Delivery of Non-Native Cargo into Mammalian Cells Using Anthrax

Publication Date (Web): April 8, 2016 ... The intracellular delivery of peptide and protein therapeutics is a major challenge due ... Modular domain s...
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Delivery of Non-Native Cargo into Mammalian Cells Using Anthrax Lethal Toxin Amy E. Rabideau and Bradley Lether Pentelute* Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ABSTRACT: The intracellular delivery of peptide and protein therapeutics is a major challenge due to the plasma membrane, which acts as a barrier between the extracellular environment and the intracellular milieu. Over the past two decades, a nontoxic PA/LFN delivery platform derived from anthrax lethal toxin has been developed for the transport of non-native cargo into the cytosol of cells in order to understand the translocation process through a protective antigen (PA) pore and to probe intracellular biological functions. Enzymemediated ligation using sortase A and native chemical ligation are two facile methods used to synthesize these non-native conjugates, inaccessible by recombinant technology. Cargo molecules that translocate efficiently include enzymes from protein toxins, antibody mimic proteins, and peptides of varying lengths and non-natural amino acid compositions. The PA pore has been found to effectively convey over 30 known cargos other than native lethal factor (LF; i.e., non-native) with diverse sequences and functionalities on the LFN transporter protein. All together these studies demonstrated that non-native cargos must adopt an unfolded or extended conformation and contain a suitable charge composition in order to efficiently pass through the PA pore. This review provides insight into design parameters for the efficient delivery of new cargos using PA and LFN.

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bioactive cargos to disrupt intracellular protein−protein interactions or to study biological processes. Furthermore, these studies support the current model of protein translocation through the PA pore and provide insight into design parameters for delivering new cargos efficiently. Several methods have been developed for the delivery of biomolecules into the cytosol of mammalian cells including cellpenetrating peptides,5−7 lipid or polymer nanoparticles,8−11 and supercharged proteins.12,13 These methods often require high concentrations of material to achieve modest effects due to inefficient endosomal escape. The delivery efficiency of cargo proteins using the PA/LFN delivery platform was previously compared to protein delivery by the transactivator of transcription of HIV-1 (TAT).14 The PA/LFN delivery platform was shown to deliver antibody mimic proteins in the cytosol about 1000 times more efficiently than the TAT peptide. Delivery efficiency of the PA/LFN delivery platform is an active area of research.

athogenic bacteria often express protein toxins capable of delivering cytotoxic payloads into the cytosol of cells.1 The cytotoxic payloads are often referred to as effector proteins and have diverse functionalities in the host cellprotease activity, modification of intracellular substrates, or interruption in cell signaling pathwaysfor the benefit of the bacterium. One example is anthrax lethal toxin from the Gram-positive bacterium, Bacillus anthracis, which has been extensively studied for the past 40 years. Thorough biophysical and biochemical studies have led to an increased understanding of each component as a discrete protein and together as a macromolecular nanomachine.2 Anthrax lethal toxin has evolved to deliver the cytotoxic payload lethal factor (LF) into the cytosol of mammalian cells (Figure 1).3,4 While native anthrax lethal toxin expressed by B. anthracis persists as a bioterrorism threat, in the past two decades the toxin has been modified to serve as a delivery platform for the transport of non-native biomolecules. The PA/LFN delivery platform consists of protective antigen (PA) and the nontoxic, N-terminal PA-binding domain of LF known as LFN (Figure 2). This review provides an in-depth analysis of the PA/LFN delivery platform for the translocation of non-native cargos (i.e., biomolecules other than the anthrax LF) into the cytosol of cells. Most notably, the PA pore has been used to deliver more than 30 proteins and polypeptides containing natural and non-natural amino acids as well as small molecule drugs. Collectively, these analyses provide insight into the promiscuity of the PA pore and demonstrate its potential for the delivery of © XXXX American Chemical Society

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ANTHRAX LETHAL TOXIN Anthrax lethal toxin is a two-component system in which lethal factor (LF; 90 kDa) is transported into the cytosol of a host cell through a complex with protective antigen (PA83; 83 kDa).2 Received: February 22, 2016 Accepted: April 8, 2016

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Figure 2. Translocation of biomolecules using PA/LFN delivery platform. The PA/LFN delivery platform was developed such that the catalytic domain of LF could be replaced with various cargo molecules to determine their translocation efficiency through PA pore or analyze their biological function in the cytosol of cells.

