Dyngo-4a | CRET Signal

Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors
Callista B. Harper1, Michel R. Popoff2, Adam McCluskey3, Phillip J. Robinson4, and Fre´ de´ ric A. Meunier1
1 Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia
2 Unite´ des Bacte´ ries Anae´ robies et Toxines, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex, France
3 Chemistry, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia
4 Children’s Medical Research Institute, University of Sydney, Sydney, New South Wales 2145, Australia

Many pathogens hijack existing endocytic trafficking pathways to exert toxic effects in cells. Dynamin controls various steps of the intoxication process used by numer- ous pathogenic bacteria, viruses, and toxins. Targeting dynamin with pharmaceutical compounds may there- fore have prophylactic potential. Here we review the growing number of pathogens requiring dynamin-de- pendent trafficking to intoxicate cells, outline the mode of internalization that leads to their pathogenicity, and highlight the protective effect of pharmacological and genetic approaches targeting dynamin function. We also assess the methodologies used to investigate the role of dynamin in the intoxication process and discuss the validity and potential pitfalls of using dynamin inhibitors (DIs) as therapeutics.

Targeting intracellular membrane trafficking to combat pathogens
The evolution of the microbial world has produced impres- sive molecular strategies to gain entry to and incapacitate specific target cells in the host. The existence of various cellular endocytic pathways provides an array of opportu- nities for microbial pathogens and viruses to access and infect our cells [1]. Once inside cells, many pathogens further hijack intracellular trafficking mechanisms to find or generate sheltered compartments to prepare for repli- cation and release. Endocytic pathways range from cla- thrin-mediated endocytosis (CME) to macropinocytosis, enabling cells to internalize essential proteins, fluids, lipids, and other signals or nutrients. These pathways require the spatiotemporal coordination of many different proteins and lipids. One protein, dynamin, is a common denominator for more of these pathways than any other enzyme [2,3]. Dynamin is a GTPase that oligomerizes around the neck of nascent endocytic compartments to pinch them off from the plasma membrane and may act in a similar manner in other initial endocytic events (Box 1). It also has other key regulatory roles in trafficking that are less well-characterized, such as vesicle formation

Corresponding authors: Robinson, P.J. ([email protected]); Meunier, F.A. ([email protected])
Keywords: dynamin; pathogens; neurotoxins; prophylaxis; trafficking; endocytosis.
from the trans-Golgi network and regulation of actin dynamics [2,4].
Recent developments have led to the design of com- pounds targeting key or converging points of these mem- brane trafficking pathways to prevent infection. Multiple endocytic entry points and subsequent trafficking events can be targeted to prevent either internalization or traf- ficking of pathogens, thereby precluding their pathogenic activities. Such drug design is distinct from compounds that directly target, for example, viral replication or bac- terial cell walls. This therapeutic approach is likely to have a broader spectrum of applications and avoid major pitfalls of other currently available treatments, such as the devel- opment of resistance, and may ultimately prove to work in synergy with more traditional approaches. Several recent studies have explored these novel options and some have successfully extended their investigations to animal mod- els (Box 2).
The development of a large number of small-molecule DIs, combined with the growing number of studies documenting the dynamin dependency of pathogenicity, paves the way for new therapeutic strategies aimed at blocking the uptake and trafficking of a broad range of pathogens (Box 2). One such study highlighted the successful use of small-molecule DIs to block the inter- nalization of botulinum neurotoxin type-A (BoNT/A) and delay the onset of botulism in mice [5]. This was the first translation of the cell-based use of DIs to an animal model of botulism. Although a growing number of studies point toward a prophylactic effect of interfer- ing with vesicular trafficking as a promising therapeu- tic strategy, it is unclear whether these pathways are suitable targets in vivo. Trafficking events have impor- tant physiological functions and it is not yet known whether targeting them in long-term therapy might lead to undesirable side effects. The following sections review the current literature to determine what is known about dynamin-dependent trafficking pathways and the pathogens that utilize them. Current limita- tions of the studies and conflicting results are discussed to determine whether it is possible to move forward in this field and develop DIs as therapeutic compounds.

Canonical endocytic pathways requiring dynamin function
Since dynamin was discovered [6,7], it has been found to play a role in various endocytic pathways, as well as other cellular functions (Figure 1 and Box 1) [2]. Dynamin acts in the initial stages of CME by catalyzing the fission of endocytic vesicles from the plasma membrane through the formation and subsequent constriction of a collar or helix around the neck of nascent endocytic vesicles [2]. GTP hydrolysis drives both the vesicle fission step and disas- sembly of the oligomerized dynamin. Dynamin assists in the release of vesicles from the plasma membrane in other types of endocytic pathway, but how this occurs is not well- characterized. Dynamin is also involved in endocytosis- independent functions, including vesicle formation from the trans-Golgi network, the cytokinesis stage of mitosis [8], the sperm acrosome reaction [9], regulation of exocyto-
sis [10] and actin dynamics [4].
A distinct characteristic of CME is the formation of clathrin-coated vesicles at the plasma membrane, a pro- cess that involves the assembly of a range of accessory and adaptor proteins, such as AP2, and is assisted by BAR domain-containing proteins that promote membrane cur- vature and recruit dynamin [3]. Dynamin operates in at least two endocytic modes of synaptic vesicle recycling in neurons [11], being required for both CME and for the activity-dependent bulk endocytosis, which involves rapid retrieval of a large portion of the plasma membrane in a clathrin-independent manner [12]. Dephosphorylation of dynamin I by calcineurin triggers the latter process [13].

