Characterization of Novel Piperidine-Based ... - ACS Publications

Subscriber access provided by UNIV OF NEWCASTLE

Article

Characterization of a novel piperidine-based inhibitor of cathepsin B-dependent bacterial toxins and viruses Stella Hartmann, Renae Lopez Cruz, Saleem Alameh, Chi-Lee Charlie Ho, Amy E Rabideau, Bradley L. Pentelute, Kenneth A. Bradley, and Mikhail Martchenko ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00053 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

Characterization of a novel piperidine-based inhibitor of cathepsin B-dependent bacterial toxins and viruses

Stella Hartmann1, ‡, Renae Lopez Cruz2, ‡, Saleem Alameh1, Chi-Lee C. Ho2, Amy Rabideau3, Bradley L. Pentelute3, Kenneth A. Bradley2,*, Mikhail Martchenko1,*

1

School of Applied Life Sciences, Keck Graduate Institute, 535 Watson Drive, Claremont, CA

91711 2

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los

Angeles, 609 Charles E Young Dr E, Los Angeles, CA, 90095 3

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave.

Cambridge, MA 02139

‡

These authors contributed equally to this work

* To whom correspondence should be addressed: [email protected] and [email protected]

ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Exploiting the host endocytic trafficking pathway is a common mechanism by which bacterial exotoxins gain entry in order to exert virulent effects upon the host cells. A previous study identified

a

small-molecule,

1-(2,6-dimethyl-1-piperidinyl)-3-[(2-isopropyl-5-

methylcyclohexyl)oxy]-2-propanol, that blocks the process of anthrax lethal toxin (LT) cytotoxicity. Here, we report the characterization of the bioactivity of this compound, which we named RC1. We found that RC1 protected host cells independently of LT concentration and also blocked intoxication by other bacterial exotoxins, suggesting that the target of the compound is a host factor. Using the anthrax LT intoxication pathway as a reference, we show that while anthrax toxin is able to bind to cells and establish an endosomal pore in the presence of the drug, the toxin is unable to translocate into the cytosol. We demonstrate that RC1 doesn’t inhibit the toxin directly, but rather reduces the enzymatic activity of host cathepsin B that mediates the escape of toxins into the cytoplasm from late endosomes. We demonstrate that the pathogenicity of Human cytomegalovirus and Herpes simplex virus 1, which rely on cathepsin B protease activity is reduced by RC1. This study reveals the potential of RC1 as a broad-spectrum host-oriented therapy against several aggressive and deadly pathogens.

KEY WORDS: Anthrax toxin, broad-spectrum, host-oriented, cathepsin B, small molecule, drug discovery


Page 2 of 37

Page 3 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A variety of pathogenic microorganisms have evolved to exploit host cellular processes that enable their entry or allow the trafficking of toxic protein complexes into the host 1. A common mechanism utilized by bacterial exotoxins, for instance, is receptor-mediated endocytosis. Anthrax lethal toxin (LT) is a binary AB toxin complex composed of protective antigen (PA), the cell binding subunit, and lethal factor (LF), the catalytic subunit. PA is able to bind to one of two host cell receptors: capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8)

2-3

. Upon binding to host cell receptors, the host endoprotease furin cleaves a

20 kDa fragment from the 83 kDa PA subunits (PA83), resulting in 63 kDa PA monomers (PA63) with exposed oligomerization sites. The receptor-bound PA63 oligomerize into a heptamer or an octamer, referred to as the PA pre-pore

4-5

. The pre-pore contains binding sites for the catalytic

subunit LF and is internalized by the host cell through clathrin-mediated endocytosis. Upon acidification of the late endosome, the PA pre-pore undergoes a conformational change into an SDS-resistant PA pore 6, which inserts into the endosomal membrane, thereby allowing the LF subunits to enter the cytosol 7. The escape of LF from endosomes into the cytoplasm is mediated by host protease, cathepsin B (CTSB) 8-9. LF is a zinc-dependent metalloprotease with diverse catalytic activities. LF cleaves the Nterminus of mitogen-activated protein kinase kinases (MAPKKs) 1-4, 6, and 7

10-11

. Disruption of

the MAPKK pathway results in reduced cytokine expression, hindering the host inflammatory response and allowing the pathogen to propagate and eventually kill the host 12-13. LF also cleaves a lethal-toxin sensitive allele of Nlrp1b, resulting in the activation of caspase-1, production of IL1β and IL-18, and induction of pyroptosis, a rapid inflammatory cell death, in murine macrophages 14-15

