Defining the Determinants of Specificity of Plasmodium Proteasome Inhibitors

Article abstract of DOI:10.1021/jacs.8b06656

The Plasmodium proteasome is an emerging antimalarial target due to its essential role in all the major life cycle stages of the parasite and its contribution to the establishment of resistance to artemisinin (ART)-based therapies. However, because of a similarly essential role for the host proteasome, the key property of any antiproteasome therapeutic is selectivity. Several parasite-specific proteasome inhibitors have recently been reported, however, their selectivity must be improved to enable clinical development. Here we describe screening of diverse libraries of non-natural synthetic fluorogenic substrates to identify determinants at multiple positions on the substrate that produce enhanced selectivity. We find that selection of an optimal electrophilic "warhead" is essential to enable high selectivity that is driven by the peptide binding elements on the inhibitor. We also find that host cell toxicity is dictated by the extent of coinhibition of the human β2 and β5 subunits. Using this information, we identify compounds with over 3 orders of magnitude selectivity for the parasite enzyme. Optimization of the pharmacological properties resulted in molecules that retained high potency and selectivity, were soluble, sufficiently metabolically stable and orally bioavailable. These molecules are highly synergistic with ART and can clear parasites in a mouse model of infection, making them promising leads as antimalarial drugs.

Full text of DOI:10.1021/jacs.8b06656

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Article
Defining the determinants of specificity of Plasmodium proteasome inhibitors
Euna Yoo, Barbara H. Stokes, Hanna de Jong, Manu Vanaerschot, TRS Kumar,
Nina Lawrence, Mathew Njoroge, Arnold Garcia, Renier van der Westhuyzen,
Jeremiah D Momper, Caroline L Ng, David A. Fidock, and Matthew Bogyo
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06656 • Publication Date (Web): 15 Aug 2018
Downloaded from http://pubs.acs.org on August 15, 2018
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Page 1 of 16
Journal of the American Chemical Society
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Defining the determinants of specificity of Plasmodium proteasome
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Euna Yoo, Barbara H. Stokes, Hanna de Jong, Manu Vanaerschot, TRS Kumar, Nina Lawrence,
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Mathew Njoroge, Arnold Garcia, Renier Van der Westhuyzen, Jeremiah D. Momper, Caroline L.
2,5
2,6
1,7,
Ng, David A. Fidock, Matthew Bogyo *
1
7
Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, Stanford, Califor-
nia 94305, USA
2
6
Department of Microbiology and Immunology and Division of Infectious Diseases, Columbia University Medical Center,
New York 10032, USA
3
Drug Discovery and Development Centre (H3D), University of Cape Town, Rondebosch 7701, South Africa
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093,
4
USA
5
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
ABSTRACT: The Plasmodium proteasome is an emerging antimalarial target due to its essential role in all the major life cycle
stages of the parasite and its contribution to the establishment of resistance to artemisinin (ART)ꢀbased therapies. However, because
of a similarly essential role for the host proteasome, the key property of any antiꢀproteasome therapeutic is selectivity. Several paraꢀ
siteꢀspecific proteasome inhibitors have recently been reported, however, their selectivity must be improved to enable clinical deꢀ
velopment. Here we describe screening of diverse libraries of nonꢀnatural synthetic fluorogenic substrates to identify determinants
at multiple positions on the substrate that produce enhanced selectivity. We find that selection of an optimal electrophilic ‘warhead’
is essential to enable high selectivity that is driven by the peptide binding elements on the inhibitor. We also find that host cell toxꢀ
icity is dictated by the extent of coꢀinhibition of the human β2 and β5 subunits. Using this information, we identify compounds with
over three orders of magnitude selectivity for the parasite enzyme. Optimization of the pharmacological properties resulted in molꢀ
ecules that retained high potency and selectivity, were soluble, sufficiently metabolically stable and orally bioavailable. These molꢀ
ecules are highly synergistic with ART and clear parasites in a mouse model of infection, making them promising leads as antiꢀ
malarial drugs.
