An Oculus to Profile and Probe Target Engagement In Vivo: How T-REX Was Born and Its Evolution into G-REX

Here we provide a personal account of innovation and design principles underpinning a method to interrogate precision electrophile signaling that has come to be known as "REX technologies". This Account is framed in the context of trying to improve methods of target mining and understanding of individual target-ligand engagement by a specific natural electrophile and the ramifications of this labeling event in cells and organisms. We start by explaining from a practical standpoint why gleaning such understanding is critical: we are constantly assailed by a battery of electrophilic molecules that exist as a consequence of diet, food preparation, ineluctable endogenous metabolic processes, and potentially disease. The resulting molecules, which are detectable in the body, appear to be able to modify function of specific proteins. Aside from potentially being biologically relevant in their own right, these labeling events are essentially identical to protein-covalent drug interactions. Thus, on what proteins and even in what ways a covalent drug will work can be understood through the eyes of natural electrophiles; extending this logic leads to the postulate that target identification of specific electrophiles can inform on drug design. However, when we entered this field, there was no way to interrogate how a specific labeling event impacted a specific protein in an unperturbed cell. Methods to evaluate stoichiometry of labeling, and even chemospecificity of a specific phenotype were limited. There were further no generally accepted ways to study electrophile signaling that did not hugely disturb physiology. We developed T-REX, a method to study single-protein-specific electrophile engagement, to interrogate how single-protein electrophile labeling shapes pathway flux. Using T-REX, we discovered that labeling of several proteins by a specific electrophile, even at low occupancy, leads to biologically relevant signaling outputs. Further experimentation using T-REX showed that in some instances, single-protein isoforms were electrophile responsive against other isoforms, such as Akt3. Selective electrophile-labeling of Akt3 elicited inhibition of Akt-pathway flux in cells and in zebrafish embryos. Using these data, we rationally designed a molecule to selectively target Akt3. This was a fusion of the naturally derived electrophile and an isoform-nonspecific, reversible Akt inhibitor in phase-II trials, MK-2206. The resulting molecule was a selective inhibitor of Akt3 and was shown to fare better than MK-2206 in breast cancer xenograft mouse models. Recently, we have also developed a means to screen electrophile sensors that is unbiased and uses a precise burst of electrophiles. Using this method, dubbed G-REX, in conjunction with T-REX, we discovered new DNA-damage response upregulation pathways orchestrated by simple natural electrophiles. We thus emphasize how deriving a quantitative understanding of electrophile signaling that is linked to thorough and precise mechanistic studies can open doors to numerous medicinally and biologically relevant insights, from gleaning better understanding of target engagement and target mining to rational design of targeted covalent medicines.

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

Molecular Mechanisms Underlying Hyaline Fibromatosis Syndrome

Julie Deuquet Ariosa

Hyaline Fibromatosis Syndrome (HFS) is a rare inherited disease that is characterized by an accumulation of an unidentified hyaline material, largely affecting connective tissues. Patients afflicted with HFS present a wide range of clinical symptoms such as nodules, papules, hyperplasia of the gingiva, joint contractions, thickness and hyperpigmentation of the skin, and osteopenia. Depending of the severity and the delay of appearance of these symptoms, the life expectancy varies between 2 years and > 30 years. Death earlier in life is due to recurrent diarrhea and chest infections of unknown origin. In 2003, the gene responsible for HFS was identified to be Capillary Morphoqenesis Gene 2 (CMG2), a gene upregulated during capillary morphogenesis in three-dimensional collagen matrices. This gene encodes a type I transmembrane glycoprotein composed of a von Willebrand (vWA) domain followed by an immunoglobulin (Ig)-like domain in its extracellular part, a single α-helix spanning transmembrane domain, and a 148 amino acids cytoplasmic tail predicted as unstructured. Even though the physiological function of CMG2 remains to be elucidated, it has been shown that this protein induces endothelial proliferation, and dictates cell polarization during embryogenesis in a zebrafish model. Besides its physiological function, CMG2 was found to be an anthrax toxin receptor, thus its alternative name ANTXR2, and has been extensively studied in this context. Genetic analysis has led to the identification of 34 CMG2 mutations in HFS patients. The molecular mechanisms underlying the disease have however not yet been identified. During the five years of my thesis, we were interested in understanding the cellular consequences of these mutations and in getting a better understanding of the physiological function of CMG2. I first showed that most of the HFS mutations in the vWA domain lead to alterations in folding of the domain, either in terms of kinetics or of structure, leading to recognition of the protein by the ER quality control (ERQC) and subsequent degradation by the ERAD pathway. A direct consequence of this retention is the loss of function at the cell surface or other cellular sites. In a second study, I studied the effect of novel HFS mutations that our collaborators and we identified in the CMG2 Ig-like domain. Since the structure of this domain was uncharacterized, we first generated a model of the domain and found that it has an Ig-like fold, despite the very low sequence homology, and harbors two disulfide bonds essential for folding and stabilization of the domain. HFS mutations localized to this domain led to aberrant intermolecular disulfide binding, ER retention and enhanced ERAD when compared to the vWA domain HFS mutations as shown in patients fibroblasts. The consequential loss of CMG2 could be partly alleviated by treating cells with proteasome inhibitor, such as Bortezomib, illustrating that ERAD components are potential therapeutic targets for HFS. As most of the HFS mutations in the ectodomain of CMG2 trigger this ER retention during the CMG2 biosynthesis, we then addressed this process by studying one of the most described post-translational modifications occurring in the ER for protein folding, namely the N-glycosylation. We found that the two potential CMG2 N-glycosylation sites, N250 and N260, are both occupied. Neither of the two sites is essential for ER exit. Glycosylation of CMG2 on N260 is however important since the expression levels of the N260A mutant were significant lower that for the WT protein, despite equivalent or higher synthesis, suggesting that glycosylation at this site is important for efficient folding. Finally, in order to better understand the CMG2 function, we investigated what the physiological ligands could be, assuming it is a receptor as suggested by its role in anthrax infection. We identified type VI collagen as a potential ligand in-vitro that activates CMG2 in-vivo, by phosphorylation of CMG2 cytoplasmic tail. This CMG2 activation upon type VI collagen binding could be a signal for ligand-triggered endocytosis, as is has been described with the protective antigen (PA) of anthrax toxin. The results of this work allow us to gain insights in the molecular mechanisms underlying HFS. Our studies indicate that the identification of the genotype-phenotype correlation of this disease is important for proper diagnosis and clinical approaches, and also that a therapeutic strategy based on proteasome inhibitors, such as Bortezomib, could be envisioned to treat some HFS symptoms in order to improve the life conditions of the patients.