into the PA prepore heptamer or octamer.24−27 The PA prepore is capable of binding up to three or four molecules of LF (Figure 1c) between two adjacent PA63 subunits with 1−2 nM affinity.28,29 The entire complex is endocytosed, and acidification of the endosome (pH ∼ 5.5) leads to a partial unfolding of LF and conformational rearrangement of the PA63 subunits to form the PA pore (∼12 Å diameter) in the endosomal membrane.26,30−34 The pH gradient generated between the endosome and cytosol (pH ∼ 7.0) leads to translocation of the unfolded, protonated LF protein via a charge state-dependent Brownian ratchet into the cell cytosol.35,36 Once in the cytosol, LF is deprotonated, allowing it to refold and function as a Zn2+ protease to cleave mitogen activated protein kinase kinases (MAPKK), ultimately resulting in cell death.37 Biochemical and biophysical studies have demonstrated that the structural components of anthrax lethal toxin relate to protein translocation. Mutagenesis analyses, kinetic studies, computational models, and a recent crystal structure have revealed how the N-terminal, PA binding domain of LF (1−254LF; LFN) interacts with two adjacent subunits of the PA prepore.29,38−40 Specifically, there are key electrostatic and hydrophobic interactions between the first α-helix and β-sheet of LFN and a deep amphipathic cleft on the surface of PA (alpha clamp) that bind LF such that the N-terminal region of the protein is partially unfolded and poised for translocation from the N- to C-terminus (Figure 1d).29,41 A recent 2.9 Å resolution cryogenic electron microscopy structure was solved for the PA pore that supports the current model for protein translocation (Figure 1e).42 The structure confirmed mutagenesis studies that predicted a narrow ring of solvent-exposed Phe427 residues in the lumen of the channel since Phe427 mutations permit pore formation but can inhibit translocation.43,44 The ring of Phe residues, called the Phe clamp, is the most restrictive part of the pore and interacts with hydrophobic stretches. The structure also revealed negatively charged residues surrounding the Phe clamp, which are hypothesized to deprotonate the translocated protein and guide unidirectional translocation.

Figure 1. Anthrax lethal toxin comprised of two discrete components lethal factor (LF) and protective antigen (PA). (a) Cytosolic delivery via anthrax lethal toxin is achieved by anthrax receptor recognition by PA83 then activation to form PA63 by a cell-surface furin family protease (1). Seven or eight PA63 molecules self-assemble to form the PA prepore (2). Then LF binds (3), and the entire complex is endocytosed into the endosome (4). Acidification triggers pore formation and translocation of LF into the cytosol (5). (b) Crystal structure of LF reveals two domains (PDB: 1J7N)PA binding domain (green; LFN) and catalytic domain (gray). (c) PA83 is composed of four domains (blue domain 1 initiates prepore formation, pink domain 2 and orange domain 3 are involved in forming the pore itself, and purple domain 4 is the receptor-binding domain; PDB: 1ACC). (d) LFN binds to two adjacent PA subunits (PDB: 3KWV). (e) PA pore structure reveals the restrictive Phe clamp (within domain 2) at the center of the pore (PDB: 3J9C).

After nearly 40 years of mechanistic and structural analyses, a model for protein translocation via anthrax lethal toxin has emerged (Figure 1a). In the presence of a divalent metal ion, PA83 is recognized by and binds to either of two cell surface receptors, tumor endothelial marker 8 or capillary morphogenesis gene 2 (TEM8 or CMG2, respectively) with nanomolar or picomolar affinity, respectively.15−17 The 1000-fold disparity in binding affinity has been attributed to nonconserved residues of CMG2 that interact with domain 2 of PA.18 Both TEM8 and CMG2 have been shown to regulate angiogenic processes.19−22 Furthermore, the anthrax receptors are expressed on most human cells at approximately 2000−50 000 receptors per cell.23 Once PA83 (Figure 1b) is receptor-bound, a furin family protease proteolytically activates the protein by cleaving the Nterminal 20 kDa portion (PA20), leaving PA63 to oligomerize

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FUSION AND CONJUGATION STRATEGIES Prior to the elucidation of the LF and PA structures, researchers utilized protein fusions to explore the translocation mechanism or to analyze the biological function of bioactive payloads inside B

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Figure 3. Chemical tools to modify LFN at the N- or C-terminus. (a) Recombinant expression in bacteria (e.g., E. coli) serves as a straightforward method to obtain fusion proteins containing canonical amino acids. (b) A semisynthetic platform using native chemical ligation (NCL) allows for the ligation of N-terminal cysteine biomolecules with C-terminal thioester biomolecules activated using mercaptophenylacetic acid (MPAA). (c) Enzyme-mediated ligation using sortase A (SrtA) has been developed for the facile ligation of any cargo to LFN under nondenaturing conditions.

