Microbial Transglutaminase and c‐myc‐Tag: A Strong Couple for the Functionalization of Antibody‐Like Protein Scaffolds from Discovery Platforms

Antibody‐like proteins selected from discovery platforms are preferentially functionalized by site‐specific modification as this approach preserves the binding abilities and allows a side‐by‐side comparison of multiple conjugates. Here we present an enzymatic bioconjugation platform that targets the c‐myc‐tag peptide sequence (EQKLISEEDL) as a handle for the site‐specific modification of antibody‐like proteins. Microbial transglutaminase (MTGase) was exploited to form a stable isopeptide bond between the glutamine on the c‐myc‐tag and various primary‐amine‐functionalized substrates. We attached eight different functionalities to a c‐myc‐tagged antibody fragment and used these bioconjugates for downstream applications such as protein multimerization, immobilization on surfaces, fluorescence microscopy, fluorescence‐activated cell sorting, and in vivo nuclear imaging. The results demonstrate the versatility of our conjugation strategy for transforming a c‐myc‐tagged protein into any desired probe.     

A FRET Sensor to Monitor Bivalent SUMO–SIM Interactions in SUMO Chain Binding

The small ubiquitin‐like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern upon conjugation to substrate proteins. SUMO chain recognition is facilitated by receptor proteins that contain at least two SUMO‐interacting motifs (SIMs). Little is known about the structure of SUMO chains, both in an unliganded form and upon complexation with multi‐SIM protein partners. A FRET sensor has been developed based on a linear dimer of human SUMO‐2 as a minimal SUMO chain analogue. The synthetic acceptor and donor dyes were conjugated by maleimide and copper‐catalyzed click chemistry to each of the two SUMO subunits. FRET changes were only observed in the presence of di‐ or multi‐SIM ligands. Alteration of the short linker sequence between SIMs 2 and 3 of RNF4 showed a great tolerance, and hence, structural flexibility, of the SUMO dimer for bivalent binding of adjacent SIMs. The di‐SUMO FRET sensor reports on the binding of SIM clusters of the proteins C5orf25 and SOBP; this suggest that these can bind to adjacent subunits of a SUMO chain. The developed FRET sensor will be a useful tool to study the importance of SIM and linker sequences, as well as biochemical and structural properties of SUMO chains and multi‐SIM proteins.     

Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.     

Carrier‐bound methotrexate. II. Water‐soluble polyaspartamide methotrexate conjugates with amide links in polymer–drug spacer

In Part I of this series, conjugation of methotrexate (MTX) to water‐soluble polyaspartamide carriers was accomplished through ester link formation between an MTX carboxyl group and a carrier‐attached hydroxyl function. Contrasting with that type of anchoring link, this project utilizes amide formation as the means of drug conjugation. This is achieved through condensation of one of the drug's carboxyl groups with a carrier‐attached primary amine function. Derived from polysuccinimide by a time‐proven nucleophilic ring‐opening process in the presence of aliphatic diamines, polyaspartamide‐type carriers 1–12 comprise subunits equipped with tert‐amine or hydroxyl side group terminals for hydrosolubilization and other subunits equipped with primary amine terminals as drug‐binding sites. MTX conjugation with these carriers is effected in aprotic solvent, the reaction being mediated by 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate. The water‐soluble conjugates are fractionated and purified by size exclusion chromatography and dialysis; they are isolated by freeze‐drying in typical yields of 40–65%. In the molar MTX/NH₂ feed ratios that are chosen (generally 1.2–1.3) and with the mole fractions of drug‐binding subunits restricted to 10 and 20%, drug loading in the resultant conjugates approximates 20–30% by mass. In follow‐on study, conjugates 1‐MTX–12‐MTX thus obtained will be screened in cell culture tests for antiproliferative activity against a number of human cancer lines. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 3415–3424, 2006     

Covalent Protein Labeling by SpyTag–SpyCatcher in Fixed Cells for Super‐Resolution Microscopy

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag–SpyCatcher system as an epitope‐like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super‐resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform‐specific labeling.     