. Small molecules that block intoxication by disrupting this process serve as powerful tools

by which host cellular pathways may be further studied and novel therapeutic targets for toxinmediated diseases may be identified. Previously, Gillespie and colleagues identified multiple such small molecules that blocked intoxication by LT 16. Characterization of one of these compounds,



Page 4 of 37

EGA, revealed a novel mechanism of action in which trafficking from early to late endosomes is blocked

16

. EGA inhibits entry of multiple bacterial toxins and viruses, thus identifying a potential

new broadly active host-targeted therapeutic approach. Here, we report the characterization of small molecule referred to as “RC1”, which was identified in a high-throughput screen for inhibitors of anthrax LT

16

. RC1’s chemical name is 1-

(2,6-dimethyl-1-piperidinyl)-3-[(2-isopropyl-5-methylcyclohexyl)oxy]-2-propanol

hydrochloride

(CID 2870294, Chembridge ID# 5807685) (Fig 1a). We found that RC1 also protects host cells from a diphtheria toxin fused with the PA binding domain of LF (LFnDTA), Haemophilus ducreyi cytolethal distending toxin (Hd-CDT), Human cytomegalovirus, and Herpes simplex virus 1. In order to identify the mechanism of action of RC1, events pertaining to toxin entry and cytotoxic activity were assessed in the context of anthrax LT, as this intoxication pathway is well characterized.

RESULTS AND DISCUSSION RC1 protects host macrophages from LT-induced cell death: RC1 was previously reported to protect host cells from anthrax LT with IC50 of low µM 16. In order to identify the effective concentrations of RC1, we performed a cell-based viability assay with mouse macrophage-like RAW264.7 cell line. We found that RC1 provides a full protection from LT-mediated cell death between 12.5 and 25 µM with an EC50 of 9.3µM (Fig. 1b). The protection conferred by the compound is independent of LT concentration, suggesting that its molecular target is a host factor (Fig. 1c). Additionally, a time-course intoxication assay further showed that RC1 protects cells when it is supplied to cells either prior to LT challenge, at the time of LT challenge, or up to 30 minutes post-intoxication (Fig. 1c), demonstrating that its molecular target may be involved at a stage prior to LF entry into the cytosol, which occurs 20-40 minutes postintoxication 17. Figure 1b shows that the pre-treatment of cells for 1 hour with 12.5 µM RC1 does


Page 5 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


not affect cellular viability. In Figure 1c the exposure of cells to 12.5 µM of RC1 is shortened even further, and thus the effect of RC1 on the viability is not expected in either of conditions. During anthrax, a host is challenged with bacteremia and toxemia. We investigated whether RC1 displays anti-bacterial properties using B. anthracis surrogate, B. cereus. This bacteria was chosen for this test because it genetically resembles B. anthracis: two microorganisms share similarity in their genomes, while B. cereus lacks anthrax toxins 18. To test RC1’s anti-microbial properties, we applied 1 μL of 10 mM RC1 and its two-fold dilutions on agarmedia grown B. cereus. We discovered that 1 μL of 10 mM and 5 mM of RC1 was effective in decreasing the growth all tested microbes (Fig. 2a). However, the RC1 zones of inhibitions were significantly smaller than the one observed after 1 μL of 10 mM Levofloxacin was tested in parallel. Levofloxacin, which is an approved B. anthracis antibiotic, was used as a positive control (Fig. 2a). In these experiments, RC1 creates a concentration gradient. In order to determine the efficacious anti-microbial concentrations of RC1, we measured its effect on bacterial growth rate in liquid media. In these tests, 100 μM RC1 and its two-fold dilutions were evaluated, and the only concentrations of RC1 that decreased the growth rate of B. cereus were 100 μM and 50 μM (Fig. 2b). However, these concentrations were shown to cause cytotoxicity to host cells experiments (Fig. 1), suggesting that the anti-microbial effect of RC1 may not be physiologically relevant. This data argues that RC1 doesn’t have an effective anti-bacterial property, and thus should be developed as an anti-toxin.

