Examination of the mode of action of the almiramide family of natural products against the kinetoplastid parasite Trypanosoma brucei

Article abstract of DOI:10.1021/np300834q

Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported small-molecule disruptors of glycosome function, making the almiramides unique molecular probes for this understudied parasite-specific organelle. Additionally, examination of toxicity in an in vivo zebrafish model has shown that these compounds have little effect on organism development, even at high concentrations, and has uncovered a potential side effect through localization of fluorescent derivatives to zebrafish neuromast cells. Combined, these results further our understanding of the potential value of this lead series as development candidates against T. brucei.

Full text of DOI:10.1021/np300834q

Article
pubs.acs.org/jnp
Examination of the Mode of Action of the Almiramide Family of
Natural Products against the Kinetoplastid Parasite Trypanosoma
brucei
Laura M. Sanchez,† Giselle M. Knudsen,‡ Claudia Helbig,§ Geraldine De Muylder,‡
Samantha M. Mascuch,⊥ Zachary B. Mackey,‡ Lena Gerwick,⊥ Christine Clayton,§ James H. McKerrow,‡
and Roger G. Linington*,†
†Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California 95064, United States
‡Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California 94143, United States
§Zentrum fur Molekulare Biologie der Universitat Heidelberg (ZMBH), University of Heidelberg, Heidelberg, Germany D-69120,
̈
̈
⊥Center for Marine Biotechnology and Biomedicine, Scripps Institute of Oceanography, University of California San Diego, San
Diego, California 92093, United States
S
* Supporting Information
ABSTRACT: Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative
agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Trypanosoma brucei, the
causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the
molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These
target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane
assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis
and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported small-
molecule disruptors of glycosome function, making the almiramides unique molecular probes for this understudied parasite-
specific organelle. Additionally, examination of toxicity in an in vivo zebrafish model has shown that these compounds have little
effect on organism development, even at high concentrations, and has uncovered a potential side effect through localization of
fluorescent derivatives to zebrafish neuromast cells. Combined, these results further our understanding of the potential value of
this lead series as development candidates against T. brucei.
uman African trypanosomiasis (HAT) is a neglected
tropical disease (NTD) endemic to sub-Saharan Africa
result of the parasites multiplying by binary fission throughout
the hemolymphatic system. During this transformation, the
host can experience a variety of symptoms, which can include
skin lesions or cardiac, endocrine, or gastrointestinal problems,
causing HAT to be misdiagnosed or, more frequently,
undiagnosed. In stage two, trypomastigotes cross into the
central nervous system (CNS) and cause a wide range of
neurological dysfunctions; the disease gets its name (African
sleeping sickness) due to the fact that patients often experience
H
caused by the parasite Trypanosoma brucei. There are an
estimated 60 million people at risk and 45, 000 deaths a year
from this infectious parasite.1 HAT is transmitted when an
infected tsetse fly takes a blood meal from a host, transmitting
metacyclic trypomastigotes from the salivary gland of the tsetse
fly to the host bloodstream. These injected metacyclic
trypomastigotes then transform into bloodstream trypomasti-
gotes and are dispersed throughout the host circulatory system,
where they go on to cause persistent systemic infection.
The disease manifests itself in two stages; stage one is
typically characterized by nonspecific symptoms that occur as a
Received: November 29, 2012
Published: February 27, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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disturbances in the sleep−wake cycle in the late stage of the
disease.
Currently there are only four licensed drugs to treat the two
stages of HAT (Figure 1), most of which have been in use for
putative mechanisms of action for several of these compound
classes.
Although a number of new treatments are currently under
development, including fexinidazole (5)5,6 and SCYX-7158
(6),3 the drug pipeline for HAT remains comparatively weak.
In order to overcome this issue, there has been a focused effort
to identify trypanosome-specific enzymes or pathways that
could serve as targets for screening campaigns. In particular,
folate metabolism, glycolysis, and protein N-myristoylation
have been identified as promising targets for T. brucei.7 Along
these lines, glycolysis is recognized as a promising target
because it is essential for survival in the bloodstream form of
the parasite and is the sole source of ATP production for the
parasite once it has been transmitted to the human host.
Typically, glycolysis is a cytosolic process in eukaryotes, but
kinetoplastid parasites house this process in an unique organelle
termed the glycosome.8 This unique compartmentalization has
inspired several research programs to pursue targets in this
pathway for potential drug leads.9 However, no compound
from these studies has yet progressed to clinical trials.
We now report the discovery of the first small molecules that
are candidates for disruption of glycosomal function in
trypanosomes. Building upon previous work to develop the
almiramide class of natural products as lead compounds against
Leishmania donovani, we have shown that these compounds are
also active against the related kinetoplastid parasite T. brucei
and have employed a derivatization strategy to generate a suite
of chemical probes for target identification. Using these probes
we have shown that the almiramides bind to two related
glycosomal membrane proteins, GIM5A and PEX11, and have
examined the site localization of fluorescent almiramide
derivatives in fixed procyclic parasites. Finally, we have
examined the vertebrate toxicity of these compounds in
zebrafish using fluorescence site localization and have shown
that these compounds have little effect on development. In
addition, all embryos survived up to 168 μM, the determined
maximum tolerated dose.
Figure 1. Current therapeutics for HAT.
RESULTS AND DISCUSSION
■
more than 50 years.2 Stage one infections are treated with the
polysulfonated napthyl urea suramin (1) and the diamidine
pentamidine (2). Suramin was developed in 1921 and is
strongly negatively charged at physiological pH, precluding
transfer across the blood brain barrier (BBB) into the CNS and
limiting its use to stage one infections. Treatments for stage
two infections include eflornithine (3) and melarsolprol (4).
