Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications

Calmodulin (CaM) is a widely studied Ca^(2+)-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca^(2+) or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications

Selective Functionalization of the Protein N-Terminus with N-Myristoyl Transferase in Bacteria

Proteins are involved in myriad processes in all organisms. They provide structural support in the membrane and scaffolding of each cell; they aid in the transmission of biochemical signals within and between cells; and they play central roles in combating various disease states. The development of techniques enabling selective and site-specific functionalization of proteins is an active area of investigation, as such modifications are critical to many studies and uses of proteins. For instance, with the addition of a unique reactive handle, a protein may be conjugated to a polymer for the production of protein-based therapeutics exhibiting improved bioavailability. Alternatively, proteins may be attached to slides to prepare diagnostic microarrays, reacted with hydrogels to create functional biomaterials, or decorated with fluorophores for in vivo imaging. Site-specific protein tagging techniques have already contributed greatly to biomedical research and will continue to advance the state of the field. The focus of my thesis research has been the development of a novel site-specific protein labeling method centered on the eukaryotic enzyme N-myristoyl transferase (NMT). In a process called myristoylation, NMT appends a fatty acid to the N-terminus of numerous substrate proteins. Previous work demonstrated that NMT tolerates a wide range of chemically functionalized analogs of its natural fatty acid substrate. Here, we describe efforts that exploit various features of NMT: its ability to bind and utilize reactive fatty acid analogs, its exquisite selectivity toward its protein substrates, and its orthogonality toward those proteins naturally present in bacteria. First, in Chapter II, we discuss the development of a model system for NMT-mediated protein labeling in the bacterium Escherichia coli. We synthesized an azide fatty acid analog that can participate in bioorthogonal chemistries, and we prepared two GFP-based substrate proteins, each displaying a recognition sequence derived from a known substrate of NMT. Our experiments indicate that labeling by NMT is site-specific, quantitative, and highly selective for each engineered substrate within the bacterial milieu. As summarized in Chapter III, the model system was extended to the N-terminal labeling of two neuronal proteins, calcineurin (CaN) and calmodulin (CaM). While CaN is naturally myristoylated, CaM was engineered to achieve labeling by NMT. Experiments with CaN and CaM confirmed that our NMT-based system is quantitative and selective in its labeling of both natural and engineered substrate proteins. Extensive characterization of each protein allowed us to identify constructs that retain wild-type levels of activity even after labeling with the azide fatty acid. Three of the protein constructs reported in Chapters II and III were utilized for microarray studies, as described in Chapter IV. We achieved rapid surface immobilization of each azide-labeled protein directly from lysate, a significant advantage when considering the time and resources normally required to purify proteins for downstream applications. The experiments and methods summarized in this chapter will be adapted for high-throughput biochemical research with protein microarrays. Finally, the orthogonality of NMT toward bacterial systems was probed further for the purpose of selective labeling of individual bacterial proteins for live-cell imaging. In addition to identifying an azide fatty acid suitable for such studies, we also selected two bacterial proteins that exhibit interesting functions and localization patterns, and we developed corresponding protein constructs for NMT-mediated labeling. Progress toward the use of NMT for in vivo imaging applications in bacteria is described in Chapter V. Ultimately, our objective throughout the design and execution of these projects was to create and validate a new technique to achieve site-specific protein labeling. The particular advantages of NMT include its tolerance of reactive fatty acid analogs and engineered substrate proteins, and its lack of interaction with proteins present in the widely used E. coli expression host. We believe that the ideas and results presented in this thesis establish NMT-mediated protein labeling as a valuable tool for addition to the existing set of site-specific protein labeling methods. Development of such methods represents an important and exciting area within the field of modern chemical biology.</p

Selective Functionalization of the Protein N Terminus with N-Myristoyl Transferase for Bioconjugation in Cell Lysate

A site to behold: Robust site-specific functionalization of engineered proteins is achieved with N-myristoyl transferase (NMT) in bacterial cells. NMT tolerates non-natural substrate proteins as well as reactive fatty acid tags, rendering it a powerful tool for protein conjugation applications, including the construction of protein microarrays from lysate