RC1 acts as a broad-spectrum anti-toxin: To determine if the protective effect of RC1 is specific to LF-mediated toxicity, the ability of the compound to block intoxication by LFnDTA was tested. LFnDTA is a hybrid toxin, which contains the N-terminal PA binding site of LF (LFn) as well as a toxin domain derived from the A-chain of diphtheria toxin (DTA), which has been widely used as an LF surrogate, and challenges cells by a different mechanism 19. Nevertheless, the cytotoxic mechanisms of the two toxins are different:



Page 6 of 37

LT induces pyroptosis, whereas LFnDTA induces apoptosis through global inhibition of protein synthesis 20. We observed that 25 µM of RC1 protects cells from LFnDTA and PA (Fig. 3a), which suggests that the compound disrupts an event in their shared entry process. Because the cellular proteins required for pyroptosis and apoptosis greatly differ, it is unlikely that RC1 disrupts a mechanism that occurs downstream of toxin entry into the cytosol. In order to further test this hypothesis, HEK293 cells were intoxicated with Haemophilus ducreyi cytolethal distending toxin (Hd-CDT) in the presence of RC1. Hd-CDT enters host cells through a pathway that is distinct from LFnDTA and LT. The intracellular trafficking process of Hd-CDT is not fully understood; however, it is known that it requires transport to late endosomes, the Golgi complex, and the ER prior to entering the nucleus

21

breaks in the DNA, which triggers apoptotic cell death

. Hd-CDT then causes double-stranded 22

. Interestingly, RC1 protects cells from

Hd-CDT at low µM range (Fig. 3b). The fact that RC1 inhibits the cytotoxicity of two enzymatically unrelated toxins, LT and Hd-CDT, which both enter into host cytoplasm from late endosomes, indicates that the compound may disrupt a late endocytic trafficking process. Other pathogenic agents, including cholera toxin (CT)

23

and Pseudomonas aeruginosa

exotoxin A (PE) 24 are transported in a retrograde fashion to the endoplasmic reticulum (ER) and retrotranslocated into the cytoplasm by the host ER-associated degradation pathway

23-24

. We

observed that cytotoxicity mediated by those toxins was not blocked by RC1 (Fig. 3c). This data supports the conclusion that RC1 inhibits the entry of toxins into the cytoplasm from acidified endosomes.

RC1 Inhibits Cytosolic Entry of LF: Cellular pyroptosis depends on LF catalytic activity. To determine whether RC1 blocks proteolysis of cellular MAPKKs by LF, we assessed the cleavage of MAPKK2 by immunoblotting. While MAPKK2 was cleaved in LF-PA treated RAW264.7 cells, treatment of cells with RC1 at 12.5-50


Page 7 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


µM prevented this effect (Fig. 4a). These results show that RC1 inhibits either cytotoxicity upstream of MAPKK cleavage or blocks LF directly. In order to test whether RC1 inhibits the enzymatic activity of LF, we used a Fluorescence Resonance Energy Transfer (FRET) based assay, in which a MAPKK2 peptide containing a cleavage site for LF with a fluorogenic FITC group at the N-terminus and DABCYL quenching group at the C-terminus was used as LF substrate for in vitro assays. After cleavage by LF the fluorescence of FITC at 523 nm increases, while a known inhibitor of LF, surfen hydrate

25

,

prevents it from cleaving the substrate and producing fluorescence (Fig. 4b). We tested the ability of RC1 to inhibit the proteolytic activity of LF at the range of 2-133 μM using FRET. An uninhibited emission at 523 nm was observed when LF was able to cleave MAPKK peptide in the presence of RC1, which shows that the drug does not block the proteolytic activity of LF from 2-133 μM.

RC1 Inhibits the Enzymatic Activity of Host Cathepsin B: In order for LF to reach the cytosol, PA83 must bind to host cell receptors, be cleaved by furin into PA63, heptamerize into a pre-pore form, bind LF, and proceed to low-pH endosomes where PA-heptamers undergo an acid-dependent conformational change from pre-pore to pore

26

. We

monitored host-cell binding of PA, its proteolytic cleavage, and its heptamerization by immunoblot in the presence and in the absence of RC1. While we observed less PA83 associated with cells in the presence of RC1, the drug did not block proteolytic processing of PA to generate PA63 (Fig. 4c). These results indicate that RC1 blocks intoxication at a step downstream of PA binding and assembly on the host-cell surface. It is known that PA pores, which result from exposure to low pH within host cell endosomes, are resistant to dissociation by SDS and run as an oligomer on SDS-PAGE

26

. Surprisingly, treatment of cells with 25 μM of RC1 resulted in slightly higher,

rather than lower, abundance of SDS-resistant PA-oligomers compared to cells treated with PA only (Fig. 4c).