Eflornithine, the only new option for therapy, was developed in
the 1980s and has been used as combination therapy with
nifurtimox (used to treat Chagas disease) to decrease both daily
dose and treatment duration in the hope of curbing the
development of resistance. Unfortunately, 3 is expensive to
produce and is only effective against T.b. gambiense. The most
commonly administered drug for stage two infections is
melarsoprol, which is an arsenic-based drug, which suffers
from issues of both toxicity and resistance. It is estimated that
between 3 and 10 percent of patients suffer drug-related
mortality due to the effects of melarsoprol.3 While the
molecular targets of these therapeutics remain elusive, our
understanding of the mechanisms of antitrypanosomal drug
action has recently been advanced using a genome-scale RNA
interference target sequencing (RIT-seq) screen.4 This screen
has yielded a broad picture of the metabolic pathways and
proteins affected by the therapeutics and offers insight into the
Creation of Natural Product-Based Chemical Probes.
Almiramides A−C are marine-derived N-methylated lip-
opentapeptide natural products, originally isolated from a
Panamanian collection of Lyngbya majuscula in conjunction
with the Panama International Cooperative Biodiversity Group
(ICBG).10 Almiramides A−C (7−9) and 12 related synthetic
analogues (10−21) have previously been shown to possess
antiparasitic activities against L. donovani, the causative agent of
leishmaniasis. The close structural relationship between L.
donovani and other kinetoplastid parasites motivated us to
extend the scope of this program to include other global health
targets from this class.
In vitro screening against T. b. brucei identified several library
members with activities that were comparable to values
previously obtained for L. donovani (Table 1). Overall, variation
in functional groups at the C-terminus of the amino acid
portion had the largest effect on activity, with compounds 14−
17, which contained carboxylic acid termini, being weakly active
compared to the compounds containing either tertiary amides
(10−13) or methyl esters (18−21). This result suggested that,
while free acids were not well tolerated at this position, it may
be possible to substitute the methyl group in either the amide
or ester series for other alkyl side chains and that this could be a
viable handle for chemical probe derivatization.
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Table 1. Screening Results for Almiramides A−C (7−9) and Related Synthetic Compounds 10−23
By contrast, variations in the alkyl chain length and terminal
moieties at the N-terminus had little effect on the activities of
compounds within each series (e.g., 10−13). While this result
may appear to suggest this as a viable position for compound
derivatization, previous work with L. donovani has shown that
the unsaturated terminus of the alkyl side chain is important for
activity and that compounds with saturated alkyl chain termini
are inactive against L. donovani. We therefore elected to retain
this motif and to develop the almiramide warhead through
derivatization at the C-terminus. To this end, two almiramide−
linker conjugates (22 and 23) were synthesized using standard
solid-phase synthesis methods, followed by solution-phase
permethylation. Both of these linker derivatives retained low
micromolar activities against T. brucei (IC50 = 0.7 and 3.5 μM,
respectively), comparable to the parent molecule at 1.9 μM,
and were therefore deemed suitable for subsequent derivatiza-
tion as chemical probes.
The almiramide series possesses a chemical scaffold that is
distinct from all current and developing therapeutics for HAT.
The flexibility of permitted functional groups at the C-terminus,
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Figure 2. Parallel target identification strategies for almiramide-based affinity purification probes. Orange triangle = biotin; yellow star = photoaffinity
benzophenone moiety; gray sphere = Affi-Gel resin.
submicromolar biological activities, and facile synthetic access
to the core scaffold indicated that this series was well suited to a
target identification study against T. brucei. An affinity capture
study for target identification was therefore initiated against
bloodstream-form trypanosomes, using compound 21 as the
core structure for affinity probe design and synthesis.
Target Identification from Whole Cell Lysates. A dual
approach using either biotinylated or affinity resin-derivatized
almiramide C was pursued for affinity purification of candidate
target proteins in T. brucei.11 Biotinylated probes have been
successfully applied in the activity based protein profiling
(ABPP) targeting active site reactivity,12 such as in the cases of
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the marine natural product didemnin B, recently shown to
target the elongation factor 1-α (EF1-α),13 or the siderophore
petrobactin found to interact with the siderophore-binding
membrane-bound protein ferric petrobactin import A (FpiA).14
Our first approach was therefore to design biotinylated
almiramide derivatives to explore the molecular targets of the
almiramides in T. brucei.
Synthesis of our biotinylated affinity capture probe began
with a commercially available biotin reporter tag containing a
primary amine terminus. This reporter was coupled to the
almiramide moiety (17) using standard coupling reagents
(Supporting Information) to generate biotinylated probe 24
(IC50 = 9.9 μM). This log unit drop in activity is common for
biotinylated probes, due to the significant alterations in
physicochemical properties imparted by the addition of
biotin−linker motifs.15 Briefly, the workflow for protein affinity
capture using biotinylated probe 24 was as follows (Figure 2):
first, biotinylated probe 24 was incubated with the preclarified
whole cell lysate of T. b. brucei at room temperature for two
hours to allow the almiramide moiety to bind with target
protein(s). Second, NeutrAvidin resin was added to capture the
probe−protein complex, and the resin was washed three times
with detergent-free buffer. Finally, SDS-PAGE analysis was
performed on the biotinylated-almiramide−protein−resin
complex, followed by excision, tryptic digest, and LCMS/MS
analysis of bands of interest from the SDS-PAGE gel to identify
candidate target proteins. In order to reduce the abundance of
nonspecific, endogenously biotinylated proteins as well as high
abundance background proteins that may have some resin
affinity,16 whole cell lysates were preclarified by incubation with
NeutrAvidin resin prior to biotin affinity capture. Affinity
capture experiments with biotinylated almiramide 24 were
repeated in four independent biological replicates, and all major
and minor bands from gel lanes for both the negative control
and biotinylated probe 24 affinity capture samples were
examined in all experiments.