Dynamin plays an essential role in other modes of endocytosis. Caveolae-mediated endocytosis is defined by uncoated, flask-shaped invaginations from the plasma membrane that require the protein caveolin [14]. Although caveolae do not use the same adaptor and accessory pro- teins as CME, dynamin is required for the scission of caveolae from the plasma membrane [15]. Dynamin is involved in macro- and micropinocytosis-like endocytic pathways [16–19] and in the phagocytosis of large particles such as bacteria or viruses [19]. In the latter instance, dynamin acts at an earlier point in the pathway that is likely to control actin dynamics shaping the phagocytic cup [20]. Dynamin has also been implicated in other endocytic pathways, such as Arf6-dependent endocytosis [21], and endocytosis of the interleukin-2 receptor (IL2R) [22,23]. Despite the incomplete molecular understanding of some of these pathways, the common requirement for dynamin GTPase activity advocates for the potential use of DIs in a broad range of prophylactic treatments.

Tools to study dynamin function All research tools have strengths and limitations that must be carefully assessed to avoid pitfalls in data interpreta- tion. The available tools to determine the cellular roles of dynamin fall into three main categories: (i) genetic modifications (transgenic animals, RNAi, overexpression of mutant or wild-type plasmids); (ii) introduction of

Figure 1. Endocytic pathways present in cells that are hijacked by pathogens and toxins, and small-molecule inhibitors targeting these pathways. Depicted are defined trafficking pathways that are commonly utilized by many viruses, toxins, and other pathogens to intoxicate and infect cells. A selected number of well-characterized pathogens are shown to illustrate the specific pathway they utilize to enter cells. The illustration shows three sections, based on dynamin dependency. The first section shows dynamin-dependent pathways. Synaptic vesicle endocytosis from presynaptic nerve terminals of neurons retrieves and recycles synaptic vesicles following their exocytic fusion. The canonical pathways, clathrin- and caveolae-mediated endocytosis, along with endocytosis of the interleukin-2 receptor (IL2R) also require dynamin. The second section shows pathways likely to involve dynamin. Evidence links dynamin to Arf-6-mediated endocytosis and macropinocytosis, although this has not been conclusively established. The last section on the right shows dynamin-independent trafficking pathways, including the clathrin-independent carrier and glycophosphatidylinositol-enriched endocytic compartment (CLIC/GEEC) pathway. The site of action and pathogens targeted by small-molecule inhibitors are indicated. Inhibition of dynamin by Dyngo-4a prevents internalization of botulinum neurotoxin type-A (BoNT/A) into recycling synaptic vesicles. Furthermore, toosendanin blocks translocation of the BoNT/A light chain into the cytosol and hence overrides its paralytic effect. Clathrin-mediated endocytosis (CME) of HIV-1 can be blocked by either inhibiting dynamin (MiTMAB) or preventing the uncoating of clathrin (Pitstop). Manganese blocks the retrograde transport of Shiga toxin; small-molecule inhibitors (Retro- 1 and -2) prevent the same step in the pathway of both Shiga toxin and ricin. The PIKfyve inhibitor, YM201636, prevents fusion of the Salmonella-containing vesicle (SCV) with a late endosome, thereby blocking maturation and replication.

cell-permeable peptides that block dynamin–protein inter- actions; and (iii) small-molecule inhibitors (DIs) targeting the GTPase activity of dynamin. There are off-target issues that are uniquely associated with each of these methodol- ogies and with the differential retention of a dynamin protein with particular inactivated properties. For exam- ple, blocking GTPase activity with DIs does not prevent protein–protein interactions and vice versa for peptides, whereas the use of RNAi removes both but can lead to compensatory effects. Multiple strategies should therefore be adopted to demonstrate a specific role for dynamin function and the limitations should be taken into consid- eration when interpreting the data.
The most widely used strategy is to express a dynamin mutant with defective GTPase activity or oligomerization in cells. Over 200 studies have utilized the K44A mutation, in which a critical lysine residue required for nucleotide
binding is mutated [24]. Expression of this, or related mutants, blocks CME by a dominant-negative mecha- nism. However, transfections usually take 2–3 days for the cell to express and compensatory mechanisms can occur in response to prolonged endocytic downregulation. A further limitation of this strategy is that overexpression or knockdown of dynamin II, but not I, induces apoptosis due to failure of mitosis [8,25]. Rarely has the correct dynamin gene or splice variant been utilized, with dyna- min I typically transfected into dynamin II-containing cells. Knockdown of dynamin by RNAi has also been widely used to study the function of dynamin. Multiple small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs) are classically required to control for off-target effects, because even scrambled sequences can often im- pact on dynamin levels. More sophisticated strategies involve methodological combinations, such as knockdown