A similar observation was reported by Ha et al. 8 and Zilbermintz et al. 9, who showed that an inhibition of host lysosomal CTSB by unrelated small molecules CA-074 and Amodiaquine respectively, resulted in (i) elevated accumulation of PA pores in late endosomes, (ii) the inability of LF to be released from the late endosomes into the cytoplasm, and (iii) reduction of cellular sensitivity to LT. Upon formation of the SDS-resistant PA pores in acidic endosomes, PA pores are then transported to lysosomes for rapid degradation 27. Ha et al. demonstrated that host CTSB mediates the fusion of lysosomes with endosomes, and that this fusion is necessary for the entry of LF from the endosomes into the cytoplasm 8. In order to gain further insight into the mechanism of RC1-mediated protection of cells against anthrax LT killing, we tested whether RC1 inhibits CTSB protease activity in RAW264.7 cells using FRET reaction and CTSB-specific substrate. We observed that cells pre-treated with 25 μM RC1 for 1 hour lost 42% of CTSB enzymatic activity (Fig. 5a). As the enzymatic reaction of CTSB is affected by pH, we determined that the addition of RC1 to CTSB reactions did not change the pH (pH 5.8) of the reaction buffer. We tested the ability of 25 µM Amodiaquine for its ability to inhibit CTSB in RAW264.7 cells as a positive control. We observed that Amodiaquine, which was reported to have a comparable anti-anthrax toxin efficacy in RAW264.7 cells 9, inhibited 41% of cellular CTSB. To further validate that RC1 inhibits host CTSB directly, we tested whether RC1 inhibits CTSB protease activity of purified recombinant human CTSB using FRET. We observed that RC1 inhibits CTSB activity in a dose dependent manner without drug pre-incubations and at drug concentrations much lower than used in our cellular experiments (Fig. 5b). The extent of inhibition of purified recombinant cathepsin B similar to that seen in Fig. 5a is achieved with just 0.67-1.33 µM of RC1, but not with 1.33 µM Amodiaquine (Fig. 5a). This suggests that while RC1 is a more potent inhibitor of purified CTSB than Amodiaquine, RC1 is reaching intracellular CTSB at lower concentrations than the determined EC50 of RC1 (Fig. 1b). Interestingly, the efficacious anti-toxin range of all three molecules, RC1, Amodiaquine, and CA-074 are comparable to each other, lowto-mid μM concentrations. Similarly to RC1, Amodiaquine did not inhibit the proteolytic activity of


Page 8 of 37

Page 9 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LF 9, which argues that these molecules are not toxin-directed compounds and are not unselective protease inhibitors. Future studies will determine whether RC1 inhibits CTSB activity by directly binding to the enzyme, or by other indirect mechanisms.

RC1 Inhibits LF Translocation into the Host Cytosol: Above data shows that although RC1 results in increased PA pore abundance, LF is unable to cleave MAPKK. This observation is consistent with the elevated accumulation of PA pores in late endosomes and the inability of LF to be released from the late endosomes into the cytoplasm, as reported by Ha et al. 8 and Zilbermintz et al 9 during CTSB inactivation. In order to test whether RC1 prevents trafficking of LF into the cytosol, we studied the presence of LF in the digitoninextracted cytosolic fraction of LT treated cells. Western blot analysis revealed that while LF translocated into the cytosol in the absence of drugs, LF trafficking into the host cytosol is prevented in the presence of RC1 (Fig. 5c). The same phenomenon was observed by treating cells with Bafilomycin A1, a compound known to prevent pore formation and subsequent LF translocation by inhibiting endosome acidification

28-30

(Fig. 5c). Similarly, the treatment of cells

with LF and PA[F427H], a translocation-deficient PA mutant, resulted in absence of LF in the cytoplasm 31 (Fig. 5c). Moreover, immunoblot analyses were carried out with antibodies against Rab5 (an early endosome marker) and Erk1/2 (a cytosolic marker) in order to confirm that only the cytosolic fraction was present. Minor amounts of Rab5 were detected in the cytosolic fractions, thus indicating little contamination from early endosomes (Fig. 5c).

RC1 Disrupts A Late Stage in Endocytic Trafficking: We investigated whether RC1 disrupts the release of LF into the cytoplasm from early or late endosomes. Ha et al. reported that CTSB facilitates the CMG2-mediated delivery of LF into the cytoplasm 8. Anthrax LT bound to the TEM8 receptor has a higher pH threshold enabling PA pore



Page 10 of 37

formation and LF translocation into the cytosol from early endosomes. In contrast, when PA is bound to the CMG2 receptor, it is dependent upon late endosomes for these processes to occur 32

. To determine whether RC1 interferes with an early or late stage in endocytic trafficking, we

assayed the protective effect of RC1 on RAW264.7 cells that expressed only TEM8 (R3D-TEM8) or only CMG2 (R3D-CMG2). We found that RC1 conferred protection to R3D-CMG2 cells but not R3D-TEM8 cells from anthrax LT, suggesting that the protective effect of RC1 is specific to late endocytic trafficking processes (Fig. 5d-e). This data is consistent with the observation that release of LF from late endosome is mediated by CTSB, and that chemical inactivation of this host protein blocks the escape of toxins from late endosomes 8.