Cumulative analysis of the affinity capture results for four
independent replicate experiments using biotinylated almir-
amide 24 identified 38 unique proteins that were present in two
or more replicates. Of these, 24 were significantly enriched in
the affinity capture condition over the control, including a
strong gel band enhancement in the 29 kDa range (Figure 3A).
This band contained both GIM5A (Tbg972.9.6900) and
PEX11 (Tbg972.11.12940), which are integral membrane
proteins found in glycosomes. These two proteins were
consistently identified with high sequence coverage in replicate
biotin affinity capture experiments (Table 2), with strong visual
gel band enhancement in all cases. However, a number of
additional candidate target proteins were also identified in at
least three replicates using this approach (Table 2), precluding
unambiguous assignment of either GIM5A or PEX11 as the
unique target proteins of the almiramides. This result is
common for protein affinity capture experiments, which often
result in the identification of lists of putative target proteins for
further study. GIM5A and PEX11 are normally present in low
titer in whole cell lysates, so the identification of these two
proteins using the biotin affinity strategy suggests that they
have strong affinity for the almiramide probe, either individually
or in complex.
Figure 3. (A) Representative SDS-PAGE gel for protein affinity
capture results. PEX11/GIM5A band enhancement outlined by dashed
box. (B) Venn diagram showing relative number of candidate target
proteins pulled down using each approach. Relative circle areas
correspond to the total number of proteins pulled down using each
approach.
Table 2. Candidate Proteins from All Three Orthogonal
Protein Affinity Capture Approaches
experiment, # of n
replicates
name
TriTryp ID
biotin Affi benzo
glycosomal membrane protein
(PEX11)
Tbg972.11.12940
3
1
2
glycerol kinase, glycosomal
elongation factor 1-alpha
beta tubulin
Tbg972.9.7700
Tbg972.10.2530
Tbg972.1.1350
Tbg972.6.4080
3
3
3
3
2
2
1
1
1
1
1
1
glyceraldehyde 3-phosphate
dehydrogenase, glycosomal
Gim5A protein
Tbg972.9.6900
Tbg972.5.5300
2
2
1
1
2
1
orotidine-5-phosphate
decarboxylase
To address the shortcomings of the biotin reporter tag
approach, we also employed an orthogonal affinity chromato-
graphic method. This approach identifies target proteins by the
covalent attachment of the ligand to the surface of an aqueous-
compatible affinity matrix via a long linker, followed by
incubation of the whole cell lysate with the derivatized affinity
gel and removal of the unbound protein fraction. This approach
does not suffer from the nonspecific enrichment of endogenous
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biotinylated proteins observed with avidin capture, but relies on
the ligand being able to bind to its molecular target(s) while
anchored to the surface of the affinity resin. Affinity matrixes
have been used successfully in the past for parasite lead
compounds to discover the target for 2,4-diaminopyrimidines
against T. brucei as MAPKs and CRKs.16
reactive species is long, which can allow diffusion-mediated
nonspecific covalent bond formation, meaning that photo-
affinity probes are often somewhat more promiscuous than
other methods. Despite this limitation, bifunctional photo-
affinity biotin probes have been used successfully in a number
of recent target identification studies.17,18
Bifunctional biotinylated probe 27 was synthesized under
standard Fmoc solid-phase peptide synthesis (SPPS) conditions
in an analogous fashion to biotinylated probe 24, differing only
in the replacement of the initial phenylalanine residue with a
commercial benzophenone-derived amino acid mimic. The
probe was completed using standard SPPS and methylation
conditions as previously described (Supporting Information)
followed by coupling to a commercially available biotin−linker
conjugate to yield probe 27.
To generate the affinity resin-derivatized almiramide,
compound 17 was first coupled to a 15 Å PEG linker, to
provide sufficient physical separation between the almiramide
reporter and the surface of the affinity gel. Linker−almiramide
conjugate 25 (Figure 2) was then anchored to the surface of an
aqueous-compatible agarose resin (Affi-Gel) using standard
amide coupling conditions (Supporting Information). Although
the Affi-Gel resin has a binding capacity of 15 μmol/mL, the
resin was loaded to just 8 μmol/mL in order to allow the
almiramide warhead sufficient flexibility and steric space to bind
to target proteins. The remaining open positions on the surface
of the resin were capped with 6-aminohexanol, which was
shown in a control experiment not to retain proteins from the
whole cell lysate (Supporting Information). For comparative
analysis, an inactive analogue of almiramide affinity resin 26 was
prepared at the same loading capacity, using a molecule
containing the same methylation pattern and length of
lipophilic terminus as compound 21, but containing a poly
alanine peptide portion in place of the Phe-Ala-Val-Val-Val
almiramide core (Supporting Information).
The workflow for the affinity resin capture experiments was
as follows (Figure 2): the parasite cell lysate was preclarified
with a control resin capped with 6-aminohexanol and incubated
with either almiramide affinity resin 25 or inactive analogue
resin 26. Following incubation, the affinity resins were washed
with detergent-free buffer and analyzed by SDS-PAGE. The
affinity capture was conducted twice (n = 2) and compared
with control resin-captured proteins to include only those
proteins that were present in at least one replicate experiment,
but not present in control lanes. This analysis revealed 12
proteins that were unique to the almiramide-affinity capture
resin. These included both PEX11 and GIM5A, which were
originally identified as candidates in the previous biotin capture
experiments, lending further credence to the hypothesis that
the almiramides target glycosomal function. However, a further
six proteins were also common to both the biotin and Affi-Gel
approaches (Figure 3 and Table 2), which precluded the
identification of a unique target protein using these data alone.