INTRODUCTION
human counterpart. Understanding differences between the
parasite and human proteasome ligandꢀbinding preferences is
therefore of paramount importance. We recently used a screen
of natural peptide substrates coupled with high resolution
cryoꢀelectron microscopy (cryoꢀEM) and single particle analyꢀ
sis to identify a first generation of covalent irreversible inꢀ
hibitors of P. falciparum proteasome that are over 100 times
more potent for the parasite relative to the human
proteasome . Our original lead vinyl sulfone molecule, WLLꢀ
vs (Figure 1) also showed two orders of magnitude selectivity
for killing parasites compared to primary human cells, sugꢀ
gesting it has a relatively good ‘therapeutic window’ where it
could be dosed without toxicity to the host. In addition, a reꢀ
cent study has described a highly selective and potent inhibitor
of the Plasmodium proteasome based on the epoxyketoneꢀ
based carmaphycin B scaffold that was originally isolated
Plasmodium falciparum (Pf) is a protozoan parasite that
causes the most severe form of malaria in humans. P. falcipa-
rum malaria remains a significant burden, leading to approxiꢀ
mately 400,000 deaths annually with most being children unꢀ
15
16
1
der the age of five . Emerging resistance to the highly effecꢀ
tive drug artemisinin (ART) and the partner drugs used in
ARTꢀbased combination therapies (ACTs) is particularly worꢀ
risome as these drugs are used worldwide as firstꢀline therapy.
Many of the older drugs have already been compromised by
13
2
ꢀ6
the emergence and spread of resistance . Therefore, there is
an urgent need for new drugs with novel mechanism of action
and broad therapeutic potential, not only to widen the scope of
treatment, but also to overcome existing multidrug resistance.
The Plasmodium proteasome has been validated as a promꢀ
ising target for antiꢀmalarial drugs because inhibitors of this
essential enzyme attenuate parasite growth at all stages of its
17
from extracts derived from cyanobacterium Symploca sp
7ꢀ12
(Figure 1). This analog containing a Dꢀamino acid at the P3
position shows similar potency and overall selectivity as the
WLL vinyl sulfone but was not tested in mouse models of
infection. However, while both classes of reported selective
inhibitors effectively kill parasites at low nanomolar concenꢀ
life cycle . More importantly, recent work has shown that
proteasome inhibitors have a high degree of synergism when
13ꢀ14
combined with ART . However, the main challenge for the
development of proteasome inhibitors as antiꢀmalarial agents
is optimization of selectivity for the parasite enzyme over its
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trations, both retain micromolar potency for the host proꢀ identical in structure to the original WLLꢀvs inhibitor except
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teasome, suggesting that additional optimization will be reꢀ
quired to avoid longꢀterm toxicity issues. In addition, due to
the unique disease epidemiology of malaria, there is a need to
optimize lead drug candidates for sufficient stability, blood
exposure levels and oral uptake. These are all criteria that have
not been addressed for any of the currently published Plasmo-
diumꢀspecific proteasome inhibitors.
for the residue in the P3 position, which was varied through a
series of commercially available bulky aromatic nonꢀnatural
amino acids (compounds 1ꢀ6, Table 1). To assess the potency
and selectivity of the compounds, we treated ring stage P.
falciparum W2 infected red blood cell cultures with comꢀ
pounds either for 1 h followed by washout (1 h EC ) or treatꢀ
5
0
ed with no wash out, followed by measurement of the parasite
viability 72 hours later (72 h EC ). We also performed the
50
same treatment on nonꢀconfluent, replicating human foreskin
fibroblast (HFF) cells to assess overall host cell toxicity. We
chose HFF because they are primary cells that are likely to be
more sensitive to toxicity of compounds compared to immorꢀ
talized cell lines that are often used to assess host cell toxicity
(i.e. HepG2 cells). We found that small substitutions on the
para position of the aromatic ring of phenylalanine (methyl
and fluoro) dramatically reduced potency and selectivity. On
the other hand, a compound containing the large 1ꢀ
naphthylalanine (compound 1) exhibited comparable activity
to the parent WLLꢀvs (EC = 10 nM) and had slightly imꢀ
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Figure 1. Chemical structures of the Plasmodiumꢀselective
proteasome inhibitors WLLꢀvs and the recently reported Carꢀ
maphycin B analog. EC50 values and selectivity index values
are shown for each compound. HFF= human foreskin fibroꢀ
blasts, EC = half maximal effective concentration
50
proved selectivity. Interestingly, the 2ꢀnaphthyl group caused
a dropꢀin potency (compound 2; EC = 90 nM), which also
50
50
translated into a lower overall selectivity. The homophenylalꢀ
anine (Hfe) substitution (compound 3), however, resulted in
substantially increased potency (EC = 3 nM) coupled with a
This study reports our efforts to define the determinants of
specificity of Plasmodiumꢀspecific proteasome inhibitors and
use this information to further enhance the specificity of lead
molecules and tune their pharmacological properties. We acꢀ
complished this goal by synthesizing and screening a library
of fluorogenic peptide substrates that contain a range of diꢀ
verse nonꢀnatural amino acids selected to be optimal for bindꢀ
50
fiveꢀfold increase in selectivity (therapeutic index of 570)
compared to WLLꢀvs and carmaphycin B analog (Table 1).