combined with either overexpression or DIs, or knockdown followed by functional recovery via rescue with an RNAi- resistant plasmid.
Another way to manipulate dynamin function in cells is to introduce synthetic peptides that block protein–protein interactions, by use of microinjection or either penetratin or myristoylation tags to render the peptides cell perme- able. This has the advantage of rapid action (minutes), but suffers from limited specificity due to the peptidic sequences commonly used. One widely used peptide pre- vents dynamin from interacting with its partner syndapin, which blocks bulk endocytosis but not CME [26]. Another synthetic peptide, commonly called P4, is based on the dynamin proline-rich domain region and blocks the dyna- min–amphiphysin interaction, which in turn inhibits syn- aptic vesicle endocytosis [27,28].
A series of dynamin knockout (KO) mice has been developed that has provided important insights into the protein’s endocytic function. Dynamin II KO is embryoni- cally lethal; however, derived mouse embryonic fibroblasts (MEFs) have proved to be important research tools [29,30]. Off-target issues are considerations in interpreting KO studies, because the localization of some of dynamin’s binding partners is severely disrupted, indicating incom- plete specificity [31].
The final strategy involves the use of DIs, which have the benefits of rapid action that is typically reversible on drug washout (Table 1). The first reported DIs were long- chain ammonium salts called MiTMABsTM [32–35]. Later, dynasore was identified from screening a large chemical library [36], although this widely used DI suffers from relatively low potency and a lack of specificity. A 40-fold improved analog of dynasore named Dyngo-4aTM was re- cently designed as a more potent tool that blocks CME and
synaptic vesicle endocytosis with fewer nonspecific inter- actions [5,9,37].
The DynoleTM, PthaladynTM, RhodadynTM, and Imino- dynTM series of DIs all have an extensive structure–activi- ty relationship [38–41]. Dyngo-4aTM and Dynole-34-2TM show the greatest in-cell potency and the least toxicity and have the greatest potential for further therapeutic devel- opments. With these DIs, it is now possible to rapidly and reversibly block dynamin by distinct mechanisms of action, thereby targeting different stages in its cycle of GTPase activity. For example, MiTMAB, OcTMAB, and room-tem- perature ionic liquids (RTILs) block dynamin recruitment to membranes, whereas the Dynoles, Dyngo-4a, and dyna- sore block dynamin function after its recruitment. Some DIs are currently being explored for their potential as antiepileptic drugs (P.J. Robinson and A. McCluskey, un- published) and anticancer drugs, including Dynole 34-2, which has a promising therapeutic window for selectively inducing cell death in cancer cells [8].
There are important considerations in experimental design and data interpretation when using DIs, some of which are summarized in Box 3. The field has now reached a state where combinations of these molecules, which come from different chemical scaffolds and target dynamin by distinct mechanisms, provides powerful tools alongside traditional approaches, with the advantage that they may be potentially developed into drugs. The future devel- opment of isoform-specific DIs is the next challenge in this field. Small molecules afford the greatest potential for development of infection prophylaxis due to their rapid and reversible action. To achieve this, some studies have tested the currently available compounds in animal dis- ease models (Box 2). However, the development of preclin- ical candidates is now required to achieve this goal.