Neither the biotin nor the affinity resin capture methods
reported above were able to address the reversibility of binding
to candidate targets, although competitive binding was
unsuccessful using almiramide addition at either the affinity
capture or the complex elution steps, indicating that almiramide
binding to target proteins must have an extremely slow off rate
if it was not irreversible. An inherent flaw with both affinity gel
and biotin reporters is their reliance on irreversible binding or
slow turnover of the warhead to target protein(s). To force the
equilibrium toward complete binding, a bifunctional probe was
designed that contained both a photoaffinity motif and a biotin
functional group. This was accomplished by replacement of the
phenylalanine residue with a benzophenone substituent (27).
Bifunctional probes can be valuable for the examination of
systems involving noncovalent protein interactions, because
they can simultaneously capture and enrich target proteins with
weak binding that would not be observed under standard biotin
affinity capture conditions. The disadvantage of benzophenone
photoreactive probes is that the lifetime of photoactivated
The workflow for 27 is shown in Figure 2 and closely
resembles the methodology used for biotinylated probe 24,
with the exception of one additional key step. Following
incubation of 27 with the preclarified cell lysate, the
photoaffinity probe was activated by irradiation with UV light
(366 nm, 3 min) to generate a short-lived carbene species (1−2
μs in benzene).19 This highly reactive carbene will rapidly form
a covalent attachment to neighboring proteins, creating a
permanent bond between target proteins and the biotin
reporter tag. The photoaffinity capture experiment was
repeated twice (n = 2) and identified 73 proteins that were
present in at least one replicate, but were not present in the
streptavidin-only control lane. Identification of a larger number
of candidates compared with the biotin-only affinity capture
experiments is typical for photoaffinity experiments with
benzophenone probes; however this list did include a number
of candidate proteins that were common to the other two
strategies used for protein affinity capture in this study.
To integrate the results of all three orthogonal affinity
capture approaches, we sorted the results by frequency of
observation across all methods and ordered this list based on
the frequency of appearance in replicate experiments for each
method (Figure 3 and Table 2). Using this approach seven
candidate proteins were identified that were present in all three
methods. Of these, five are associated with the glycosome, while
the remaining two (β-tubulin and EF1-α) are highly abundant
proteins that are likely not relevant to the mechanism of action
of the almiramides. All of the glycosomal proteins were present
in at least one replicate from all three affinity capture methods
and at least two of three replicates in the biotin affinity capture
experiments.
Glycosomes are globular organelles roughly 0.2−0.3 μM in
diameter, surrounded by a single phospholipid bilayer,20 and
have been found in all kinetoplastid parasites so far examined.
Uniquely, glycosomes compartmentalize the first seven steps of
glucose metabolism and two of glycerol metabolism:21 due to
the unusual regulatory characteristics of the enzymes
concerned, this compartmentation prevents runaway “turbo-
explosion” glycolysis and cell death.22 Other glycosomal
pathways include ether lipid metabolism, the pentose
phosphate pathway, and purine salvage; they vary somewhat
depending on the organism and life-cycle stage.23,24 Each T.
brucei parasite contains roughly 60 glycosomes, which in the
bloodstream form of the parasite represents the sole source of
energy production.20 By contrast, the insect procyclic form of
the parasite is able to utilize more substrates for energy
production, such as the ability to catabolize amino acids.25
Inhibition of glycosomal function has been proposed as an
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Figure 4. Synthetic route to fluorescent almiramide derivatives and control compounds for fluorescence imaging.
attractive target for therapeutic intervention because the target
has no mammalian homologue and is essential for parasite
survival. However, to date no compounds have been identified
that directly disrupt the viability of glycosome structures.26−29
The proteins and signals involved in glycosomal protein import
and membrane biogenesis are clearly related to those of
peroxisomes and glyoxysomes, allowing glycosomes to be
classified as microbodies.30,27 Approximately 32 “PEX” proteins
(or peroxins) are involved in microbody biogenesis: some are
found in all microbodies so far examined, whereas others are
specific to subgroups of organisms. The three most abundant
integral membrane proteins in the glycosomal membrane are
the glycosomal integral membrane proteins GIM5A and
GIM5B, and PEX11.31 All three of these proteins are members
of the PEX11 family, although sequence identity with
mammalian and yeast PEX11 proteins is low. Both GIM5
and PEX11 have been shown to be essential to the bloodstream
form of T. brucei.31,32 GIM5A and GIM5B have also been
shown to form heterodimers in vivo;32 each protein has two
transmembrane domains of 24−32 amino acids, and the
orientation is such that the C- and N-termini, which are much
better conserved within Kinetoplastidae than the trans-
membrane domains, protrude into the cytosol.33 Over-
expression of either GIM5 or PEX11 alters glycosome
morphology and numbers, suggesting roles in maintaining
glycosomal membrane structure. More recent results clearly
indicate that PEX11 family proteins are required for
elongation34 and proliferation35 of peroxisomes, including the
generation of membrane curvature during peroxisomal fission.36
It is also possible that PEX11 proteins might form a pore to
allow transit of low molecular weight metabolites.37 In procyclic
forms, depletion of GIM5A in the presence of GIM5B results in
depletion of ether lipids,32 supporting a specific role of GIM5A
for this particular pathway.
Remarkably, all five candidate target proteins identified in the
protein pull-down experiments are glycosome-associated:
GIM5A, PEX11, and three matrix enzymes, GAPDH, glycerol
kinase, and orotidine-5-phosphate decarboxylase.31 This is
strongly indicative of glycosomal function as the target of the
almiramides, and we speculated that they may target glycosome
biogenesis by disrupting the functions of GIM5 and PEX11. To
test this hypothesis, we explored a number of approaches to
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validate this mechanism of almiramide function. We stained
almiramide-treated trypanosomes with anti-aldolase antibody,
but it rapidly became clear that all drug concentrations that
affected growth caused such drastic morphological alterations
that there was no chance of detecting a specific effect on
glycosomes.