pentaglycine tag onto molecules containing the LPXTG motif (Figure 3c).47 Chen et al. recently evolved SrtA enzymes to have ∼50−140-fold increase in LPETG substrate coupling activities such that ligation reactions can be carried out under nondenaturing conditions in less than 1 h with high yields.48 SrtA-mediated ligation has been used to attach peptides containing non-natural functionalities and small molecules on the C-terminus of LFN and LFN-DTA (DTA represents the A chain of diphtheria toxin) as well as mixed chirality peptides to the N-terminus of LFN. Recombinant expression, NCL, and enzyme-mediated ligation each install a natural, amide linkage between LFN and the cargo of interest. While this is critical for initial analysis of translocation efficiency, there are a variety of other bioconjugation techniques that can be employed to fuse cargos onto LFN. Common examples include site-specific incorporation of non-natural amino acids, disulfide linkage, maleimide conjugation, or click chemistry.49 Furthermore, combinations of techniques can be used to produce the desired construct. Recently, Ling et al. demonstrated that SrtA can accommodate peptide thioester substrates, which can be subsequently used for NCL to form protein conjugates separated by non-natural sequences such as LFN-DTA in which the two proteins were joined together by a D-peptide linker.50

the cytosol. The earliest work relied on recombinant expression to create fusions with the catalytic domains of select protein toxins. For protein fusions comprised of the 20 canonical amino acids, recombinant expression is often the simplest and most high yielding technique (Figure 3a). Nevertheless, recombinant expression is not adequate for probing the delivery of nonnative cargos that contain non-natural functionalities such as amino acids with inverted chirality, noncanonical side chain residues, or modified backbone structures. In order to synthesize non-native cargos, semisynthetic and enzymatic techniques have been employed including native chemical ligation (NCL) and enzyme-mediated ligation using sortase A (SrtA). Advantages of incorporating non-natural functionalities into protein fusions include stabilization to intracellular degradation, use of affinity handles, and perturbed binding affinities to target molecules. Pentelute et al. developed a semisynthetic approach using NCL to ligate non-natural peptides onto the N-terminus of LFN in order to explore the specificity of PA with regard to translocation initiation (Figure 3b).45 While NCL facilitates the site-specific ligation of peptides, oftentimes it is performed under denaturing conditions to achieve optimal substrate concentrations.46 As a result, ligated products are often purified by reverse phase high performance liquid chromatography (RPHPLC), followed by refolding. While LFN has been found to refold well, some cargos may not refold properly, resulting in inactivity. Enzyme-mediated ligation using the catalytic domain of sortase A from Staphylococcus aureus (SrtA) has allowed for the specific attachment of non-natural biomolecules under nondenaturing conditions. SrtA functions as a cysteine protease and transpeptidase to ligate substrates containing an N-terminal

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METHODS TO STUDY TRANSLOCATION The delivery of protein and peptide cargos with non-natural functionalities has provided insight into the specificity of the PA pore, efficiency of the PA/LFN delivery platform, and a deeper understanding of the mechanism for protein translocation. Translocation efficiency of cargos at the C-terminus of LFN (or LFN-DTA) has been analyzed using three common laboratory C

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Figure 4. Representative results from standard translocation assays. (a) A plot for a planar lipid bilayer experiment that shows blocking of ion conduction and translocation of LFN.45 Blaustein and co-workers developed this powerful in vitro approach to study translocation. This assay measures the change in ion current after the addition of PA63 prepore and LFN to the cis chamber. A proton gradient generated from a pH increase in the trans chamber permits translocation of LFN.35,36,51 (b) Protein synthesis inhibition assay measures the activity of DTA fused to LFN after translocation. After incubating cells with LFN-DTA, 3H-Leu is added to monitor the extent of protein synthesis inhibition caused by DTA. EC50 values of conjugates can be compared with the LFN-DTA control to determine translocation efficiency.14 (c) Western blot analysis of the cytosolic fraction measures the amount of material delivered into the cytosol. Cytosolic extraction is achieved using a lysis buffer containing digitonin.14

assays: planar lipid bilayer,33,43,51,52 protein synthesis inhibition or cytotoxicity analysis based on enzymatic activity,53,54 and Western blot analysis of the delivered material.55 Each approach has provided new insight into key features of translocation. As a cell-free assay, a planar lipid bilayer has been used to measure the change in ion current after LFN constructs have been added to a chamber containing PA pore embedded in an artificial membrane. Electrophysiological measurements from the bilayer experiments have provided critical information about translocation initiation and the mechanism of delivery through the PA pore (Figure 4a).35,36,43,51 In vitro, enzymatic assays have been developed using reporter protein cargos, providing a sensitive measure of translocation into eukaryotic cells. One specific enzymatic assay relies on the activity of the A chain of diphtheria toxin (DTA), which inactivates elongation factor 2 (EF-2) through adenosine diphosphate (ADP) ribosylation using the nicotinamide adenine dinucleotide (NAD+) cofactor and inhibits cytosolic protein synthesis.56 Also known as the protein synthesis inhibition assay, this assay monitors the activity of DTA to assess the delivery of assorted cargos fused to LFN-DTA into cells by 3H-Leu incorporation

(Figure 4b). The Pseudomonas exotoxin A (PE) catalytic domain, which inhibits protein synthesis by a similar mechanism as DTA, is also commonly used as a reporter protein.56 Cytotoxicity assays using tetrazolium salts have also been used to measure the translocation efficiency of toxic enzymatic domains. Western blot analysis has been utilized as a third method to measure translocation efficiency. After translocation, the cytosolic fraction of cells is lysed using digitonin, a nonionic detergent used to permeabilize the cytosolic membrane, then analyzed by Western blot (Figure 4c).57 Immunostaining for LF, DTA, or biotin (if present) provides a semiquantitative measure of translocated material. Further development of more sensitive assays is underway in order to accurately measure endosomal escape efficiency through the PA pore and quantify the amount of material delivered into the cytosol.