Table 1. Classes of DIs targeting dynamin
Inhibitor Compound scaffold Mode of action a (mM) IC50(RME) b (mM) IC50(SVE) Specificityc
Dynamin I vs II (mM) Refs
Dyngo-4aTM Hydrazide G domain: allosteric site Not tested d
16 1.2 Dynamin I selective
0.38 0.05 vs 2.6 0.12 [5]
Dynole-34-2TM Indole G domain: uncompetitive with GTP 5.0 0.9 105 Non-selective [37,40]
Pthaladyn-23 Phthalimide G domain: GTP competitive Not active 12.9 5.9 Dynamin I selective
17.4 5.8 vs 63 33 [39]
Rhodadyn-C10 Rhodanine Unknown 7.0 2.2 Not reported Non-selective
9.1 1.2 vs 30.3 8.2 [41]
MiTMABTM
OcTMABTM Long-chain amines
and ammonium salts PH domain: competitive with lipid
and noncompetitive with GTP e
20.9 3.2
16.0 4.2 10.5
Not reported Non-selective [37,98]
RTIL-13 Norcantharidin PH domain 9.3 1.9 6.9 Not tested [35,37]
IminodynsTM 22
23 Iminochromene G domain: uncompetitive with GTP 10.7 4.5
74.6 8.8 99.5 1.7
40.4 0.8 Non-selective
0.45 0.05 vs 0.39 0.15
0.26 0.08 vs 0.29 0.11 [38]
Bis-T-22 Dimeric tyrphostins G domain, allosteric site Not reported Not reported Non-selective [34]
Dynasore Hydrazide Unknown: noncompetitive inhibition f
~15 d
79.3 1.3 ~15 (non-selective) [5,36]
Sertraline Naphtha-enamine Mixed inhibition with respect to GTP
and lipid binding Not reported Not reported Non-selective [91]
aTransferrin uptake in U2OS cells.
bFM4-64 uptake in synaptosomes.
cIn vitro inhibition of GTPase activity.
dEndocytosis of BoNT/A-Hc in cultured neurons. eEndocytosis of epidermal growth factor in COS-7 cells. fEndocytosis of transferrin in HeLa cells.

pathogens, which need to access or to pass through differ- ent tissues to promote intoxication. For example, BoNT must first transcytose the intestinal cell layer before being taken up in motor nerve terminals [43]. Therapeutic inter- vention therefore requires targeting the relevant patho- genic pathway.

Determining the dynamin dependency of pathogen internalization
The list of bacteria, fungi, viruses, and bacterial toxins known to be endocytosed via dynamin-dependent path- ways is rapidly expanding (Table 2). The field has frequent- ly yielded apparently conflicting results for several reasons stemming from variations in the pathogen strain, cell lines, or technology used to interfere with dynamin function. Although knockdowns or DIs are excellent tools with which to determine the dynamin requirement at various stages of the infection process, they should not be solely relied on, and detailed pathway mapping is required to conclude at which step dynamin might be acting. There are various pitfalls in the use of a single method to assess dynamin dependency. For example, dynamin dominant-negative constructs increase endosomal pH [42] and use of high dynasore concentrations up to 200 mM greatly increases the probability of off-target effects. Many studies rely on a single dynamin-inhibition approach. It is clear that the internalization pathway utilized by various pathogens is influenced by cell type and strain. Such diversity is unsur- prising considering the opportunistic nature of many
Determining the dynamin dependency of virus internalization
Although dynamin is required for the endocytosis of mul- tiple viruses, studies have often reached conflicting con- clusions on the endocytic pathway utilized. It is frequently unclear whether dynamin is required for endocytosis or for the subsequent trafficking steps during later stages of the infection cycle. Viruses may use either dynamin-dependent or -independent modes of endocytosis or a combination of the two. Although dynamin might be important during the initial virus internalization, it could also, or instead, be required for trafficking of vesicles within the cell or during replication or viral particle secretion from the cell. These steps are not often distinguished, but data are often ana- lyzed from the perspective of infection per se, which is an outcome of multiple complex trafficking and signaling pathways. In several instances, the specific pathway is difficult to determine, such as for the murine norovirus 1, which does not require clathrin, caveolae, or macropi- nocytic or phagocytic mechanisms for its internalization into cells, but is blocked by dynamin inhibition and choles- terol disruption [44]. These types of issues must be consid- ered when determining the molecular machinery involved in the internalization and infectivity of viruses.
Experimental conditions can affect the internalization pathway taken by viruses. An example is the addition of fetal calf serum to culture medium, which can turn the trafficking pathway of influenza type-A from dynamin- dependent CME to dynamin-independent macropinocyto- sis [45]. Viruses can also use different pathways in differ- ent cell types. For example, adeno-associated virus (AAV) infection is at least partially dynamin-dependent in HeLa cells [46], but utilizes the dynamin-independent clathrin- independent carrier/glycophosphatidylinositol-enriched endocytic compartment (CLIC/GEEC) pathway in HEK- 293T cells [47]. Further complicating matters, whereas dynasore caused a mild reduction in HeLa cell infection, this was not observed with the K44A dynamin dominant- negative construct [47], leaving open the question of dyna- min’s involvement.
Studies on the endocytic pathways taken by viruses have generated some contradictory conclusions.
One issue is that the initial route of viral entry may play a role in its infectivity. HIV infection of macrophages occurs through a dynamin-dependent variation of a macropinocytosis pathway [17]. When HIV enters cells by CME, dynamin inhibition blocks both internalization and also the later fusion with endosomes [48–51], with similar effects observed using the clathrin inhibitor Pitstop-2TM [49]. Dyna- min inhibition may therefore provide a potential prophylactic treatment for HIV, but the trafficking pathway it takes in pathologically relevant cell types needs to be assessed.

