Geoffray Leriche

Cleavable linkers in chemical biology

Interest in cleavable linkers is growing due to the rapid development and expansion of chemical biology. The chemical constrains imposed by the biological conditions cause significant challenges for organic chemists. In this review we will present an overview of the cleavable linkers used in chemical biology classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.

Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles

Exquisite chemoselectivity for cysteine has been found for a novel class of remarkably hydrolytically stable reagents, 3-arylpropiolonitriles (APN). The efficacy of the APN-mediated tagging was benchmarked against other cysteine-selective methodologies in a model study on a series of traceable amino acid derivatives. The selectivity of the methodology was further explored on peptide mixtures obtained by trypsin digestion of lysozyme. Additionally, the superior stability of APN-cysteine conjugates in aqueous media, human plasma, and living cells makes this new thiol-click reaction a promising methodology for applications in bioconjugation.

A FRET-based probe with a chemically deactivatable quencher

A new concept of a chemically deactivatable quencher is proposed for a FRET-based probe that turns-on its fluorescence by either an enzymatic cleavage or a chemical reagent (sodium dithionite). This concept allowed us to quantify the caspase-3 cleavage activity in solution and to reveal unreacted probes in cell experiments.

Nondenaturing chemical proteomics for protein complex isolation and identification.

The isolation and identification of proteins by chemical proteomics relies on the use of a chemical probe that targets and allows the extraction of a specific class of protein. This technology allows the full proteomic study of a drug’s secondary targets as well as the study of primary targets and their associated complexes. Efficient recovery of the target protein is often carried out under harsh and denaturing conditions, which can lead to contamination by nonspecific materials and the loss of protein partners, structural information, and protein function. To reduce protein contamination, linkers that can be cleaved chemically in biological media have been introduced by using the properties of the azo function, disulfide bond, vinyl sulfide, or acylhydrazone. In particular, structural optimization of the azo function significantly improved the reduction kinetics with sodium dithionite. The use of mild reducing conditions circumvents harsh and denaturing conditions during chemical proteomic experiments and affords opportunities for the direct functional or structural analysis of isolated biological targets and their associated macromolecular complexes. In this study, we designed a linker, 1, that contains 2-(4’-hydroxy-2’-alkoxy phenylazo)benzoic acid (HAZA) as an optimized cleavable site possessing on one side a biotin affinity tag for enrichment and on the other side an alkyne moiety that can be conveniently linked to various molecular probes by a click reaction. HAZA linker 1 can be cleaved in less than ten seconds with 1 mm sodium dithionite. The use of this cleavable linker enabled us to capture and release native functional proteins complexes (Scheme 1). In order to target a specific protein in a complex mixture, a chemical probe is linked to 1 (step A). The biological target and associated proteins are pulled out from a complex cell lysate through affinity purification with capture beads (step B). The immobilized protein complexes are then released by chemoselective elution with sodium dithionite (step C). Eluted target protein and associated proteins can be separated by SDS-PAGE (step D) and identified by mass spectrometry (step E). At this stage, a functional study of the eluted complex can be performed (step F). We used the bacterial type II topoisomerase, DNA gyrase, as the target protein and its inhibitor, the aminocoumarin novobiocin, as the molecular hook. This model system, a 320 kDa A2B2 tetrameric multidomain enzyme has already been validated by previous experiments. DNA gyrase introduces negative supercoils into closed, circular, duplex DNA in an ATP-dependent fashion by cleaving both strands of a DNA duplex and transporting a second duplex through the double-strand break. This supercoiling activity is essential for DNA replication, transcription, and recombination. DNA gyrase is also able to relax supercoiled DNA in an ATP-independent manner. The DNA gyrase B subunit ATP binding site is targeted by the antibiotic novobiocin. An affinity probe 2 was synthesized by coupling a novobiocin azide group with the HAZA linker by click chemistry (Scheme 2). The reductive cleavage of this probe with 6 mm of dithionite was achieved in less than 20 s, as determined by UV spectroscopy. Cleavage was also confirmed by MS analysis (Scheme 2 and Figure S1 in the Supporting Information). We first evaluated the effect of sodium dithionite on protein integrity to confirm that linker cleavage would not affect the protein structure. DNA gyrase subunit B was thus incubated under typical dithionite concentrations, 4] and analyzed by native gel electrophoresis. We observed that denaturation of the protein occurred at concentrations of dithionite above 10 mm (Figure S3). Interestingly, this shows that the dithionite concentrations commonly used to reduce azo-arene-based linkers (3 25 mm) would not be compatible with nondenaturing protocols. In order to obtain the highest quantity of released protein while retaining a nondenaturing procedure, we investigated the capture and release of recombinant purified DNA gyrase B (Gyr-B) by optimizing the dithionite concentration and elution time. For this study, probe 2 was first incubated with streptavidin magnetic beads for 1 h at room temperature. Excess probe was removed by several washes in a magnetic separator, then Gyr-B was added to the resuspended magnetic beads and incubated for 1.5 h at room temperature. Excess proteins were discarded, and the remaining proteins were cleaved off by using freshly prepared dithionite solutions at different concentrations up to 6 mm for 15 min at room temperature (Figure S4). Optimal cleavage was achieved by [a] Dr. G. Budin, G. Leriche, Dr. A. Wagner Laboratory of Functional Chemo-Systems UMR 7199 74 Route du Rhin, 67401 Illkirch-Graffenstaden (France) Fax: (+ 33) 368-854-306 E-mail : wagner@bioorga.u-strasbg.fr [b] Dr. M. Moune-Dimala, J. Papillon, Dr. V. Lamour, Dr. L. Brino Laboratory of Structural Biology and Genomics Institute of Genetics and Molecular and Cellular Biology Inserm U964 UMR7104 CNRS UdS B. P. 10142, 67404 Illkirch-Graffenstaden (France) [c] Dr. J.-M. Saliou, Dr. S. Sanglier-Cianf rani, Prof. A. Van Dorsselaer Laboratoire de Spectrom trie de Masse BioOrganique Institut Pluridisciplinaire Hubert Curien IPHC CNRS, UMR 7178, Universit de Strasbourg UDS ECPM, 25 Rue Becquerel, 67087 Strasbourg (France) [d] Dr. V. Lamour Laboratory of Biochemistry and Molecular Biology Hautepierre–Strasbourg Hospitals Avenue Moli re, B. P. 49, 67098 Strasbourg (France) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000574.

Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea‐Inspired Tetraether Lipids

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies.

Spiro Diorthoester (SpiDo), a Human Plasma Stable Acid-Sensitive Cleavable Linker for Lysosomal Release

pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for selective release of therapeutics selectively at targets and orthoesters have been demonstrated to be good candidates for such linkers. Following an HPLC screening, a Spiro Diorthoester (SpiDo) derivative was identified as a potent acid-labile group for the development of pH-sensitive targeted systems. After incorporation of this linker into activatable FRET-based probe and side-by-side comparison to a well-known alkylhydrazone linker, this SpiDo linker has shown a fast and pH sensitive hydrolysis for mild acidic conditions, a pH sensitive lysosomal hydrolysis, and high stability in human plasma.

Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes

This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.

Hydrophobic Nanoparticles Reduce the β-Sheet Content of SEVI Amyloid Fibrils and Inhibit SEVI-Enhanced HIV Infectivity

Semen-derived enhancer of virus infection (SEVI) fibrils are naturally abundant amyloid aggregates found in semen that facilitate viral attachment and internalization of human immunodeficiency virus (HIV) in cells, thereby increasing the probability of infection. Mature SEVI fibrils are composed of aggregated peptides exhibiting high β-sheet secondary structural characteristics. Herein, we show that polymers containing hydrophobic side chains can interact with SEVI and reduce its β-sheet content by ∼45% compared with the β-sheet content of SEVI in the presence of polymers with hydrophilic side chains, as estimated by polarization modulation-infrared reflectance absorption spectroscopy measurements. A nanoparticle (NP) formulation of this hydrophobic polymer reduced SEVI-mediated HIV infection in TMZ-bl cells by 60% compared with the control treatment. Although these NPs lacked specific amyloid-targeting groups, thus requiring high concentrations to observe biological activity, the use of hydrophobic interactions to alter the secondary structure of amyloids represents a useful approach to neutralizing the SEVI function. These results could, therefore, have general implications in the design of novel materials that can modify the activity of amyloids associated with a variety of other neurological and systemic diseases.

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character.

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum-Stable Membranes with Low Leakage

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.