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DELIVERY OF NON-NATIVE CARGOS Natural Proteins. Early experiments of protein translocation through PA pore monitored the delivery of protein fusions as a method to characterize the structural requirements D

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Figure 5. Translocation efficiency analyzed for various peptides and proteins. (a) Representative protein and peptide cargos that translocated efficiently through the PA pore include DTA, PE, affibody (L and D forms), GB1 (L and D forms), DARPin, HA4, AKFRPDSNVRG peptide (L and D forms), biotinylated AKFRPDSNVRG peptide, and a doxorubicin-peptide conjugate. (b) Representative protein and peptide cargos that did not translocate efficiently (gray box) through the PA pore include DTA containing N58C+S146S disulfide, AKFRPDSNVRG cyclic peptide, docetaxelpeptide conjugate, and peptide containing three cysteic acid residues.

listeriolysin O (LLO),61,62 Legionella pneumophila flaggellin protein,63 actin cross-linking domain (ACD) of RTX from Vibrio cholerae,64 Rho inactivation domain (RID) from Vibrio cholerae,65 and a Ras/Rap1-specific endopeptidase (RRSP) from Vibrio vulnificus.66,67 Delivery of a cytotoxic T lymphocyte LLO epitope was analyzed for its immune response activity in mice as a potential method for developing vaccines against pathogens. 61 In order to study the biochemical and physiological consequences of inflammasome stimulation, von Moltke et al. delivered a Legionella pneumophila flagellin protein to stimulate the inflammasome and monitored eicosanoid release.63 Satchell and co-workers utilized the PA/LFN delivery platform to study the functions of various effector domains from multifunctional autoprocessing repeats in toxin (RTX) domains (MARTX) expressed by pathogenic bacteria. The bioactivity of the actin cross-linking domain (ACD) from Vibrio cholerae was analyzed after translocation into HEp-2 cells. Cordero et al. demonstrated that ACD directly catalyzes the covalent cross-linking of actin providing insight into the mechanism of cell death caused by Vibrio cholerae.64 Sheahan et al. analyzed the inactivation of Rho GTPases by the Rho Inactivation Domain (RID) from Vibrio cholerae after delivery

of initiating and sustaining translocation. These studies utilized recombinant expression to generate protein fusions of LFN with the A chain of diphtheria toxin (DTA),53,54,58 Pseudomonas exotoxin A (PE),59 A chain of Shiga toxin (STA),53 dihydrofolate reductase (DHFR),58 and β lactamase (Figure 5a).60 All together these studies demonstrated that regions near the N-terminus of LFN are important for initiating translocation into eukaryotic cells.41 The proteins, however, must be unfolded or in an extended conformation for efficient translocation, which was verified by the impeded translocation of LFN-DTA containing an artificial disulfide (Figure 5b) and LFN-DHFR bound to methotrexate.58 The efficient translocation of DTA, STA, PE, DHFR, and β lactamase provided the first indication that the PA pore can accommodate nonnative cargo proteins. After translocation, the measured enzymatic activity for each protein cargo indicated that these proteins were folded correctly in the cytosol and recognized their intracellular substrates. Since the development of the PA/LFN delivery platform, various protein cargos have been delivered into cells to understand their biological function. Examples include a cytotoxic T lymphocyte epitope from Listeria monocytogenes E