Table 2. Pathogens entering cells through dynamin-dependent endocytosis
Toxins Associated disease Cell type studied Method Proposed endocytic
pathway Refsa
Anthrax toxin from Bacillus
anthracis Anthrax HeLa T/Ib DynI K44A DNc CME [73]
Toxin A (TcdA) and B (TcdB)
from Clostridium difficile Antibiotic-associated
diarrhea HeLa
HT-29 DynII K44A DNc
dynasore CME [72]
a-Toxin (TcnA) from
Clostridium novyi Gangrene HeLa Dynasore CME [72]
BoNT/A from Clostridium botulinum Botulism NG108-15
Hippocampal neurons Murine model DynII K44A DNc
Dynasore Dyngo-4a Synaptic vesicle recycling [5,69]

C2 toxin from
C. botulinum Gastrointestinal hemorrhagic and
necrotic lesions HeLa T/Ib DynII K44A DNc cell line IL2R [23,75]
Diphtheria toxin from
Corynebacterium diphtheriae Diphtheria HeLa Dynasore CME [72]
Iota toxin from
Clostridium perfringens Enterotoxemia HeLa T/Ib DynII K44A DNc
cell line IL2R [23]
Lethal toxin (TcsL) from Clostridium
sordellii Gangrene Enterotoxemia HeLa Dynasore CME [72]
Leukotoxin from
Mannheimia haemolytica Bovine respiratory
disease BL-3 DynII siRNA (partial
inhibition) CME (partial) [74]
TeNT from Clostridium tetani Tetanus Motor neurons Hippocampal neurons P4 peptide DynII K44A DNc CME (motor neurons) Synaptic vesicle recycling (central nervous system
neurons) [70,71]

Bacteria and yeasts Associated disease Cell type studied Method Proposed endocytic
pathway Refsa
Chlamydia trachomatis and psittaci Chlamydia HeLa Dyn K44A DNc DynII siRNA Actin- and Rac-dependent (dynamin dependency not
confirmed) [86,87]
Listeria monocytogenes Listeriosis JEG-3
HepG2 Dyn siRNA
Dynasore CME [79,84]
Yersinia pseudotuberculosis Pseudotuberculosis (yersinia) HeLa Dyn siRNA
Dynasore CME [79]
Staphylococcus aureus Golden staphylococcal
infections HeLa Dyn siRNA
Dynasore CME [79]
Candida albicans Candidiasis JEG-3
HEK-293 DynII WT
Dyn siRNA CME [83]
Uropathogenic
Escherichia coli (UPEC) Urinary tract infection 5637 Dynasore CME [82]
Group A Streptococcus Pharyngitis Keratinocytes Dynasore
Dyn siRNA CME [80]
Viruses Associated disease Cell type studied Method Proposed endocytic
pathway Refsa
Adeno-associated virus Not pathogenic HeLa Dyn K44A DNc Not confirmed [46]
Adenovirus Respiratory infection HeLa A549 KB SAEC T/Ib Dyn K44A DNc cell line
Dynasore
DynII K44A DNc
DynII siRNA CME [99]

African swine
fever virus (ASFV) African swine fever Vero
WSL Dyn K44A DNc
Dynasore CME [100]
Avian reovirus Avian reovirus disease Vero
DF-1 Dynasore
DynII siRNA Caveolae-mediated
endocytosis [101]
Bluetongue virus-1 Bluetongue disease/
catarrhal fever BHK-21 Dynasore Dynamin-dependent
macropinocytosis [102]
Coxsackievirus B3 (CVB3) and A9 (CVA9) Viral myocarditis/central nervous system infection/ respiratory disease HeLa T/Ib Dyn K44A DNc cell line
Dynasore
DynII K44A DNc
DynII siRNA Not well defined (Arf6-dependent/B9
cholesterol-dependent) [103,
A549 104]

Dengue virus (DENV) 1, 2, 3, and 4 Dengue fever Vero
Mosquito C6/36 BHK-21 Dynasore Antibody CME (major pathway) [62]

Ebola virus Ebola hemorrhagic fever SNB19 HuVEC
Hff Vero
Monocytes
Dendritic cells DynII K44A DNc
Dynasore Multiple pathways [16,54]

Echovirus (EV1) from
Picornaviridae Echovirus disease CV-1 DynII K44A DNc Caveolae-mediated
endocytosis [105]
Equine infectious
anemia virus (EIAV) Equine infectious anemia ED DynI K44A DNc
DynII K44A DNc Strain dependent
(CME major pathway) [106]
Hantaan virus Korean hemorrhagic fever HeLa T/Ib Dyn K44A DNc
cell line CME [107]
Hepatitis B virus (HBV) and hepatitis C virus (HCV) Hepatitis B Hepatitis C HepaRG PHH Huh7.5.1 S/Ed DynII K44A DNc cell line
Dynasore
DynII shRNA Caveolae-mediated endocytosis (HBV) [108,
109]