EPFL2011

Peptide-Polymer Conjugates for Therapeutic Applications

Ana Maria Raduta

Over the last ten years the attention of the pharmaceutical industry has shifted away from small molecules to focus on new biologics such as antibodies, proteins and therapeutic peptides. Herein, peptides have emerged as a particularly interesting category that fills a niche between small molecule chemicals and the larger proteins. Although they have great potential for drug development, their use is still limited due to their intrinsic low systemic stability, rapid renal elimination and poor membrane permeability. Various formulations have been developed to take advantage of their therapeutic properties, one of the most promising strategies being chemical conjugation to a polymer. Peptide-polymer conjugates are hybrid materials that consist of one or more synthetic polymers covalently attached to one or more peptide fragments. The aim of this thesis was to provide an insight into the requirements for the design of peptide-polymer conjugates for therapy. Chapter 1 illustrates the state of the art in the development of peptides for therapeutic applications, exemplifies the different architectures and structures of peptide-polymer conjugates and discusses ongoing efforts to improve and diversify the range and applications of these conjugates. Chapter 2 describes a novel synthetic strategy to prepare PHPMA based peptide-polymer conjugates via thiol chemistry starting from poly(pentafluorophenyl methacrylate). By varying the feed composition, the extent of side chain modification could be controlled. Further modification of the reactive copolymer scaffolds led to the site specific attachment of model peptides. This method was used to synthesize stable as well as pH and redox sensitive peptide-polymer conjugates and the release of the peptide from the polymer backbone was studied in acidic and redox conditions. In Chapter 3, the aim was to construct a peptide-polymer conjugate that could serve to promote intracellular delivery and drug release but also play a role in the intracellular trafficking of the drug. To this end, coiled coil peptides E3/K3 was attached to a PHPMA polymer backbone and was used to facilitate release from the backbone at acidic pH and further endosomal escape. Initially, the membrane lytic properties of E3/K3 and three newly designed E3/K3X motifs on model membranes were investigated as a function of pH. Secondly, the potential application of E3/K3 peptide-polymer conjugate for cytoplasmic delivery was evaluated in vitro. Chapter 4 explores the synthesis and in vitro therapeutic activity of synthetic peptide-polymer conjugates focusing on the inhibition of an oncogenic transcription factor (AP-1). To this end, AFosW peptide, a peptide binder that can interfere with AP-1 formation was attached to a polymer backbone via a redox or pH sensitive linker using a strategy developed in Chapter 2. Fluorescent microscopy and flow cytometry provided an insight into the cellular uptake of these conjugates. Their ability to interfere with AP-1 formation in vitro was quantified. Chapter 5 describes the synthesis of a second generation of peptide-polymer conjugates for AP-1 inhibition that along the FosW dominant negative peptide also incorporates a motif that facilitates endosomal escape and a nuclear localization sequence. To investigate the potential application for therapy, the conjugates were evaluated and compared with regard to their ability to inhibit AP-1 formation in living cells.

EPFL2015