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into HEp-2 cells using the PA/LFN delivery platform.65 The bioactivity of a toxic domain within MARTX from Vibrio vulnif icus whose function was previously unknown was characterized using the PA/LFN delivery platform. Antic et al. delivered the domain into HeLa cells and demonstrated that the Ras/Rap1-specific endopeptidase (RRSP) effector domain cleaves the Switch 1 region of Ras and Rap1 proteins and thus interferes with downstream signaling in the MAPK pathway.66,67 Engineered Protein Variants. Antibodies serve as powerful tools for medical diagnostics and the treatment of disease. The use of antibodies, however, is limited to outside the cell due to their inability to cross the plasma membrane into the cytosol and the presence of disulfide cross-links. Recently, single-domain, cysteine-free scaffold proteins have been developed as antibody mimics. The scaffolds include monobody from the tenth type III domain of human fibronectin (10FN3),68,69 affibody from immunoglobulin binding protein A,70,71 designed ankyrin repeats protein (DARPin),72 and the B1 domain of protein G (GB1).73 Researchers have recently analyzed the translocation efficiency of these scaffolds, which can be evolved to bind extracellular receptors or intracellular targets with high affinity.14,74 Liao et al. recently analyzed the delivery and intracellular activity of select affibody and monobody antibody mimics using the PA/LFN delivery platform.14 Specifically, the researchers analyzed the delivery and associated bioactivity of a tandem monobody designed by Koide and co-workers to bind the Src homology 2 domain (SH2) of Bcr-Abl (HA4−7c12; Kd 12 nM)75,76 and an affibody designed to bind Raf (ABRaf; Kd 100 nM) developed by Nygren and co-workers.77 The HA4−7c12 tandem monobody conjugate was translocated in chronic myeloid leukemia cells (K562), and intracellular binding to BcrAbl was confirmed by coimmunoprecipitation. The inhibition of Bcr-Abl kinase activity and induction of apoptosis was observed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), which detects DNA fragmentation by labeling the termini. The ABRaf affibody conjugate was translocated in human embryonic kidney 293T (HEK 293T) cells, and the interruption of the mitogen activated protein kinase (MAPK) pathway was monitored. ABRaf was found to significantly reduce the phosphorylation levels of Erk1/2 after activation with epidermal growth factor (EGF). Taken together, the HA4−7c12 and ABRaf delivery data indicate that antibody mimics can be efficiently delivered into the cell cytosol through the PA/LFN delivery platform, refold after translocation, and perturb intracellular protein−protein interactions (PPIs). The GB1 and DARPin antibody mimics have been shown to translocate efficiently into the cytosol of cells.14 These scaffolds have yet to be delivered into cells using PA pore to perturb PPIs; however, the delivery of DARPin constructs with different thermostabilities has been analyzed. Plückthun and co-workers recently demonstrated that very stable DARPin constructs (i.e., ≥ 90 °C melting temperatures) cannot translocate efficiently through the PA pore.74 The researchers addressed the thermostability of DARPin by engineering constructs with reduced stability, amenable for delivery via PA pore. A similar observation was made for 10FN3 (unpublished results) in which wild-type 10FN3 (Tm ∼ 88 °C) translocated less efficiently than HA4, a mutant 10FN3 construct with Tm ∼ 75 °C. Together, these observations indicated conjugates with significant thermally stability can have reduced translocation efficiencies.

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REPROGRAMMING THE PA/LFN DELIVERY SYSTEM

Stabilization of Protein Conjugates. The ubiquitin (Ub)/proteasomal system plays a major role in protein degradation in eukaryotic cells. The N-end rule described by Varshavsky and co-workers states that the N-terminal amino acid of a protein impacts the protein’s intracellular stability with regard to proteasomal degradation.78 Since LFN follows the Nend rule, strategies have been developed to increase cytotoxic activity and decrease immunogenicity.79 Leppla and co-workers demonstrated that reductive methylation to dimethylate the epsilon amino group of lysine residues improves cytoxicity of LFN-PE conjugates by stabilizing the proteins to intracellular degradation.80 LFN-PE conjugates including those prone to degradation (i.e., contain N-terminal His residue) were reductively methylated at all 36 lysine residues using borane dimethyl amine and formaldehyde. After translocation into several eukaryotic cell lines, cytotoxicity and Western blot assays revealed each methylated conjugate was stabilized to degradation. A disadvantage of this approach is nonspecific methylation, which means that for substrates requiring lysine for catalysis or structural integrity, reductive methylation cannot be used for stabilization. Stabilization of proteins to proteasomal degradation using one N-terminal D-amino acid was recently demonstrated using the PA/LFN system.81 Incorporation of N-terminal D-amino acids onto LFN was achieved through either SrtA ligation or NCL. Proteasomal degradation of LFN-DTA constructs containing L- or D-amino acids was investigated using the protein synthesis inhibition assay as well as Western blot analysis. In both assays, while constructs containing N-terminal L-amino acids followed the N-end rule, constructs with Nterminal D-amino acids were stabilized to degradation. Rabideau and Pentelute demonstrated that a protein containing one Nterminal D-amino acid is not ubiquitinated. In order to support that this phenomenon was not protein-specific, a hindered disulfide cleavable linker was incorporated and similar observations of protein stabilization were made for DTA and DARPin. This work updated the N-end rule to include D-amino acids as stabilizing amino acids. Mirror Image Polypeptides. The biological impact of polypeptides containing amino acids with non-natural side chains, backbone composition, mirror image chirality, and many others remains relatively unexplored.82 Biomolecules composed of mirror image amino acids are of particular interest for their unique biological stability and nonimmunogenic properties. The translocation efficiency of mixed chirality fusions to LFN through PA pore was recently analyzed.83 Protein synthesis inhibition assays indicated that mirror image polypeptides and proteins translocate as efficiently into the cell cytosol as their L- counterparts. Western blot analysis indicated that an unstructured L-peptide cargo on the C-terminus of LFN resulted in rapid degradation of the protein conjugate in the cell cytosol; however, a conjugate containing a D-peptide cargo of the same sequence was found to be stabilized to degradation. Capping the C-terminus with two D-amino acids (D-cap) stabilized the L-peptide cargo. Evidently, the incorporation of a short D-cap at the C-terminus of an unstructured polypeptide can provide stabilization to intracellular degradation. Delivery of bioactive cargos into the cell cytosol is an attractive application for translocation. Using the PA/LFN delivery platform, a D-peptide MDM2 antagonist (TAWYANF*EKLLR, where F* is p-CF3-D-Phe)84 was recently delivered F