Herpes simplex virus Herpes simplex HeLa HaCaT Human
keratinocytes
Murine epidermis DynII K44A DNc
Dynasore MiTMAB Dynamin-dependent macropinocytosis [53]

HIV AIDS HeLa TZM-bl
Macrophages T/Se and T/Ib Dyn G273D cell line Dyn K44A DNc Dynasore
MiTMAB CME [17,48,
50,51]

Influenza virus Influenza DF-1 Dynasore Dyn siRNA CME (major pathway) [45,110]
A549
HeLa
CNE-2Z
E-36
Junin arenavirus (JUNV) Argentine hemorrhagic fever Vero DynII K44A DNc CME [111]
Murine norovirus-1 (MNV-1) Murine norovirus disease RAW264.7
Murine macrophages Dynasore
DynII K44A DNc Not well defined (dynamin- and
cholesterol-dependent) [44]
BPV1 Carcinomas/hemangio- endotheliomas of the
bladder HEK293 Dynasore CME [66]
C33A

HPV31 and HPV16 Genital warts/cervical cancer HaCaT DynII K44A DNc
Dynasore HPV16 CME
HPV31 caveolae- mediated endocytosis [66,112]
HEK293
C33A

Canine parvovirus Parvo Mv1 T/Ib DynI K44A DNc
cell line CME [113]
Poliovirus Poliomyelitis HBMEC DynII K44A DNc Caveolae-mediated
endocytosis [114]
HRV Common cold HeLa Rhabdomyosarcoma
cells Dyn K44A DNc Dynasore
T/Ib Dyn K44A DNc Strain dependent (dynamin-dependent) [55,56,
115]

Rotavirus Gastroenteritis MA104 Dyn K44A DNc Strain dependent
(dynamin-dependent) [60]
Semliki forest virus (SFV) Semliki forest virus disease HeLa T/Ib DynI K44A DNc DynI K44A DNc
Dynasore CME [67,115]
Simian virus 40 (SV-40) Rarely causes disease
but associated with cancer CV-1 DynII K44A DNc Self-induced invaginations (likely partially dynamin-
dependent) [58]
Sindbis virus Sindbis fever HeLa T/Ib DynI K44A DNc Not defined (dynamin-
dependent) [115]
Tiger Frog virus Cytopathic HepG2 Dynasore Caveolae-mediated
endocytosis [116]
Vaccinia virus Not pathogenic HeLa Dynasore Macropinocytosis (likely
dynamin-dependent) [117]
VSV Vesicular stomatitis BSC-1 Dynasore
DynII WT CME (also requires actin) [118]
aFor journal space considerations, some references were omitted from this table.
bTetracycline-inducible dynamin K44A dominant-negative cell line.
cDynamin K44A dominant-negative mutant.
dStably expressing cell line.
eTemperature-sensitive dynamin cell line.

A second issue is determining which endocytic route leads to pathology. In the case of herpes simplex virus (HSV), which infects both skin cells and sympathetic neurons in vivo, the virus uses an Arf6-/dynamin- dependent pathway in HeLa cells [52], but dynamin- dependent macropinocytosis-like endocytosis in oth- er cells such as epidermal cultures [53]. Macropino- cytosis is therefore likely to be the pathological endocytic pathway. However, the route of viral infection in cultured neurons is dynamin-indepen- dent. Therefore, it may be premature to conclude that DI therapy represents a clinically viable option in this situation.
A third issue stems from the use of viruses that are not fully active. For example, Zaire Ebola virus (ZEBOV) can use multiple entry pathways, but the main pathway for the replication-competent virus is dynamin-independent macropinocytosis in Vero cells [54].
A fourth issue involves the pleiotropic effects of dynamin inhibition, which can lead to potentially misleading conclusions. Different strains of human rhinovirus (HRV) are taken up via multiple endocytic pathways, but commonly use CME [55,56]. However, infection with HRV2 can be blocked via an off-target effect of the K44A mutant, which increases pH in the endosomal system, thereby indirectly preventing infection [42].
A fifth issue is that some viruses, such as simian virus 40 (SV40), are capable of inducing specialized path- ways within cells to gain access to the intracellular environment [57]. SV40 was originally shown to enter cells via a caveolae-dependent pathway, but was later found to enter MEFs derived from caveolin- 1 knockout mice and induce tubulation in the absence of cellular activity [57]. It remains unclear how these SV40-containing invaginations pinch off from the plasma membrane, but internalization relies on actin, tyrosine kinases, and cholesterol [57]. Al- though dynamin is not likely to be involved in its uptake [57], overexpression of K44A does prevent SV40 internalization in CV-1 cells derived from the natural host of the virus [58]. The pinching off of the tubules may involve actin and cholesterol, as previously shown for the scission of Shiga toxin- induced tubules [59].
A sixth issue is the fact that different strains of the same virus may have the ability to use distinct endocytic pathways to gain cellular entry. For example, although most strains of rotavirus use CME, the rhesus rotavirus does not [60]. Similarly, dengue virus (DENV1–4) enters most cell types by CME, but DENV2 uses a clathrin- and caveolae- independent pathway in Vero cells [61,62].
Finally, dynamin may play a post-uptake role during viral infection. Dynamin is involved in other cellular functions, such as vesicle formation from the trans- Golgi network and actin dynamics [2]. These can be associated with both intracellular vesicle trafficking and the secretory pathway, which are used by several viruses following their replication [63]. Dynamin has
been implicated in the infectivity of several viruses at later stages of viral infection, such as budding [64]. Most studies have not determined the step at which dynamin acts during infection, yet this is important for determining the potential of DIs as therapeutics, affecting the time window of action. Vesicular stomatitis virus (VSV) requires dynamin and its tyrosine phosphorylation for release of the glycopro- tein, VSV-G, from the trans-Golgi network [65]. Although bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 (HPV16) enter cells via CME [66], HPV16 has also been suggested to use a dynamin-independent pathway similar to macro- pinocytosis [67]. Addition of dynasore after BPV1 and HPV16 decreases the infection but it is not known whether this is due to blocking further endocytosis or a later step. Dynamin inhibition also blocks secretion of hepatitis B surface antigens [64], suggesting that DIs have the potential to be therapeutically effective even when administered after pathogen exposure.

