Gonçalo J. L. Bernardes

Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology

Chemical modification of proteins is a rapidly expanding area in chemical biology. Selective installation of biochemical probes has led to a better understanding of natural protein modification and macromolecular function. In other cases such chemical alterations have changed the protein function entirely. Additionally, tethering therapeutic cargo to proteins has proven invaluable in campaigns against disease. For controlled, selective access to such modified proteins, a unique chemical handle is required. Cysteine, with its unique reactivity, has long been used for such modifications. Cysteine has enjoyed widespread use in selective protein modification, yet new applications and even new reactions continue to emerge. This Focus Review highlights the enduring utility of cysteine in protein modification with special focus on recent innovations in chemistry and biology associated with such modifications.

Site-selective protein-modification chemistry for basic biology and drug development

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

Advances in Chemical Protein Modification

O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovacion, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.

CORM-3 Reactivity toward Proteins: The Crystal Structure of a Ru(II) Dicarbonyl-Lysozyme Complex

CORM-3, [fac-Ru(CO)(3)Cl(κ(2)-H(2)NCH(2)CO(2))], is a well-known carbon monoxide releasing molecule (CORM) capable of delivering CO in vivo. Herein we show for the first time that the interactions of CORM-3 with proteins result in the loss of a chloride ion, glycinate, and one CO ligand. The rapid formation of stable adducts between the protein and the remaining cis-Ru(II)(CO)(2) fragments was confirmed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), Liquid-Chromatography Mass Spectrometry (LC-MS), Infrared Spectroscopy (IR), and X-ray crystallography. Three Ru coordination sites are observed in the structure of hen egg white lysozyme crystals soaked with CORM-3. The site with highest Ru occupancy (80%) shows a fac-[(His15)Ru(CO)(2)(H(2)O)(3)] structure.

A Small‐Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors

Antibody-drug conjugates are a very promising class of new anticancer agents, but the use of small-molecule ligands for the targeted delivery of cytotoxic drugs into solid tumors is less well established. Here, we describe the first small-molecule drug conjugates for the treatment of carbonic anhydrase IX expressing solid tumors. Using ligand-dye conjugates we demonstrate that such molecules can preferentially accumulate inside antigen-positive lesions, have fast targeting kinetics and good tumor-penetrating properties, and are easily accessible by total synthesis. A disulfide-linked drug conjugate with the maytansinoid DM1 as the cytotoxic payload and a derivative of acetazolamide as the targeting ligand exhibited a potent antitumor effect in SKRC52 renal cell carcinoma in vivo. It was furthermore superior to sunitinib and sorafenib, both small-molecule standard-of-care drugs for the treatment of kidney cancer.

Methods for converting cysteine to dehydroalanine on peptides and proteins

Dehydroalanine is a synthetic precursor to a wide array of protein modifications. We describe multiple methods for the chemical conversion of cysteine to dehydroalanine on peptides and proteins. The scope and limitations of these methods were investigated with attention paid to side reactions, scale, and aqueous- and bio-compatibility. The most general method investigated—a bis-alkylation–elimination of cysteine to dehydroalanine—was applied successfully to multiple proteins and enabled the site-selective synthesis of a glycosylated antibody.

Carbon‐Monoxide‐Releasing Molecules for the Delivery of Therapeutic CO In Vivo

The development of carbon-monoxide-releasing molecules (CORMs) as pharmaceutical agents represents an attractive and safer alternative to administration of gaseous CO. Most CORMs developed to date are transition-metal carbonyl complexes. Although such CORMs have showed promising results in the treatment of a number of animal models of disease, they still lack the necessary attributes for clinical development. Described in this Minireview are the methods used for CORM selection, to date, and how new insights into the reactivity of metal-carbonyl complexes in vivo, together with advances in methods for live-cell CO detection, are driving the design and synthesis of new CORMs, CORMs that will enable controlled CO release in vivo in a spatial and temporal manner without affecting oxygen transport by hemoglobin.

From disulfide- to thioether-linked glycoproteins.