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ACS Chemical Biology Table 1. More than 30 Different Non-Native Cargos Have Been Delivered into the Cytosol of Cellsa non-native cargo Pseudomonas exotoxin A (PE) diphtheria toxin, A chain (DTA)

fusion and conjugation strategies

cell type

A chain of Shiga toxin (STA) listeriolysin O epitope (LLO) β lactamase dihydrofolate reductase (DHFR) actin cross-linking domain (ACD) of RTX from Vibrio cholerae Legionella pneumophila flagellin protein Rho inactivation domain (RID) reductively methylated PE green fluorescent protein (GFP; at C-terminus of LF) KEKEKNKDENKRKDEER (ligated to N-terminus of LFN) Ras/Rap1-specific endopeptidase (RRSP) cytolethal distending toxin B (CdtB)

CHO CHO-K1, RAW264.7, MC3T3, RBL-1, VERO, L6 CHO-K1 mouse, P815 (H-2d) mouse spleen, CHO, HeLa CHO-K1, L6 HEp-2 Mouse HEp-2 HN6 BHK CHO-K1 HeLa, HCT116, MDA-MB-231, HEK 293T RAW264.7, CHO-K1, HeLa, HN6

AlexaFluor545 (conjugated to K581 of LF) affibody B1 domain of protein G (GB1) tenth human fibronectin type three domain (10FN3) designed ankyrin repeats protein (DARPin) HA4−7c12 tandem monobody AKFRPDSNVRG peptide akfrpdsnvrG (all D) peptide AKFRPDSNvrG (Dcap) peptide tawyanf*ekllr (all D; f* is p-CF3-D-Phe) peptide affibody (all D) GB1 (all D) biotinylated affibody (all D) [β-Ala]KFRPDSNVRG peptide [N-Me-Ala]KFRPDSNVRG peptide [Prop-Gly]KFRPDSNVRG peptide AK[F3−Phe]RPDSNVRG peptide AK(Cys)FRPDSNVRG peptide LRRLRAC(Doxorubicin) peptide-drug conjugate LRRLRAC(MMAF) peptide-drug conjugate AKFRPDSNVRGK(Biotin) peptide X-LFN-DTA (X is any L or D-amino acid) LFN-Gal4:antisense oligonucleotide complex

J774A.1, BHK-21 CHO-K1, HEK 293T CHO-K1 CHO-K1 CHO-K1 K562 CHO-K1 CHO-K1 CHO-K1 U87-MG CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1, HEK 293T, HeLa HeLa, Vero

LFN-PKR:siRNA complex

HeLa, Vero

DTA with N58C+S146C disulfide DHFR with methotrexate mCherry (at C-terminus of LF) KEKEKNKDENKRKXXXR (X is cysteic acid; ligated to N-terminus of LFN) DARPin, Tm > 90 °C AKFRPDSNVRG cyclic peptide akfrpdsnvrG (all D) cyclic peptide LRRLRAC(Docetaxel) peptide-drug conjugate

CHO-K1 L6 BHK CHO-K1

recombinant recombinant or enzymatic or NCL recombinant recombinant recombinant recombinant recombinant recombinant recombinant recombinant, chemical recombinant NCL recombinant recombinant, NHS bioconjugation site specific click enzymatic enzymatic enzymatic enzymatic or recombinant enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic, click enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic, NCL recombinant; noncovalent complex recombinant; noncovalent complex recombinant recombinant recombinant NCL

HEK 293T CHO-K1 CHO-K1 CHO-K1

recombinant enzymatic enzymatic enzymatic

a

citation 59 50, 53, 58 53 61, 62 60, 98 58 64 63 65 99 88 89 66, 67 86 87 14 14 14 14, 74 14 83 83 83 83 83 83 83 85 85 85 85 85 85 85 85 81 100 100 58 58 88 89 74 85 85 85

Efficiently translocating cargos are not highlighted, and inefficiently translocating cargos are shown in bold.