Evaluation of anti-viral property of RC1: In light of the observations that RC1 acts as broad-spectrum anti-toxin, we investigated whether the therapeutic breadth extends to viruses and microbes. In addition to mediating endocytosis of pathogenic agents, CTSB has also been shown to proteolytically process viral proteins that are necessary for the viral replication. Such viruses are Herpes simplex virus 1 Cytomegalovirus

33

and Human

34

. The entry of these viruses into host cells differs from that of anthrax LT:

Herpes simplex virus 1 is transported in a retrograde fashion to the endoplasmic reticulum (ER) and retrotranslocated into the cytoplasm by the host ER-associated degradation pathway

35

;

Human Cytomegalovirus strain AD169 entry into fibroblasts involves fusion at the plasma membrane and does not require low endosomal pH

36

. We determined that RC1 reduced the

pathogenicity of both viruses (Table 1) with EC50 of 12 μM. This efficacious concentration of RC1 is comparable to that of anti-LT (Fig. 1b), and this data supports the conclusion that RC1 inhibits the entry of pathogenic agents into the cytoplasm by inhibiting host CTSB. Host protein therapeutics have the potential to protect against multiple infectious organisms. Broad-spectrum host-oriented drugs can be especially useful to block numerous unrelated pathogenic agents that exploit the same host proteins for entry and pathogenesis.


Page 11 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Broad-spectrum host-oriented drugs can be identified by targeting host proteins exploited by numerous unrelated pathogenic agents. The discovery and development of novel drugs against biological threat agents such as B. anthracis is an important concern worldwide. A host can be co-exposed to numerous pathogens, thus patients need to receive numerous pharmaceuticals, in order to protect against multiple infectious organisms. Unfortunately, each co-administered drug may have its own side-effects. Therefore, therapies capable of inhibiting multiple pathogenic agents are needed. Broad-spectrum anti-pathogen drugs can be identified by targeting host proteins exploited by numerous unrelated pathogenic agents. In this work we show that RC1 protects host cells by inhibiting the host target rather than the pathogen, which defines RC1 as a host-oriented broad-spectrum anti-toxin and anti-viral compound. For that reason, RC1 is less likely to be circumvented by pathogen mutations that lead to microbial drug resistance. The fact that RC1 is effective against anthrax toxin when cells are treated before or at the time of intoxication helped us identify the host protein target and the step in toxin endocytosis inhibited by the drug (Fig. 1c). However, animal studies are needed to determine whether RC1 is effective prophylactically and post-challenge. We show that RC1 acts as a broad-spectrum host-oriented anti-toxin and anti-viral by inhibiting host CTSB. This is further supported by data illustrating that RC1 protects cells from intoxication by PA + LFnDTA and Hd-CDT. Because the catalytic effects and pathogenic mechanisms of LF, LFnDTA, and Hd-CDT greatly differ from each other, it is unlikely that RC1 inhibits these toxins directly. In fact, the event that is known to be common among the intoxication pathways of LT, LFnDTA, and Hd-CDT is early to late endosome trafficking. Thus, we propose that RC1 inhibits CTSB known to mediate the endosomal process of these toxins. Moreover, our data implicates CTSB in Hd-CDT pathogenicity, as such CTSB role hasn’t been reported to date. Delivery of LF into the cytoplasm occurs through the formation of the heptameric PA63pore, which becomes SDS-resistant in acidified endosomes 26. The SDS-resistant PA63 complex is then transported to lysosomes for rapid degradation

37-38


. Consistent with Ha et al.