Next, we created a suite of fluorescent probes for imaging.
The BODIPY fluorophore was selected because BODIPY’s
spectral properties surpass those of fluorescein, tetramethylr-
hodamine, Texas Red, and longer-wavelength dyes,38 and
BODIPY dyes are relatively nonpolar with an electrically
neutral chromophore. Together, these properties minimize dye-
induced perturbations of parent molecule properties and reduce
the biological impact of introducing this chromophore to test
compounds.39 The synthesis of these probes was accomplished
by extension of the methodology described above. Briefly,
almiramide−linker compound 22 was deprotected with LiOH
to give the corresponding free acid 28 (Figure 4) and coupled
to commercially available BODIPY FL EDA (32) under
standard coupling conditions to give fluorescent derivative 33.
Nonfluorescent control compound 31 was synthesized using a
similar approach by coupling 28 to N-(3-phenylpropionyl)-1,2-
ethanediamine 30. Finally, a BODIPY control compound
lacking the almiramide warhead was prepared by coupling
BODIPY FL EDA (32) to commercially available hydro-
cinnamic acid (34) under standard coupling conditions (Figure
4).
Localization of Fluorescent Almiramides in Trypano-
somes. As a prelude to fluorescence imaging, all compounds
were screened for activity against T. b. brucei. The BODIPY
control 36 was inactive against the parasite up to the highest
tested concentration (50 μM), while fluorescent almiramide
derivative 33 retained an activity of 3.1 μM, indicating both that
the fluorophore possessed no intrinsic activity against the
parasite and that derivatization of the almiramide moiety with
the fluorescent tag did not eliminate its biological activity. To
visualize the localization of the almiramide derivative within the
parasite, we tested a multitude of different conditions. After
brief in vivo labeling with high drug amounts, live parasites were
fluorescent, but the signal was lost following fixation, which is
necessary to detect subcellular structures. In vivo labeling with
lower amounts, for longer times, was problematic because of
toxic effects, and the drug did not bind to anything in
formaldehyde-fixed preparations. The only successful protocol
involved EtOH fixation of dried procyclic trypanosomes,
followed by permeabilization using Tx100 and incubation
with either 5 or 50 μM 33. DAPI (nuclear stain) and anti-
aldolase (glycosome stain) were used for co-localization. Figure
5A shows that compound 33 (5 μM) was distributed
throughout the cytosol with localized brighter regions.
Compound 33 did not localize to the nucleus, nor to the
kinetoplast DNA, which can be seen as a small bright region
below the nucleus in the DAPI images. At the higher
concentration (50 μM, Figure 5B) the almiramide probe
localized preferentially to some non-glycosome structures that
lay in the parasite posterior, between the nucleus and flagellar
pocket. This location is suggestive of structures of the secretory
pathway, but we have not tested this in more detail. Glycosomal
membranes are, like those of peroxisomes, probably initially
formed by the ER, and some PEX11 family proteins are
involved.35 A preferential association of almiramide with a
minor portion of GIM5 or PEX11, within the ER membrane,
therefore cannot be ruled out. Overall, however, the results of
Figure 5. Fluorescence site localization results for BODIPY-labeled
almiramide probe 33. (A) Uptake of probe 33 at 5 μM and
distribution of staining with respect to the nucleus and kinetoplast
(large and small staining regions, respectively, stained in DAPI
channel). (B) Distribution of probe 33 with respect to glycosome
distributions at 50 μM. Glycosomes stained with red anti-aldolase
antibody.
these experiments were inconclusive, since it is possible that
under the conditions used the almiramide derivative was
binding in a nonspecific fashion.
Genetic Validation of Protein Targets. Unfortunately,
previous attempts to express GIM5A and GIM5B in Escherichia
coli have proved unsuccessful,26 precluding in vitro binding
experiments. To complement the results from the pull-down,
we therefore attempted to test the effect of the almiramides on
GIM5 function in vivo. If the almiramides target GIM5,
overexpression of GIM5 ought to confer almiramide resistance.
Overexpression of GIM5 or PEX11 in procyclic trypanosomes
is, however, toxic by itself.31,33 We transfected bloodstream-
form trypanosomes with a vector containing a tetracycline-
inducible GIM5B gene,27 hoping that perhaps we could counter
this toxicity using the almiramides. Several clones were
obtained, but in these forms, no overexpression could be
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detected by Western blotting (data not shown), and no effect
on almiramide toxicity was observed. It is possible that GIM5B
expression is already too high for additional expression to have
any impact. Alternatively, it is possible that even “leaky”
expression of extra GIM5B, in the absence of inducer, is lethal
in bloodstream forms, precluding selection of appropriate
clones. Although the fact that both over- and underexpression
of GIM5 and PEX11 severely inhibit parasite growth makes
these proteins attractive potential drug targets, it also represents
a serious technical limitation on genetic approaches to verify
them as the targets of potential inhibitors.
Zebrafish Toxicity and Site Localization Imaging.
Current frontline therapeutics for HAT are antiquated and
suffer from issues of toxicity and development of resistance.
The almiramides and their analogues represent a unique
chemical scaffold for the treatment of HAT that is distinct from
any of the current therapeutic options. However, to be of value
as candidates for further development, a large number of
potential liabilities must be examined, including toxicity in
whole animal models. Zebrafish are gaining prominence for
exploring ADMETox properties of early stage leads in hit-to-
lead drug development projects40 because of their straightfor-
ward husbandry requirements, their small size, which allows
testing with small amounts of test compounds, and their ability
to report on a wide variety of developmental and neurological
characteristics.