All of these issues highlight the need to adopt careful experimental design and data interpretation and to ensure that the chosen cellular model is as close as possible to the native cellular target of the virus.

Determining the dynamin dependency of toxin internalization
Most viruses use dynamin-dependent CME to enter cells, albeit not exclusively, and this may also be true for many toxins that are typically produced by invading pathogenic bacteria. Only a limited number of studies have been undertaken on toxin internalization.
BoNT types A–G are responsible for botulism, using synaptic vesicle recycling to gain access into motor nerve terminals and ultimately cause flaccid paralysis [68]. BoNT/A was the first of its class to be shown to enter cells via a dynamin-dependent, synaptic vesicle endocytosis pathway [5,69]. Blocking dynamin function not only pre- vents internalization but also delays intoxication. Other BoNTs are taken up in synaptic vesicles via an activity- dependent mechanism, so it seems likely that they also use dynamin-dependent pathways. Tetanus neurotoxin (TeNT), another clostridial neurotoxin, is internalized at motor nerve terminals via CME [70], but does not act there. Instead it is retrogradely transported and, following trans- cytosis, reaches the spinal cord interneurons where it is internalized into synaptic vesicles. The selective blockade of the release of inhibitory neurotransmitter promotes spastic paralysis [71]. The clostridial toxins A and B from Clostridium difficile, a-toxin from Clostridium novyi, and
lethal toxin from Clostridium sordellii are blocked by
dynamin and clathrin inhibitors [72]. CME is also utilized by diphtheria toxin [72].
Other toxins use combinations of CME and other path- ways. A number of toxins require initial binding to lipid rafts before entering cells via clathrin- and dynamin-de- pendent mechanisms, such as TeNT [70], anthrax toxin [73], and leukotoxin [74]. C2 toxin from Clostridium botu- linum and Iota toxin from Clostridium perfringens are likely to utilize a clathrin-independent pathway required

for trafficking of the IL2R [23]. IL2R is the only endogenous protein known to use this RhoA-dependent, dynamin-de- pendent pathway [22]. CME could play a role in the internalization of C2, because inhibition of clathrin func- tion causes a delay in intoxication [75]. It is possible that C2 uses the IL2R internalization pathway as a primary route and CME as a secondary route.
Cholera and Shiga toxin require both dynamin-depen- dent and -independent pathways to intoxicate cells. Chol- era toxin can use multiple pathways, including those originating from dynamin-dependent or -independent en- docytosis. When dynamin is inhibited, cholera toxin inter- nalization is only slightly reduced in various cell types, indicating that it uses other high-capacity pathways. For this reason, cholera toxin has been used as a marker for the CLIC/GEEC pathway [76]. This CLIC/GEEC pathway is also utilized by the VacA toxin from Helicobacter pylori to enter cells before trafficking through the endosomal sys- tem [77]. Shiga toxin, like SV40, induces tubulation in a dynamin-independent manner through the pentavalent binding of the toxin to gangliosides and induction of mem- brane curvature. Although dynamin can be involved in the pinching off of these tubules from the plasma membrane, this can be regulated by an actin- and cholesterol-depen- dent process alone [59]. However, dynamin is required for retrograde trafficking of Shiga toxin from endosomes to the trans-Golgi network [78].
As for viruses, there is a need to carefully design experi- ments to pinpoint the mechanism used by toxins to gain entry, ensuring relevant choice of a cellular model and examination of the complete trafficking pathway.