cytosol of a eukaryotic cell, where it subsequently bound the target protein and disrupted a critical PPI. Non-Natural Peptides. The translocation of proteins containing non-natural moieties is of particular interest for their bioactivity, stability, or affinity properties. Conjugates containing peptide cargo with β-alanine, N-methyl alanine, propargylglycine, or 2,4,5-trifluorophenylalanine modifications were found to translocate efficiently into CHO-K1 cells by

into the glioblastoma U87-MG cell line, which overexpresses MDM2.83 A biotinylated form of the LFN conjugate (Kd 12.3 ± 4.3 nM) toward MDM2 was found to interact with MDM2 after delivery into U87-MG cells. Moreover, after delivery into U87-MG cells, the conjugate was found to disrupt the p53/ MDM2 pathway, as evidenced by upregulation of MDM2, p53, and p21 protein levels. For the first time, the PA/LFN delivery platform was utilized to deliver a bioactive D-peptide into the G

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ACS Chemical Biology Western blot and protein synthesis inhibition assays.85 Furthermore, the same conjugates with biotinylated C-termini were also found to translocate efficiently, as indicated by costaining of DTA and streptavidin on the Western blot. These results demonstrated efficient translocation of the biotin moiety and provided further support for the delivery of intact conjugates into the cytosol. Moreover, subtle non-natural modifications to the peptide cargos do not affect their translocation efficiency through the PA pore. There are several other examples of translocating biotinylated cargo, which collectively confirm the efficient translocation of biotinylated protein conjugates and the accessibility of biotin as an affinity handle.74,83,86 Cyclic Peptides. The cyclization of peptides is a useful approach to develop proteolytically stable therapeutics with large surface areas for protein binding. The delivery of L and Dforms of a cyclic peptide comprised of 11 amino acids was recently explored using the PA/LFN delivery platform.85 According to the protein synthesis inhibition assay and Western blot analysis, the cyclic peptide conjugates were unable to translocate through the PA pore. Rabideau et al. hypothesized that the cyclic peptides’ constrained conformation and inability to unfold contributed to their inefficient translocation (Figure 5b). Further investigation is required to fully understand the potential of cyclic peptides as cargo of the PA/LFN delivery platform. Small Molecule Drugs. To further probe translocation through PA pore, the delivery of small molecule drugs has been explored. Small molecule drugs have diverse properties, functionalities, and three-dimensional structures. The translocation of three common chemotherapeutics, doxorubicin, docetaxel, and monomethyl auristatin F (MMAF), was analyzed by Rabideau et al.85 Analysis by protein synthesis inhibition assay as well as Western blot of the cytosolic fraction indicated that small molecule drugs can translocate through the PA pore, but with limitations. Of the three molecules tested, docetaxel was unable to translocate into the cytosol through PA pore. We hypothesized that constrained or rigid molecules such as docetaxel cannot unfold or adopt a conformation amenable to translocation through the about 12 Å pore (Figure 5b). Modified Delivery System. The PA/LFN delivery platform has been modified in some cases for the delivery of fluorescent cargos such as small molecule probes or proteins. Using the genetic code expansion technique, Zheng et al. installed an alkynyl-pyrrolysine residue within LF at position K581 and then used click chemistry to site-selectively label the protein with AlexaFluor545.87 The researchers monitored endocytic trafficking of the labeled protein in BHK fibroblast cells. Zornetta et al. demonstrated the PA-mediated delivery of green fluorescent protein (GFP) into the cytosol fused to LF.88 The delivery of mCherry, however, proved inefficient. The researchers hypothesized that the difference in translocation was due to a higher resistance of mCherry to unfolding. The delivery of fluorescent proteins requires further investigation with respect to the cargo protein’s melting temperature and the fate of the chromophore during translocation. The PA pore is cation-selective, thus favoring the passage of protonated or neutral species. While the N-terminal 28 residues are critical for initiating translocation, the incorporation of cysteic acid (pKa −1.9) in this region halts translocation (Figure 5b).89 N-terminal fusions of polycationic stretches (e.g., Lys, Arg, His) have been investigated for the delivery of DTA using PA only. Blanke et al. and Sharma and Collier showed

that DTA fused to polycationic residues rather than LFN can be delivered into cells through PA pore.90,91 Wright et al. recently utilized PA-mediated delivery to investigate the delivery of peptide nucleic acids (PNA).92 Reporter cells containing luciferase transgenes with mutant splice sites were treated with PA and antisense PNA(Lys)8 oligomers, which bind to the mutant splice sites. The researchers demonstrated that the PNA oligomers were delivered into cells, corrected the splice defect, and induced luciferase expression. Recently, Dyer et al. demonstrated PA/LFN-mediated delivery of Gal4:antisense oligonucleotide and protein kinase R (PKR):siRNA noncovalent complexes into HeLa and Vero cells through knockdown of Syntaxin5.74 Together, these studies suggest that the PA/LFN delivery platform can be further modified for the translocation of charged cargos. Retargeting PA. The PA/LFN delivery platform has been modified for the targeted translocation of material into cells such as tumor cells that overexpress proteases or specific receptors. Leppla and co-workers engineered PA to be activated by urokinase plasminogen activator (uPA) or matrix metalloproteases (MMPs).93 Replacement of the furin-cleaved PA20 domain with sequences recognized by MMPs or uPA allowed for targeted PA-mediated delivery into specific tumor cells that expressed such proteins. Liu et al. demonstrated in vivo that tumors treated with the LF through targeted PA proteins experienced a greater antitumor activity than those treated with LF through wild-type PA.94 Collier and co-workers recently retargeted PA to recognize non-native receptors such as human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) by mutating two key residues responsible for binding the anthrax receptors and adding a new targeting domain to the C-terminus of PA.95−97 Retargeting PA to cells that overexpress specific receptors is a method to increase the amount of material delivered into the cell. Investigations are underway to study the delivery efficacy and bioactivity of delivered non-native cargos using retargeted PA proteins.