Both the unlabeled compound 21 and fluorescent derivative
38 were tested against zebrafish with the objectives of
identifying toxic side effects of the compound series and
determining whether the compounds localized to any organs or
specific regions of the fish. To this end, compound 21 was
incubated with zebrafish embryos (48 h postfertilization) for 24
h, and embryo viability was then assessed to establish the
maximum tolerated dose (MTD). After 24 h of incubation with
21, the MTD was 168 μM, giving a wide therapeutic index
considering that this compound series was active against T. b.
brucei in the low micromolar range. These doses also agreed
well with the previously reported LD50 values against Vero
cells.10
Figure 6. Fluorescence imaging for (A) control compound 37 and (B)
almiramide probe 38 in juvenile zebrafish. Images display lateral region
at the intersection of the head and the upper spine. Zebrafish eye
visible as a large dark region in the center-left of each image. The
fluorescent staining indicates binding of almiramide to neuromast cells.
These data are also presented as a Z-stack video in the Supporting
Information (Video S1).
cin, and gentamycin.41,42 To test the affinity of probe 38 for
neuromasts in zebrafish, we examined the effect of pretreatment
with neomycin on neuromast staining. From this study
embryos that were untreated with neomycin, but treated with
250 μM 38 displayed the blue rosette patterning along the
lateral line. Embryos that were untreated with neomycin, but
treated with fluorescent control compound 37 displayed no
rosette patterning, and embryos that were pretreated with
neomycin (500 μM) followed by treatment with 250 μM 38
displayed no fluorescent uptake. These results indicate that 38
has affinity for neuromasts at high concentrations. Additional
experimentation is necessary to determine whether this uptake
is specific to 38 or the nonspecific result of the exposure of
these cells to the external environment, as the high probe
concentration might suggest. Regardless, the almiramides
should be closely monitored for potential ototoxicity as the
scaffold is further developed. If such a side effect is observed,
ototoxicity can be prevented by blocking the c-Jun-N-terminal
kinase (JNK) with the peptide D-JKNI-1.43
Using these results to determine appropriate concentrations
for imaging probes, we tested two different almiramide−
fluorophore conjugates for fluorescence staining and site
localization. Imaging using 48 h postfertilization (hpf) embryos
revealed high levels of fluorescence in rosette structures along
the lateral line of the zebrafish. Changing the protocol to use
120 hpf embryos revealed strong fluorescence in the intestinal
tract of the zebrafish, likely the result of fish “gulping” water
during compound treatment. With fluorescent probe 33 no
fluorescence was observed after 3 h of exposure. However at
high doses of probe 38 (250 and 500 μM), bright blue rosettes
were observed along the lateral line of the fish (Figure 6). This
patterning was not observed with the other fluorescent
analogue or with fluorophore control compounds alone.
From the structure and patterning of the observed fluorescence,
it appeared that 38 may have affinity for zebrafish neuromasts
at very high doses. Neuromasts are sensory organs located on
the epithelium of the skin of the fish and are composed of
different cell types, including hair cells, which allow the fish to
sense water vibrations. In humans, neuromasts are analogous to
the hair cells found in the cochlea, which is located in the inner
ear. The hair cells play a role in auditory function. Human hair
cells and neuromasts have a well-documented sensitivity to the
amino glycoside antibiotics, for example, neomycin, tobramy-
The relatively high concentration of 21 at which toxicity is
observed in zebrafish embryos is in stark contrast to the low
concentrations necessary to cause T. b. brucei death. This
differential activity bodes well for the development of the
almiramides as antiparisitics. However, evaluation in higher
vertebrates will be necessary to ensure that toxic effects to the
host from these molecules remain minimal.
In conclusion, this work represents the discovery and
characterization of the first example of small molecules that
may disrupt glycosomal function in T. brucei. The combined
results from the affinity capture experiments and fluorescent
microscopy experiments strongly support the conclusion that
the almiramides target the glycosomal membrane. Although we
were unable to confirm this by immunofluorescent staining or
reverse genetics, both approaches suffered from serious
technical limitations, rendering the results inconclusive.
Glycosomes are an encouraging potential target for kinetoplas-
tid drug discovery because glycolysis is not compartmentalized
in mammalian cells, making this target parasite-specific. More
work is still needed to uncover the precise molecular target of
the almiramides, but given that GIM5A and GIM5B have not
been successfully expressed, and in vitro binding experiments
are therefore precluded, alternative methods for target
identification will be required. Along with GIM5A, PEX11 is
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the different affinity approaches. For the biotinylated capture, 20 μM
of compound 24 dissolved in DMSO was added to the preclarified
lysate and inverted at rt for 2 h. NeutrAvidin beads were prepared as
follows: 400 μL of slurry was transferred to a 15 mL Falcon tube, and
10 mL of cold detergent-free buffer (50 mM HEPES, pH 7.4, 100 mM
NaCl, 1 mM EDTA, 1 mM EGTA, Halt protease inhibitor cocktail)
was added to wash the resin, followed by centrifugation for 3 min at
2500 rpm to pellet. The wash was removed, the pellet resuspended
with 10 mL of cold detergent-free buffer, and the process repeated.
Upon completion of the affinity binding step, the lysate−24 mixture
was transferred to a 1.5 mL Eppendorf tube containing the prepared
NeutrAvidin beads and inverted for 2 h at rt to capture the protein−
compound complexes. Following affinity capture, the resin was
centrifuged, and the supernatant was removed. The resin−protein−
24 complex was carefully washed with 1 mL of cold detergent-free
buffer and spun down, the wash removed, the washing protocol
repeated, and all liquid removed from the resin. Samples were stored
overnight at −20 °C until gel analysis could be performed.