Determination of Cellular Processing Rates for a Trastuzumab-Maytansinoid Antibody-Drug Conjugate (ADC) Highlights Key Parameters for ADC Design

Antibody-drug conjugates (ADCs) are a promising class of cancer therapeutics that combine the specificity of antibodies with the cytotoxic effects of payload drugs. A quantitative understanding of how ADCs are processed intracellularly can illustrate which processing steps most influence payload delivery, thus aiding the design of more effective ADCs. In this work, we develop a kinetic model for ADC cellular processing as well as generalizable methods based on flow cytometry and fluorescence imaging to parameterize this model. A number of key processing steps are included in the model: ADC binding to its target antigen, internalization via receptor-mediated endocytosis, proteolytic degradation of the ADC, efflux of the payload out of the cell, and payload binding to its intracellular target. The model was developed with a trastuzumab-maytansinoid ADC (TM-ADC) similar to trastuzumab-emtansine (T-DM1), which is used in the clinical treatment of HER2+ breast cancer. In three high-HER2-expressing cell lines (BT-474, NCI-N87, and SK-BR-3), we report for TM-ADC half-lives for internalization of 6–14 h, degradation of 18–25 h, and efflux rate of 44–73 h. Sensitivity analysis indicates that the internalization rate and efflux rate are key parameters for determining how much payload is delivered to a cell with TM-ADC. In addition, this model describing the cellular processing of ADCs can be incorporated into larger pharmacokinetics/pharmacodynamics models, as demonstrated in the associated companion paper.Hertz Foundation. Graduate FellowshipNational Science Foundation (U.S.). Graduate Research Fellowship ProgramPfizer Inc.National Cancer Institute (U.S.) (Koch Institute Support (core) grant P30-CA14051

A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10[superscript 5]–10[superscript 10] doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC[subscript 50] values of 4–12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody–drug conjugates.Hertz Foundation (Fellowship)National Science Foundation (U.S.). Graduate Research Fellowship ProgramPfizer Inc.National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051

Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies

As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolismprocesses that are critical to ADC efficacy and toxicityimaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a “clickable” ADC bearing an azide-functionalized linker-payload (LP) poised for “click” reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems

Determination of Cellular Processing Rates for a Trastuzumab-Maytansinoid Antibody-Drug Conjugate (ADC) Highlights Key Parameters for ADC Design

Antibody-drug conjugates (ADCs) are a promising class of cancer therapeutics that combine the specificity of antibodies with the cytotoxic effects of payload drugs. A quantitative understanding of how ADCs are processed intracellularly can illustrate which processing steps most influence payload delivery, thus aiding the design of more effective ADCs. In this work, we develop a kinetic model for ADC cellular processing as well as generalizable methods based on flow cytometry and fluorescence imaging to parameterize this model. A number of key processing steps are included in the model: ADC binding to its target antigen, internalization via receptor-mediated endocytosis, proteolytic degradation of the ADC, efflux of the payload out of the cell, and payload binding to its intracellular target. The model was developed with a trastuzumab-maytansinoid ADC (TM-ADC) similar to trastuzumab-emtansine (T-DM1), which is used in the clinical treatment of HER2+ breast cancer. In three high-HER2-expressing cell lines (BT-474, NCI-N87, and SK-BR-3), we report for TM-ADC half-lives for internalization of 6–14 h, degradation of 18–25 h, and efflux rate of 44–73 h. Sensitivity analysis indicates that the internalization rate and efflux rate are key parameters for determining how much payload is delivered to a cell with TM-ADC. In addition, this model describing the cellular processing of ADCs can be incorporated into larger pharmacokinetics/pharmacodynamics models, as demonstrated in the associated companion paper.Hertz Foundation. Graduate FellowshipNational Science Foundation (U.S.). Graduate Research Fellowship ProgramPfizer Inc.National Cancer Institute (U.S.) (Koch Institute Support (core) grant P30-CA14051

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila

Cytosolic nucleotidase II (cN-II) from Legionellapneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DatabaseThe coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers and . The accession number of GMP complexed LpcN-II is . Structured digital abstract <list list-type=''bulleted'' id=''febs12727-list-0001''> andby() andby() Structured digital abstract was added on 5 March 2014 after original online publication