8

and


Page 12 of 37

Zilbermintz et al. 9, RC1-inhibition of CTSB had no effect on SDS-resistant PA63-heptamer formation but, rather, protected it from lysosomal degradation (Fig. 4c). Late endosomes and lysosomes are known to fuse to form hybrid organelles, which then undergo back fusion

39-41

. Pathogenic agents exploit back-fusion processes to escape from

endocytic organelles before degradation by lysosomal proteases start. It was shown that CTSB plays an important role in the entry of LF into the cytosol. CTSB is a lysosomal cysteine protease known to mediate the degradation and processing of lysosomal proteins

42

. CTSB has been

previously shown to promote the cytoplasmic delivery of LF 8-9. Moreover, several studies suggest that LF associated with CMG2, but not TEM8, is released into the cytoplasm through an endocytic trafficking route involving CTSB-dependent lysosomal fusion 8, 43-44. LF is delivered from intraluminal vesicles into the cytoplasm likely through back fusion. LF enters the cytoplasm by translocating from the luminal compartment of the multivesicular late endosomes through a not-yet-defined back-fusion process, which in turn relies on CTSB activity 45-46

. This study shows that intracellular PA-pores accumulate in the presence of RC1 (Fig. 4c),

that RC1 reduces MAPKK cleavage by LF (Fig. 4a) without inhibiting LF directly (Fig. 4b), and that RC1 inhibits the enzymatic activity of CTSB (Fig. 5a-b). This suggests that CTSB activity is required for the delivery of LF from endolysosomes into the cytoplasm (Fig. 5c), and that the cytoplasmic entry of LF occurred from the late endosome (Fig. 5d-e). PA is exposed to lysosomal proteases and undergoes a rapid degradation. This data agrees with the notion that LF is packaged into endosomal carrier vesicles and is protected from the proteolytic degradation 46. It is possible that RC1 may have multiple targets in a host cell. This phenomenon would define RC1 as a polypharmacological drug, wherein the drug is acting on multiple targets of either a unique or multiple disease pathways. In fact, numerous chemical structures related to RC1 have been observed to inhibit filamin A 47-48, a host protein known to mediate the process of endocytosis 49

. However, our preliminary results indicate that this protein is not exploited by anthrax LT,

cholera toxin, nor Pseudomonas exotoxin A.


Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Several chemicals that structurally resemble RC1 have previously been reported to act as anti-viral compounds. Cheng et al.

50

identified a small molecule compound, CBS1129, that

inhibits the entry of filoviruses, such as Ebola virus. Just like RC1, CBS1129 contains piperidine and an oxy-2-propanol linker. Although Cheng et al. did not identify the target of CBS1129, another study identified a related piperazine-containing compound that inhibits Ebola virus by inhibiting host CTSB

51

. Moreover, RC1 may inhibit multiple pathogenic agents because of its

physicochemical properties. In fact, numerous approved drugs are cationic amphiphilic drugs that have been shown to accumulate inside cells and to affect several proteins/pathways exploited by pathogens during infection

52

. Future studies will determine the efficacy and safety of RC1 in

animal models of infection for CTSB-dependent pathogens.

CONCLUSION We demonstrate that RC1 acts as a host-oriented broad-spectrum antitoxin and anti-viral compound. We demonstrate that RC1 targets host cathepsin B that mediates the escape of anthrax and Haemophilus ducreyi cytolethal distending toxins into the cytoplasm from late endosomes. We further demonstrate that RC1 reduces the pathogenicity of Human cytomegalovirus and Herpes simplex virus 1, which rely on cathepsin B activity. This study reveals the potential of RC1 as a broad-spectrum host-oriented therapy against several pathogenic agents. Future studies will evaluate the potential of RC1 in animal model of infections. The efficacy of RC1 will be evaluated as a monotherapy, as well as in combination with anti-bacterial drugs, such as ciprofloxacin and levofloxacin. Moreover, therapeutic timing of RC1 will be evaluated, where the drug will be administered to animals prior, after, and at the time of the infection.



Page 14 of 37

METHODS Cell Culture RAW264.7, HEK 293T, R3D-CMG2, R3D-TEM8, and HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) from Cellgro, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamax (PSG), and incubated at 37°C and 5% CO2. R3D-CMG2 and R3D-TEM8 cells were generated as previously described methods

44

. Jurkat cells were

cultured in RPMI media supplemented with 10% FBS and 1% PSG and incubated at 37°C and 5% CO2. CHO-K1 cells were cultured in HAMs F-12 media supplemented with 10% FBS and 1% PSG and incubated at 37°C and 5% CO2.