For the biotinylated-benzophenone affinity chromatography (27),
the same procedure was utilized, with the addition of UV exposure to
induce cross-linking. At the end of incubation of 20 μM 27 with the
preclarified lysate, the sample was irradiated with a UV lamp for 3 min
to induce cross-linking.46 Then the capture procedure was performed
as described above.
For the Affi-Gel-10 affinity chromatography, the preclarified lysate
was divided equally and incubated with either 26 or 25 for 5 h at 4 °C
to capture the protein−compound complexes. The Affi-Gel samples
were then washed and prepared for gel analysis as previously
described.
Protein Identification by Mass Spectrometry. Protein identifica-
tion from affinity-purified samples was performed using peptide
sequencing by mass spectrometry. Affinity-purified samples were
eluted from capture resin, then resolved by SDS-PAGE using 10%
Tris-glycine gels (Bio-Rad) and stained by silver stain.47 Gel bands
were excised and subjected to in-gel trypsin digestion using standard
protocols (in-gel digestion protocol, UCSF). Extracted peptides were
sequenced using a QStar Pulsar quadrupole-orthogonal-acceleration-
time-of-flight hybrid mass spectrometer (Applied Biosystems),
coupled to an Ultimate HPLC with Famosautoinjector (LC Packings),
and a self-packed C18 column (New Objective Inc., 5 μm bead size,
100 μm × 150 mm). The LC was operated at 350 nL/min flow rate,
and peptides were separated using a linear gradient over 42 min from
5% to 50% ACN in 0.1% formic acid. Data were acquired in
information-dependent acquisition mode; 1 s survey scans over the
310−1400 m/z range were followed by 3 s CID fragmentation spectra
over a 60−1400 m/z range.
Data were analyzed with Analyst 2.0 software (Applied Biosystems)
with the Mascot script 1.6b20 (Matrix Science). Analyst processing
options for peak finding in a spectrum were 0.5% default threshold,
400 gain filter, and a Gaussian filter limit of 10; for TOF
autocentroiding: 20 ppm merge distance, 10 ppm minimum width,
50% percentage height, and 100 ppm maximum width. Default
parameters were used except that “no deisotoping” was selected and
precursor mass tolerance for grouping was set to 0.2. Database
searches were performed using ProteinProspector v. 5.10.1 (http://
prospector.ucsf.edu) using the Batch-Tag and Search Compare
modules.48 Searches were performed on the SwissProt databank,
downloaded March 21, 2012, and containing 535,248 entries, as well
as the T. brucei sequences in TriTrypDB v. 4.1 (http://www.tritrypdb.
org), downloaded April 23, 2012, and containing 28,250 entries. For
estimation of false discovery rate, these databases were concatenated
with a fully randomized set of entries.49 Data were searched with a
parent mass tolerance of 200 ppm and fragment mass tolerance of 300
ppm.
involved in glycosomal biogenesis and proper fission of
budding glycosomes. Affinity capture of PEX11 and GIM5A
is therefore consistent with the almiramides affecting
glycosomal biogenesis. The almiramides represent a promising
molecular tool with which to further probe glycosomal function
during parasite development. Given that the almiramides
display low vertebrate toxicity, they are encouraging candidates
for further lead development. This scaffold is synthetically
tractable, derivatizable, and amenable to large-scale production
for evaluation in murine parasitemia models. In conclusion the
almiramide scaffold represents the first potential molecular
probe for the disruption of glycosomal membranes in T. brucei
and offers a new avenue for future drug development against
African sleeping sickness.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Jasco P-2000 polarimeter using a 10 mm path length
cell at 589 nm. UV spectra were acquired on an Agilent 8453 UV−vis
spectrophotometer. NMR spectra were acquired on a Varian Inova
600 MHz spectrometer equipped with a 5 mm HCN triple resonance
cryoprobe and referenced to residual solvent proton and carbon
signals (δH 7.26, δC 77.1 for CDCl3 and δH 1.94, δC 1.4 for CD3CN).
Unless otherwise stated, reactions were performed under an argon
atmosphere using freshly dried solvents. Methylene chloride (DCM)
was dried by passing through an activated alumina column. Solvents
used for HPLC chromatography were HPLC grade and were used
without further purification.
Trypanosomes. Trypanosoma brucei brucei strain 427-221 was
grown at 37 °C, 5% CO2 in HMI-9 medium containing 10% fetal
bovine serum, 10% Serum Plus (JRH Inc.), and penicillin/
streptomycin.
Trypanosome Screening Assay. T. b. brucei 427-221 cells were
diluted to 2 × 104 per mL in complete HMI-9 medium and aliquoted
in Greiner sterile 384-well flat white opaque culture plates using a
WellMate cell dispenser from Matrix Tech. Test compounds were
serially diluted in dimethyl sulfoxide (DMSO) and added to the assay
plates with the robotic dispenser BiomekFXp liquid handler (Beckman
Coulter). Thimerosal (2 μM final concentration) was added as a
positive control, and DMSO as a negative control (1% final
concentration). Trypanosomes were incubated with compounds for
48 h at 37 °C with 5% CO2 before monitoring cell viability as
previously described.44 Briefly, the trypanosomes were lysed in the
wells by adding 25 μL of CellTiter-Glo (Promega), and the lysed
trypanosomes placed on an orbital shaker at room temperature (rt) for
2 min. After lysis, the resulting ATP-bioluminescence was measured at
rt using an Analyst HT plate reader (Molecular Devices). Percentage
inhibition of parasite growth was calculated for each well as [1 −
(RLUx − RLU+)/(RLU− − RLU+)] × 100, where RLUx, RLU+, and
RLU− are respectively the relative light units for each well and positive
(thimerosal) and negative (DMSO) controls. A screening window
coefficient, denoted Z′ factor, was used to evaluate the performance of
the assay. The Z′ factor, calculated as 1 − (3σc+ + 3σc−)/(μc+ − μc−),
where σc+, σc−, μc+, and μc− are respectively the standard deviation and
mean values of positive and negative controls, is reflective of the assay
signal dynamic range and the data variation associated with signal
measurement.45 IC50’s were calculated using Prism (GraphPad).
