" of the EPFL, demonstrate that we can drastically reduce the speed of leakage outside the vascularisation of these molecules and/or improve their retention by the nanoparticules.

EPFL2004

Image- and Fluorescence-Based Test Shows Oxidant-Dependent Damages in Red Blood Cells and Enables Screening of Potential Protective Molecules

David Crettaz, Michel Prudent, Benjamin Rappaz, Gerardo Turcatti

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape “Tirocytes”. The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.