Intoxication and Viability Assays Cells were seeded the day before the experiment at 2x103 cells in 40 µL per well onto a 384-well plate in either DMEM supplemented with 25 mM HEPES or Ham’s F12 as indicated in the figure legends. For the protection profiles, titrations of RC1 were prepared and incubated with cells for 1 hour at 37°C and 5% CO2 before intoxication with various concentrations of LT, LFnDTA + PA, or Hd-CDT at 37°C and 5% CO2 for 4, 24, or 48h as indicated in the figure legends. LF and PA were purified using previously described methods

53-54

. LFnDTA (the diphtheria toxin A chain

fused with the N-terminus of LF) was obtained as a gift from Jeremy Mogridge from the University of Toronto. For the time-of-addition assay, 12.5 µM RC1 was administered to cells at various time points relative to intoxication with 500 ng/mL LT and incubated at 37°C and 5% CO2 for 4 hours. Cell viability is defined as the percentage of surviving cells obtained relative to untreated cells (100%). The results for all aforementioned experiments were obtained by adding 10 µL of ATP Lite reagent (Perkin Elmer) to each well for 2 minutes and cell viability was measured using the Wallac


Page 15 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Victor 3V multilabel plate reader. Cell viability is reported in terms of relative luminescence units (RLU). All graphs and statistics were generated using GraphPad Prism. Maximum protection is reported when the average RLU value of an experimental condition is equal or within error of the average RLU value for the ‘Cells Only’ control. The concentration at which RC1 displays 50% of its maximum activity is referred to as the EC50. The EC50 was calculated by normalizing experimental values to a ‘Cells Only’ control then graphing the data according to a non-linear fit (variable slope). The EC50 is then reported by the software. RAW264.7 cells were seeded the day before the experiment at 2x104 cells per well onto a 96-well plate in 100 μL of media. RAW264.7 cells were pre-treated with RC1 concentrations for 1 hour at 37°C 5% CO2. RAW264.7 cells were challenged with P. aeruginosa exotoxin A or cholera toxin (purchased from List Biological Laboratories (Campbell, CA)) for 24 hours, which were also pre-treated with identical drug concentrations, such that the final toxins concentrations were 2.0 and 4.0 μg/ml respectively. Determination of RAW264.7 viability was performed by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as described 9. Cell viability is defined as the percentage of surviving cells obtained relative to cells treated with DMSO (100%).

Bacteria and media Bacillus cereus (ATCC 14579) was grown in lysogeny broth (LB) at 37°C. Subsequently, the liquid microbial culture was grown from a single colony by shaking at 200 RPM for a period of 16 hours prior to the beginning of the experiment. In agar media assays, the absorbance at optical density at 600 nm of microbial overnight cultures were determined for each of our experiments. The absorbance values were then converted to cells/mL using McFarland’s scale

55

. Six hundred million and twenty-five million

bacterial and fungal cells, respectively, were added to 25 cm petri dishes. One μL of 10 mM RC1



was placed directly on the agar surface using a pipette. The zones of inhibition were assessed as in 56 . In addition, we utilized the software ImageJ (https://imagej.nih.gov/ij/) to digitally quantify the magnitude of every zone of inhibition. In liquid assays, the overnight grown culture was diluted in fresh medium to 0.1 OD600 and aliquoted in 100 μL into a 96-well plate, followed by the addition of various concentrations of RC1. The same volumes of media were added to control wells. The plates were grown with constant shaking at 37°C and the absorbance was measured by a SpectraMax Plus Microplate Reader (Molecular Devices) every 1000 seconds for a period of 12 hours.

Western Blot Assays RAW264.7 macrophages were seeded at 2x106 cells in 2 mL per well onto a 6-well plate in DMEM supplemented with 10% FBS, 1% PSG, and 25 mM HEPES. On the next day, cells were incubated with RC1 at varying concentrations as indicated in the figure captions for 1 hour at 37°C and 5% CO2. Cells received PA or LT and were incubated for the time indicated in the figure captions. Cells were lysed using NP-40 lysis buffer (1% NP-40, 5% glycerol, 1mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 50 mM Tris-HCl pH 7.9 in H2O) with HALT protease/phosphatase inhibitor (Thermo Scientific). Cell lysate protein concentration was determined by the Bio-Rad Protein Assay. Samples were separated via SDS-PAGE through a 7.5% or 10% polyacrylamide gel and transferred to a PVDF membrane for Western blotting. The membrane was blocked in 5% milk in tris-buffered saline with tween (TBST; 5M NaCl, 1M TrisHCl pH 7.4, Tween 20% in H2O) for 1 hour at room temperature, then incubated overnight at 4° C with rabbit anti-PA antibody (Covance), anti-MAPKK-2 antibody (Santa Cruz Biotechnology), or anti–β-tubulin antibody (Sigma Aldrich) diluted to 1:4000 or 1:1000, respectively, in 5% milk in TBST. The membrane was then washed three times in TBST. The membrane was then treated with goat-anti-rabbit secondary antibodies conjugated with horseradish peroxidase (Invitrogen)