Target Protein Affinity Capture. Preparation of Trypanosome
Lysate. Cells were resuspended (approximately 200 μL of 100 million
cells pellet−1) in a buffer containing 50 mM HEPES (pH 7.4), 100
mM NaCl, 0.5% NP40, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
and 1% Tween20, supplemented with Halt protease inhibitor cocktail
(ThermoFisher Scientific), and lysed using a Dounce homogenizer,
with pestle B, 70 strokes. DNA was pelleted by centrifugation.
Affinity Chromatography. The cleared lysate was incubated with
either control Affi-Gel resin or NeutrAvidin resin for 1 h at 4 °C to
remove endogenously biotinylated proteins. The lysate was then
divided equally between Falcon tubes to provide replicate material for
For database searching, peptide sequences were matched as tryptic
peptides with no missed cleavages and carbamidomethylated cysteines
as a fixed modification. Variable modifications included oxidation of
methionine, N-terminal pyroglutamate from glutamine, loss of
methionine, and N-terminal acetylation. For reporting of protein
identifications from this database search, score thresholds were
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selected that resulted in a protein false discovery rate of approximately
1%. The specific Protein Prospector parameters were as follows:
minimum protein score of 22, minimum peptide score of 15, and
maximum expectation values of 0.02 for protein and 0.05 for peptide
matches. Protein identification results from specific affinity purification
experiments are reported with a spectral count as an approximation of
protein abundance, along with percent sequence coverage and an
expectation value for the probability of the protein identification.50,51
Trypanosome Fluorescence Imaging. About 2 × 106 procyclic
trypanosomes were resuspend in 2 μL of modified Eagle’s medium for
procyclics (MEM-PROS) medium supplemented with 10% fetal calf
serum,52 spread on a glass slide, allowed to dry, and then fixed with
70% EtOH. The cells were rehydrated in 1× PBS/0.2% Triton for 20
min, washed three times with PBS, and incubated for 20 min with
PBS/0.5% gelatin and then with anti-aldolase (1 h, 1:500) in the same
PBS−gelatin mix. After two washes with PBS, the cells were incubated
for 1 h with 5 μM 33 or 36, plus 1:500 Alexa 594 α-rabbit and 100 ng/
mL DAPI. The stained spreads were washed three times with 1× PBS,
dried, then mounted in 90% glycerol/10% 10× PBS, and imaged with
a Leica DMRXA microscope at 100× oil magnification.
Zebrafish Studies. Animals. Adult zebrafish (Danio rerio)
obtained from Aquatica Tropicals were maintained at 27 2 °C in
plastic recirculating aquaria (Aquaneering Inc.) on a 12 h light:12 h
dark cycle. Fish were fed two to three times daily with a flake mix (57%
Aquatox Flake, 19% Spirulina Flake, 8% Hikari Micropellet, 8%
Cyclop-eeze, 4% Golden Pearl 300−500, and 4% Golden Pearl 500−
800) and live brine shrimp. All animal experimentation was conducted
according to NIH and University of California, San Diego, guidelines
under IACUC-approved protocol S05560.
Breeding and Embryo Collection. Two adult male and two adult
female zebrafish were introduced into a two-way fish breeder
(Petsmart, Inc.) without tank flow the evening prior to breeding and
separated by a plastic divider overnight. The divider was removed the
following morning, and after 1 h embryos were transferred to Petri
dishes containing egg water medium consisting of filtered water
supplemented with 60 μg/mL Instant Ocean salts and 0.001%
methylene blue.53
Almiramide Toxicity. Embryos at 48 and 120 h postfertilization
were used to determine the maximum tolerated dose of almiramide. A
10 mM stock of 22 in DMSO was diluted to a concentration of 500
μM in egg water lacking methylene blue. Twofold serial dilutions were
performed in a 96-well plate, and final concentrations tested were 500,
250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, and 0 μM. Two embryos
were exposed to each concentration. Survival, defined as the presence
of a heartbeat, was assessed using an inverted microscope at 3 and 24 h
following compound addition. Survival of 22-exposed animals was
compared to animals exposed to the benzopyrone coumarin prepared
in an identical manner.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 831-459-3014. E-mail: rliningt@ucsc.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank K. Sharaf for assistance with synthetic preparation of
some starting materials, A. Hamdoun for access to confocal
microscope facilities, and J. Campanale for technical assistance.
Mass spectrometry was performed in the UCSF MS facility (A.
L. Burlingame, Director) supported by 8P41GM103481. This
work was funded by the International Cooperative Biodiversity
Group (ICBG) program in Panama (Grant ICBG TW006634,
R.G.L. and L.G.), NIH1R56AI085177-01A1 (R.G.L.), the
Sandler Family Foundation (J.H.M.), and an NSF graduate
research fellowship (L.M.S.).
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S
* Supporting Information
1
Experimental procedures and H and 13C NMR spectra for all
synthetic compounds. Z-stack video of rosette staining pattern
in zebrafish and spectral count files for all protein
identifications. This material is available free of charge via the
Internet at http://pubs.acs.org.
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