We also made a version of compound 3 in which we replaced
the P2 leucine with a methylserine (Ser(Me); compound 7) in
order to enhance solubility and metabolic stability. This nonꢀ
natural residue was chosen based on its use in orally available
18
ing based on our prior highꢀresolution electron microscopy
inhibitors of the human proteasome . Compound 7 showed
16
(
EM) structure of the P. falciparum proteasome . Using this
slightly increased overall selectivity compared to 3 but it lost
potency in parasite killing (Table 1). Finally, our prior EM
structure suggested that the S1 pocket on the β2 and β5 subuꢀ
nits of the parasite proteasome could accommodate larger resꢀ
idues than the human enzyme. As a result, we also synthesized
biased substrate library, we identified nonꢀnatural amino acid
residues in multiple positions on the substrate that could be
used to build a set of secondꢀgeneration vinyl sulfone inhibiꢀ
tors with substantially improved selectivity over the original
WLLꢀvs lead molecule. This selectivity for parasites was priꢀ
marily derived from reduced activity against the human β2
subunit even after prolonged exposure times. We also examꢀ
ined the role of the reactive electrophile in controlling inhibiꢀ
tor specificity. These studies demonstrate that specificity gains
resulting from selection of optimal peptide sequences in the
backbone were lost when using the more reactive epoxyketone
and boronate ‘warheads’ which retained significant reactivity
towards the human β2 subunit resulting in host cell toxicity.
Finally, optimization of solubility and microsome stability
resulted in a lead molecule with favorable in vivo properties
and some oral bioavailability. This lead molecule showed
strong synergy with ART and was able to clear parasites in a
mouse model of infection, making it a promising lead for use
in optimizing oral formulations and progressing to IND enaꢀ
bling studies.
compounds 8ꢀ10 (Table 1) that contained
a
paraꢀ
methylphenylalanine residue in the P1 position. These comꢀ
pounds, while highly potent for the parasite enzyme, also beꢀ
came more potent for the human enzyme resulting in an overꢀ
all drop in selectivity. We therefore chose to focus on the scafꢀ
fold from compound 7 for further optimization.
One of the most commonly used tools to systematically deꢀ
fine the specificity of proteases makes use of libraries of fluoꢀ
19ꢀ20
rogenic substrates . Screening can be accomplished using
individual substrates assayed in parallel or through the use of
positional scanning substrate combinatorial libraries (PSꢀ
SCLs) that contain a mixture of peptides where one or more of
the positions are individually fixed while the remaining posiꢀ
21ꢀ23
tions are randomized . These types of substrate libraries
have also been used to identify optimal binding groups that
can then be converted into inhibitors by replacing the reporter
fluorophore with a reactive electrophile in an approach termed
24ꢀ26
substrate activity screening (SAS) . Furthermore, the use of
RESULTS
nonꢀnatural amino acids in PSꢀSCLs has been shown to result
in highly selective substrates and subsequent inhibitors of varꢀ
Identification of non-natural amino acids that enhance
selectivity for the Plasmodium proteasome. Our previous
studies of the P. falciparum proteasome demonstrated that
bulky aromatic residues in the P3 position of a substrate proꢀ
vide a key determinant for driving selectivity towards the parꢀ
27ꢀ29
ious protease targets . We therefore decided to use substrate
libraries to more systematically screen for optimal residues
that could be translated into inhibitors that have enhanced
selectivity for the parasite proteasome. We synthesized a liꢀ
brary of fluorogenic substrates with the 7ꢀaminoꢀ4ꢀ
1
3
asite enzyme and away from the human enzyme . We thereꢀ
fore synthesized a set of 6 tripeptide vinyl sulfones that were
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resin to facilitate rapid and highly parallel synthesis using
carbamoylmethylcoumarin (ACC) reporter group attached to a
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6
Table 1. Potency and selectivity of P1 and P3 bulky aromatic VS inhibitors. EC50 values for each of the inhibitors 1−10 and the
Carmaphycin B analog in 72 h and 1 h treatment of P. falciparum W2 strain at ring stage and nonꢀconfluent HFFs.