The presence of carbohydrate structures on proteins has been estimated to occur in 50% of eukaryotic cells and is linked to several biological events, such as the regulation of cell signaling, cellular differentiation, and immune response. In nature, glycoproteins are found as heterogeneous mixtures, which complicates their characterization and functional determination. Better access to homogeneous glycoproteins and their mimics is likely to improve our understanding of their roles. Naturally occurring protein and peptide glycans are predominantly linked to an asparagine or serine/threonine residue, and many glycopeptide syntheses are based on the introduction of mimics of such tethers. It was not until 1971 that a natural S-glycosidic linkage was identified on a peptide: L0te and Weiss isolated octaand decapeptides in which galactose and glucose, respectively, were attached to the side chain of an N-terminal cysteine residue. Several methods have since been developed for the synthesis of Slinked glycopeptides: the conjugate addition of a glycosyl thiol to a dehydroalanine-containing peptide, the reaction of a glycosyl thiol with a b-bromoalanine moiety, and the rearrangement of an allylic selenenylsulfide. However, to date no chemical method has been applied to the synthesis of S-linked glycoproteins. Importantly, S-linked glycopeptides display enhanced chemical and enzymatic stability relative to their native congeners. A process for desulfurizing disulfide-linked glycoproteins to provide thioether-linked homologues would allow ready access not only to this class of natural products but also to novel glycoproteins. We have described previously the use of glycomethanethiosulfonates (glycoMTS), glycophenylthiosulfonates (glycoPTS), and glycoselenenylsulfides (glycoSeS) as efficient chemoselective reagents for the synthesis of disulfide-linked glycopeptides and glycoproteins. Herein we present the synthesis of thioether-linked glycoconjugates from these readily synthesized disulfide-linked precursors. Important examples of the contraction of disulfide and peroxide linkages upon treatment with sources of P are known; however, the generality of such transformations remains to be established. Our research in this area was motivated by a then surprising reaction of the sugar disulfide 1 with tributylphosphine to afford thioglycoside 2 in 74% yield (Scheme 1). Inversion at the anomeric center suggested