Drug Delivery to the Malaria Parasite Using an Arterolane‐Like Scaffold

Antimalarial agents artemisinin and arterolane act via initial reduction of a peroxide bond in a process likely mediated by ferrous iron sources in the parasite. Here, we report the synthesis and antiplasmodial activity of arterolane‐like 1,2,4‐trioxolanes specifically designed to release a tethered drug species within the malaria parasite. Compared with our earlier drug delivery scaffolds, these new arterolane‐inspired systems are of significantly decreased molecular weight and possess superior metabolic stability. We describe an efficient, concise and scalable synthesis of the new systems, and demonstrate the use of the aminonucleoside antibiotic puromycin as a chemo/biomarker to validate successful drug release in live Plasmodium falciparum parasites. Together, the improved drug‐like properties, more efficient synthesis, and proof of concept using puromycin, suggests these new molecules as improved vehicles for targeted drug delivery to the malaria parasite.     

Introducing Glycolinkers for the Functionalization of Cytotoxic Drugs and Applications in Antibody–Drug Conjugation Chemistry

Antibody–drug conjugates (ADCs) are promising alternatives to naked antibodies for selective drug‐delivery applications and treatment of diseases such as cancer. Construction of ADCs relies upon site‐selective, efficient and mild conjugation technologies. The choice of a chemical linker is especially important, as it affects the overall properties of the ADC. We envisioned that hydrophilic bifunctional chemical linkers based on carbohydrates would be a useful class of derivatization agents for the construction of linker–drug conjugates and ADCs. Herein we describe the synthesis of carbohydrate‐based derivatization agents, glycolinker–drug conjugates featuring the tubulin inhibitor monomethyl auristatin E and an ADC based on an anti‐EGFR antibody. In addition, an initial in vitro cytotoxicity evaluation of the individual components and the ADC is provided against EGFR‐positive cancer cells.     

Enhancing Antibody Response against Small Molecular Hapten with Tobacco Mosaic Virus as a Polyvalent Carrier

Virus nanoparticles (VNPs) have been applied as carrier proteins for effective vaccine development. In this paper, we report the usage of tobacco mosaic virus (TMV) as a carrier for the display of the small molecule estriol (E3), a weakly immunogenic hapten. A highly efficient copper (I)‐catalyzed azide–alkyne cycloaddition reaction (CuAAC) was performed for the conjugation of E3 onto TMV capsid at tyrosine (Tyr) 139, by which the antigen density could be controlled. The immune properties of these constructs were evaluated in mice. We found that a strong and long‐term antibody response was elicited by conjugating a high density of small molecular haptens on TMV through an oligo(ethylene glycol) (OEG) linker, likely due to the effective activation of B‐cells. This study suggests that TMV can serve as a promising platform to induce strong humoral immune responses and that the optimized conjugation strategy was critical to produce high quality antibodies.     

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.     

PEG-proteins: Reaction engineering and separation issues

Poly(ethylene glycol)-conjugated (or PEGylated) proteins are an increasingly important class of therapeutic proteins that offer improved in vivo circulation half lives over their corresponding native forms. Their production involves covalent attachment of one or more poly(ethylene glycol) molecules to a native protein, followed by purification. Because of the extremely high costs involved in producing native therapeutic proteins it is important that subsequent PEGylation processes are as efficient as possible. In this paper, reaction engineering and purification issues for PEGylated proteins are reviewed. Paramount considerations for PEGylation reactions are specificity with respect to the conjugation site and overall yield. Batch PEGylation reaction methods are discussed, along with innovative methods using packed bed or “on-column” approaches to improve specificity and yield. Purification methods are currently dominated by ion exchange and size exclusion chromatography. Other methods in common use for protein separations, including hydrophobic interaction chromatography, affinity chromatography and membrane separations, are rarely used in PEGylated protein purification schemes. A better understanding of the effects of PEGylation on the physicochemical properties of proteins (isoelectric point, surface charge density and distribution, molecular size and relative hydrophobicity) and interactions between PEGylated proteins and surfaces is needed for the future development of optimal purification processes and media.     