Determining the dynamin dependency of bacterium, parasite, and fungus internalization
Dynamin inhibition blocks the internalization or intoxi- cation of various bacteria, parasites, and fungi. Some of these pathogens also require clathrin, although it is un- likely to function in classical CME due to the large size of bacteria. Several types of bacteria can promote their own internalization by binding to protein receptors and acti- vating signaling cascades within the host cell. Typically, clathrin is recruited and phosphorylated, followed by recruitment of actin and dynamin-mediated internaliza- tion [79–82]. Similarly the fungus Candida albicans invades cells via a non-typical clathrin-, actin-, and dyna- min-dependent pathway [83]. Listeria monocytogenes is also internalized via a similar pathway [79], although beads coated with listeriolysin O, the toxin produced by L.
monocytogenes, are internalized in a dynamin-dependent
but clathrin-independent manner [84]. Because this toxin is required for the internalization of the bacterium, it is possible that the beads or the cell type could affect the mode of internalization. The different strains of Chla- mydia utilize different mechanisms to enter cells, relying on actin and Rac [85]. Chlamydia trachomatis and Chla- mydia psittaci require dynamin function [86,87], al- though it is unclear whether dynamin is involved in later stages of infection and replication [87]. Dynamin is also required for cell invasion and parasitophorous vacuole formation by host cells during infection by the parasite Trypanosoma cruzi [88].
Using DIs as a therapeutic strategy to treat/prevent virus and toxin infections We have outlined an extensive number of studies exploring the way in which pathogens use dynamin-dependent traf- ficking pathways to enter and intoxicate cells. Together, the findings provide a compelling argument for developing DIs as a potential therapeutic strategy. They also raise the possibility that it may not be essential to distinguish between entry and intoxication for therapeutic develop- ment, although a potential opportunity exists to treat patients at later stages following exposure.
Is it too early to consider DIs in infection prophylaxis? Several studies suggest otherwise. The ability of Dyngo-4a to block intoxication with BoNT/A in cultured hippocampal neurons has been confirmed in a murine study. Treatment of mice with the DI delayed the onset of botulism and highlighted the fact that treated mice exhibited few side- effects due to the action of Dyngo-4a [5]. However, mice treated with Dyngo-4a display greatly reduced sperm fer- tility due to reduced dynamin function in acrosomal exo- cytosis [9]. Another study investigating the ability of dynasore to treat atherosclerosis did not report any side effects after treatment of mice with dynasore for 16 weeks [89]. To the best of our knowledge, no other studies have reported on DIs in vivo.
Is dynamin inhibition the best clinical target? At this
stage, it appears to be a better prophylactic candidate than alternatives such as clathrin inhibition using Pitstops, due to the broader involvement of dynamin in multiple cla- thrin-independent endocytic modes. The state of dynamin- based drug development is now mature and has the poten- tial to drive more animal-based studies, particularly given the relative safety of dynamin-based therapy in animal models to date. The concept of using DIs for infection prophylaxis does not depend on 100% inhibition of dyna- min function or of endocytosis. Typically, drugs are able to reduce the activity of a pathway sufficiently to slow the infection process, allowing the body’s immune system to clear the toxin or pathogen before infection or intoxication reaches significant levels. Therefore, the goal of DI therapy should be to reduce endocytosis sufficiently to be therapeu- tic, but not abolish endocytosis.
DIs are beginning to be used in the treatment of several disorders. Bisphosphonate drugs are prenylation inhibi- tors currently used to treat osteoporosis, but recent studies have found that these drugs can also be used to prevent viral infection. One study linked this to inhibition of dyna- min II rather than direct interaction with the virus [90]. Because these drugs are already approved for use in humans, this indicates that DIs can be used for other disorders. Similarly, some clinically important selective serotonin reuptake inhibitors are also DIs [91]. There is not yet any indication that dynamin inhibition contributes to their antidepressant action, but this use suggests that inhibiting dynamin in humans may have only a few side effects. In fact, heterozygote dynamin I knockout mice are healthy, viable, and fertile, suggesting no apparent effects from a partial systemic reduction in dynamin I levels [92]. Together these studies suggest that there is indeed poten- tial for future therapy based on targeting dynamin (with DIs), and other key components of vesicular trafficking.

Acknowledgments
This work was supported by a grant from the Australian Research Council and from the National Health and Medical Research Council of Australia; F.A.M. and P.J.R. are fellows of the National Health and Medical Research Council of Australia. C.B.H. is supported by an Australian Postgraduate Award. We thank Dee McGrath for her help with graphic design, James Daniel for help with Box 3, and Rowan Tweedale and Sally Martin for their critical comments and suggestions.

Disclaimer statement
A.M. and P.J.R. hold trademarks and patent applications concerning several of the published dynamin and clathrin inhibitors discussed in this review and supply compounds for commercial distribution through Abcam (Cambridge, UK).

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