A Traceless Vascular‐Targeting Antibody–Drug Conjugate for Cancer Therapy

Monoclonal antibodies have demonstrated considerable utility in the clinical treatment of cancer, but unmodified immunoglobulins are rarely curative, especially when used as single agents. Thus, there is considerable interest in arming antibodies with bioactive payloads (e.g., drugs, radionuclides, cytokines), to improve their potency and selectivity, thus increasing activity at the tumor site while sparing normal tissues. Significant progress has beenmade in the past few years in the area of antibody–drug conjugates (ADCs) for the selective delivery of cytotoxic drugs to tumors. As a result of these investigations, new agents with pronounced clinical activities have been developed, including SGN-35 (an ADC directed against CD30-positive hematological malignancies) and trastuzumab-DM1 (which has shown activity in metastatic breast cancer). As conventional drug conjugation strategies yield heterogeneous ADC preparations, intense efforts are being devoted to the development of methods for site-selective modification of therapeutic antibodies, thus leading to products with improved performance and batch-tobatch reproducibility. Furthermore, comparative evaluations of intact immunoglobulins in IgG format and other recombinant antibody formats for ADC development have been conducted. It is generally assumed that ADCs may need to be internalized by the tumor cells for the active release of cytotoxic drugs. Once ADCs are internalized and the drug is released in the intracellular compartments, a cross-fire effect (corresponding to the migration to neighboring cells) may occur, as has been reported for the treatment of tumors consisting of a mixture of antigen-positive and antigennegative cells. However, monoclonal antibodies specific to tumor cell antigens often exhibit limited diffusion into the solid tumor mass by several mechanisms, including slow extravasation and antibody trapping by perivascular tumor cells (the so-called antigen barrier). In view of the fact that the formation of new blood vessels (angiogenesis) is a rare process in a healthy adult but a characteristic feature of virtually all types of aggressive cancers, it would be reasonable to develop vascular-targeting ADCs. Unlike the use of cell-specific markers, vascular targeting offers comprehensive tumor coverage, as the majority of cancers express splice isoforms of tenascin-C and of oncofetal fibronectin. In addition, vascular targeting helps address the issue of heterogeneity of antigen expression within the tumor mass (i.e., tumor cells which are positive or negative for the antigen). Despite the remarkable potency of cytotoxic compounds targeting the tumor vasculature and the strong dependence of growing neoplastic masses on florid angiogenesis, only limited efforts were directed in the past towards the investigation of ADCs that target tumor vascular antigens. We have recently shown that the antibody-based delivery of photosensitizers to tumor blood vessels and irradiation may induce complete and long-lasting cancer eradication, in a process that also involves the action of natural killer cells. Thus, there appears to be a strong rationale for the targeted delivery of cytotoxic agents to the tumor neovasculature for cancer therapy. Given that antibodies are large molecules compared to cytotoxic agents, potent drugs need to be used to generate ADCs that can be administered at reasonably low doses and that are compatible with industrial development activities at acceptable cost of goods. Herein, we aimed at generating a novel class of chemically defined vascular-targeting ADCs that release cytotoxic drugs with a mechanism that does not require antibody internalization. We reasoned that ADCs based on linkerless antibody modification with a potent thiolcontaining drug would allow the formation of homogeneous products by the formation of a mixed disulfide. These agents could release the cytotoxic payload in the extracellular space, when tumor cell death is initiated and releases high concentrations of reducing agents (e.g., cysteine, glutathione) to the surrounding environment. Provided that a sufficiently large amount of ADC can be delivered to the subendothelial extracellular matrix, the drug release process would be amplified as tumor cell death progresses. Dolastatins are a group of small peptides isolated from the Indian ocean hare Dolabella auricularia that bind to tubulin subunits and inhibit new microtubule assembly and depolymerize existing microtubules, thus blocking cell cycle [*] Dr. G. J. L. Bernardes, Dr. S. Tr!ssel, I. Hartmann, Dr. J. Scheuermann, Prof. D. Neri Department of Chemistry and Applied Biosciences Swiss Federal Institute of Technology (ETH Z!rich) Wolfgang-Pauli Str. 10, 8093 Z!rich (Switzerland) E-mail: dario.neri@pharma.ethz.ch Dr. G. Casi, Dr. K. Schwager Philochem AG, Libernstrasse 3, 8112 Otelfingen (Switzerland) [**] G.J.L.B. is an EMBO and Novartis Foundation Research Fellow. We thank Katrin Gutbrodt for her help in histology and immunofluorescence experiments, Dr. Kathrin Zuberb!hler and Nadine Pasche for their help during therapy experiments, and Dr. Eveline Traschel for treating tumor-bearing mice with IgG(F8). Financial contribution from ETH Z!rich, Swiss National Science Foundation, SwissBridge/Stammbach Stiftung, Kommission f!r Technologie und Innovation (KTI) and Philochem AG is gratefully acknowledged. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201106527. Angewandte Chemie

Spontaneous CO Release from RuII(CO)2–Protein Complexes in Aqueous Solution, Cells, and Mice†

We demonstrate that RuII(CO)2–protein complexes, formed by the reaction of the hydrolytic decomposition products of [fac-RuCl(κ2-H2NCH2CO2)(CO)3] (CORM-3) with histidine residues exposed on the surface of proteins, spontaneously release CO in aqueous solution, cells, and mice. CO release was detected by mass spectrometry (MS) and confocal microscopy using a CO-responsive turn-on fluorescent probe. These findings support our hypothesis that plasma proteins act as CO carriers after in vivo administration of CORM-3. CO released from a synthetic bovine serum albumin (BSA)–RuII(CO)2 complex leads to downregulation of the cytokines interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α in cancer cells. Finally, administration of BSA–RuII(CO)2 in mice bearing a colon carcinoma tumor results in enhanced CO accumulation at the tumor. Our data suggest the use of RuII(CO)2–protein complexes as viable alternatives for the safe and spatially controlled delivery of therapeutic CO in vivo.