Development of specific "drug-like property" rules for carboxylate-containing oral drug candidates

The carboxylate moiety is an important pharmacophore in the medicinal chemist's arsenal and is sometimes an irreplaceable functionality in drug–target interactions. Thus, practical guidance on its use in the most optimized manner would be a welcome addition to rational drug design. Key physicochemical and ADMET‐PK properties from a dataset of drugs containing a carboxylate (COOH) moiety were assembled and compared with those of a broader, general drug dataset. Our main objective was to identify features specific to COOH‐containing oral drugs that could be converted into simple rules delineating the boundaries within which prospective COOH‐containing chemical series and COOH‐containing drug candidates would be reasonably expected to possess properties suitable for oral administration. These specific “drug‐like” property rules include molecular weight, the number of rotatable bonds, the number of hydrogen bond donors and acceptors, predictions of lipophilic character (calculated log P and log D values), topological polar surface area (TPSA), and the pK_a value of the carboxylate moiety. Similar to the various sets of criteria that have emerged over the past decade and which have significantly reshaped the way medicinal chemists think about preferred drug chemical space, we propose these specific COOH “drug‐like” property rules as a guide for the design of superior COOH‐containing drug candidates and as a tool to better manage the liabilities generally associated with the presence of a COOH moiety.     

Responsive polymeric nanoparticles for controlled drug delivery

Small‐molecule drugs often have limited solubility, display rapid clearance or poor selectivity that leads to undesired side‐effects. Although prodrug strategies can improve solubility and lower toxicity, activation ‘on demand’ as well as targeted transport of prodrugs remains a challenge in drug delivery. Responsive polymeric nanoparticles can help meet these challenges with the encapsulation or conjugation of drugs, allowing release at the target site upon triggering by an internal or external stimulus. The adaptable design of polymeric nanoparticles allows them to play a vital role in achieving a specific and desired response following application of a specific stimulus. Here, the most recent progress in responsive polymeric nanoparticles is reviewed with a focus on the chemical properties of the utilized polymers. © 2017 Society of Chemical Industry     

A Luminescent Cyclometalated Iridium(III) Complex Accumulates in Mitochondria and Induces Mitochondrial Shortening by Conjugation to Specific Protein Targets

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)₂(phen‐ITC)](PF₆) (Ir‐ITC; Hpq=2‐phenylquinoline, phen‐ITC=5‐isothiocyanate‐1,10‐phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir‐ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir‐ITC with live‐cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two‐dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage‐dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir‐ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.     

Recent Chemical Approaches for Site-Specific Conjugation of Native Antibodies: Technologies toward Next-Generation Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as “magic bullets”. These conjugates offer the ability to target drug delivery to specific cells, based on cell-specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site-specific conjugation. Herein, various site-specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue-selective labeling for generating ADCs, disulfide rebridging, and affinity-peptide-mediated site-specific chemical conjugation technologies, are reviewed and described.     

Modeling the Kinetics of Protein Conjugation Reactions

Over the past decades, the pharmaceutical industry has shown a continuous infatuation for therapeutic proteins. In order to constantly improve their efficacy, chemical reactions involving the conjugation of the therapeutic protein with various chemical reagents have been developed. Little efforts have been put forth to simulate the kinetics of protein conjugation and the literature dealing with kinetic models is rather scarce as compared to the abundant references about conjugation reactions in general. In this work, techniques to measure the kinetics of reaction, model the conjugation between add‐on molecules and proteins, and evaluate the model parameters are discussed.     

Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.     

Dendrimer as nanocarrier for drug delivery

Dendrimers are novel three dimensional, hyperbranched globular nanopolymeric architectures. Attractive features like nanoscopic size, narrow polydispersity index, excellent control over molecular structure, availability of multiple functional groups at the periphery and cavities in the interior distinguish them amongst the available polymers. Applications of dendrimers in a large variety of fields have been explored. Drug delivery scientists are especially enthusiastic about possible utility of dendrimers as drug delivery tool. Terminal functionalities provide a platform for conjugation of the drug and targeting moieties. In addition, these peripheral functional groups can be employed to tailor-make the properties of dendrimers, enhancing their versatility. The present review highlights the contribution of dendrimers in the field of nanotechnology with intent to aid the researchers in exploring dendrimers in the field of drug delivery.     

A “Quenchergenic” Chemoselective Protein Labeling Strategy

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular “quenchergenic” aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.     

A “Clickable” MTX Reagent as a Practical Tool for Profiling Small‐Molecule–Intracellular Target Interactions via MASPIT

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ‐selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three‐hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration‐dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non‐regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full‐length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.