Page 16 of 37

Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diluted 1:2000 in 5% milk in TBST for 1 hour at room temperature. The membrane was washed with TBST and then treated with HRP peroxidase buffer and luminol enhancer (Bio-Rad). Films were developed following a 1-minute exposure to the membrane. The PA bands were quantified using NIH Image J software (https://imagej.nih.gov/ij/) and normalized by the quantification of the background bands or tubulin. In the LF cytosol translocation experiments, RAW264.7 cells received 20 µM or 40 µM RC1 and were incubated for 1 hour prior to treatment with 100 nM LFN, a 32 kDa N-terminal LF region that includes PA-binding domain, and 20 nM PA83 for 6 hours. After treatment, the cells were washed with PBS and lifted with 0.25% trypsin-EDTA for 5 minutes to remove surface bound proteins, and then washed twice with PBS. Cells were lysed in digitonin buffer for 10 min on ice (50 ug/mL digitonin in 75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, and 250 mM sucrose plus protease inhibitor cocktail), centrifuged for 5 minutes at 13,000 rpm and analyzed by Western blot. The following antibodies were used: goat anti-LF (bD-17, Santa Cruz Biotechnology, TX), goat anti-LF (bD-17; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Erk1/2 (Cell Signaling), and rabbit anti-Rab5 (Cell Signaling). In some conditions, 200 nM of pore formation inhibitor bafilomycin a1 or a translocation-deficient PA mutant, PA[F427H], were used as controls. ERK1/2 and Rab5 were used as cytosolic and endosomal markers, respectively. PA[F427H] was expressed in Escherichia coli BL21 (DE3) cells at New England Regional Center of Excellence/Biodefense and Emerging Infectious Diseases (NERCE).

FRET MAPKKide LF activity For RC1 testing in 96-well plates, the reaction volume was 250 μL per well, containing 20 mM HEPES pH 7.2, 5 μM MAPKKide conjugated with DABYL and FITC (List Biological Laboratories, Inc), and 3.3 μM of JHCCL compound. The reaction was initiated by adding LF to a final concentration of 6 μg/mL. Kinetic measurements were obtained at 37ºC every 40 sec for 40 min



using a fluorescent plate reader. Excitation and emission wavelengths were 490 nm and 523 nm, respectively, with a cutoff wavelength of 495 nm. Rates of reactions were quantified by the Microsoft Excel LINEST function and normalized for LF-MAPKKide reaction without RC1.

Cathepsin B Activity Assays CTSB activity in total cell lysates was determined using an InnoZyme ™ CTSB activity assay kit (EMD Milipore) and performed according to the manufacturer’s instruction. Cellular CTSB activity with and without RC1 was tested by pre-treating cells with 33 μM of RC1 for 1 hour, followed by lysing cells and testing CTSB activity with a fluorescently labeled substrate (Arg-Arg peptide labelled with FITC-DABCYL). The activity of 0.5 ng/μL of purified human CTSB (ACRO Biosystems) was mixed with and without RC1 concentrations without pre-incubation and detected with a fluorescently labeled substrate. Fluorescence intensity indicating CTSB activity was measured at an excitation wavelength of 370 nm and emission wavelength of 450 nm (Molecular Devices, Spectra Max 384 PLUS), and analyzed by the Microsoft Excel LINEST function and normalized for the reaction without RC1.

Viral Tests Tests were performed using viral cytopathic effect (CPE) inhibition. Four concentration CPE inhibition assays were performed. Confluent or near-confluent cell culture monolayers in 96-well disposable microplates were prepared. HFF cells were maintained in DMEM supplemented with 10% FBS and 1% PSG. For antiviral assays the same medium is used but with 10% FBS reduced to 2% or less and supplemented with 50 μg/mL gentamicin. RC1 was prepared at four log10 final concentrations, 0.1, 1.0, 10, and 100 μM. Five microwells were used per dilution: three for infected cultures and two for uninfected toxicity cultures. Controls for the experiment consisted of six microwells that were infected (virus controls) and six that were untreated (cell controls). The assay


Page 18 of 37

Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was initiated by first removing growth media from the 96-well plates of cells. Then the test compound was applied in 0.1 mL volume to wells at 2X concentration. Virus, normally at

Characterization of Novel Piperidine-Based ... - ACS Publications

Recommend Documents