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SUMMARY AND OUTLOOK The PA/LFN delivery platform permits the facile delivery of biomolecules with diverse structures and functionalities into the cytosol of eukaryotic cells (Figure 5 and Table 1). The PA pore is relatively promiscuous for the delivery of non-native cargo on the C-terminus of LFN. Investigations by several groups have demonstrated that once translocation is initiated by LFN, there are a few guidelines that cargos must follow in order to gain efficient cytosolic entry through the PA pore. First, the cargo must be able to adopt an unfolded or extended conformation in the endosome. Second, non-natural moieties such as nonnatural backbone structures and side chain modifications like mirror image or modified amino acids do not disrupt the translocation process. Third, cargos containing moieties with low pKa values that cannot be protonated in the endosome may inhibit translocation. Taken together, these design principles can be employed for the delivery of previously unexplored cargos such as oligonucleotides or post-translationally modified proteins. There are several questions that remain unanswered regarding the PA/LFN delivery platform. The amount of material delivered into the cytosol varies based on the cell type (i.e., number of anthrax receptors expressed), incubation time (i.e., rate of endocytosis and receptor recycling), concentration of PA and LFN constructs, and translocation efficiency of the H

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ACS Chemical Biology

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LFN construct. While Western blot analysis provides a semiquantitative analysis of the amount of material delivered into the cytosol, a more accurate and sensitive method will provide researchers with a better glimpse of translocation efficiency and a more precise measure of the amount of material delivered into the cytosol. Furthermore, exploration into the efficacy and immunogenicity of the PA/LFN delivery platform in vivo will provide insight into the platform’s potential therapeutic use in multicellular organisms.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was generously supported by MIT start-up funds, the MIT Reed Fund, NSF CAREER Award (CHE-1351807), and a Damon Runyon Cancer Research Foundation award for B.L.P. and a NSF Graduate Research Fellowship for A.E.R. We would also like to thank X. Liao for helpful conversations and R. J. Collier (Harvard) for his continued support.

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KEYWORDS Anthrax toxin: bipartite bacterial toxin expressed by Bacillis anthracis comprised of a transporter protein (protective antigen) and an effector protein (lethal factor and/or edema factor) Lethal Factor (LF): effector protein of anthrax toxin, which is a zinc protease in the cytosol that cleaves mitogen activated protein kinase kinases, leading to cell death Protective Antigen (PA): pore-forming protein of anthrax toxin, which binds to TEM8 or CMG2 cell surface receptors, forms a prepore that is endocytosed, and rearranges to form a pore in the endosomal membrane for protein translocation Translocation: delivery of proteins from the endosome into the cytosol through PA pore LFN: N-terminal, PA-binding domain of LF that lacks the enzymatic domain and efficiently translocates through PA pore Non-native cargo: cargo (protein, peptide, or small molecule) other than LF or EF capable of translocating through PA pore Phe clamp: most restrictive part of PA pore that is comprised of seven Phe427 residues from each PA subunit and drives protein translocation Sortase A (SrtA): cysteine protease and transpeptidase expressed by Staphylococcus aureus that ligates substrates containing C-terminal LPTXG motif to substrates with Nterminal oligoglycine Native Chemical Ligation (NCL): chemical ligation technique used to ligate a substrate containing a C-terminal thioester to a substrate with an N-terminal cysteine

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Reviews

ACS Chemical Biology Pseudomonas exotoxin A enzymatic domain to the cytosol of tumor cells via cleavable ubiquitin fusions. mBio 4, e00201-13. (100) Dyer, P. D. R., Shepherd, T. R., Gollings, A. S., Shorter, S. A., Gorringe-Pattrick, M. A. M., Tang, C. K., Cattoz, B. N., Baillie, L., Griffiths, P. C., and Richardson, S. C. W. (2015) Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity. J. Controlled Release 220, 316−328.

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DOI: 10.1021/acschembio.6b00169 ACS Chem. Biol. XXXX, XXX, XXX−XXX