72 h EC50 (ꢀM)
1 h EC50 (ꢀM)
No.
Cap
R
3
R
2
R
1
Pf
HFF
SI
Pf
HFF
SI
WLL
0.015
± 0.005
1.52
± 0.41
0.147
± 0.009
65.50
± 15.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
101
446
-
vs
0
.010
1.80
± 0.39
0.061
± 0.011
1
178
19
570
9
nd
nd
nd
nd
±
±
0.002
0.090
1.67
± 0.26
0.495
± 0.058
2
3
4
5
6
7
8
9
0.016
0.003
1.88
± 0.46
0.048
± 0.006
75.65
± 12.68
1576
21.5
nd
± 0.0006
0.191
± 0.036
1.67
± 0.32
6.27
± 2.64
135
± 42.2
0.062
1.01
± 0.16
2.03
± 0.34
16
nd
nd
±
±
±
±
0.013
1.207
> 100
nd
nd
0.093
0.022
13.38
± 4.41
0.32
± 0.035
600
138
172
464
> 250
nd
0.005
0.015
2.08
± 0.46
0.091
± 0.028
nd
0.002
0.002
0.31
± 0.098
0.008
± 0.002
11.4
± 2.2
1443
1536
±
0.0002
0.009
4.36
± 1.04
0.081
± 0.013
124
± 42.3
10
± 0.002
Carmaphycin B
analog
0
.030
2.75
± 0.50
0.342
± 0.074
92
> 50
±
0.008
SI; selectivity index, nd; not determined
30
standard solidꢀphase peptide synthesis methods (Figure
A). As a test library, we synthesized MorꢀLeuꢀLeuꢀP1ꢀACC
keeping the other positions fixed to the template sequence. We
then measured processing of each substrate by purified P. fal-
ciparum and human proteasomes and normalized values to the
control substrate, MorꢀHfeꢀSer(Me)ꢀLeuꢀACC (Figure 2CꢀE).
Scanning of the P1 library demonstrated that small aromatic
and aliphatic residues (Thi, Fur, Pra, Nva, allylGly) have inꢀ
creased activity and selectivity relative to leucine. Among
these improved residues, 2ꢀthienylalanine (Thi) showed the
strongest and most selective activity for the P. falciparum
proteasome (Figure 2C). We also found that aliphatic side
chains (propyl, butyl) are well tolerated in the P2 and this poꢀ
sition could be used to tune the physicochemical properties.
Screening of the P2 library of substrates showed that only the
2
(
Mor = morpholinoacetyl) in which we scanned through the
natural amino acids (minus methionine) at P1 position.
Screening of this set of substrates using purified P. falciparum
13
and human proteasomes confirmed our prior findings that the
parasite proteasome has a preference for aromatic residues at
P1 such as Phe, Tyr, Trp, and His (Figure 2B).
We then synthesized a series of focused substrate libraries
containing tripeptides based on the optimized template seꢀ
quence in compound 7, MorꢀHfeꢀSer(Me)ꢀLeu (capꢀP3ꢀP2ꢀ
P1). For each library, we varied a single position (P1, P2 and
P3) through 45 – 50 nonꢀnatural amino acids (Figure S2) while
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cyanobutyl (Cba) side chain displayed enhanced substrate specificity for Pf proteasome over human enzyme compared to
1
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0
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0
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Profiling the substrate specificity of the P. falciparum 20S proteasome using fluorogenic substrate libraries. (A)
Schematic of coumarinꢀbased fluorogenic substrate libraries containing nonꢀnatural amino acids at each of the P1, P2, and P3 posiꢀ
tions showing processing by the protease to produce a fluorescent signal. (B) Plot of the slope over the linear range of the reaction
(RFU/s) of each substrate containing the indicated P1 amino acid residue for the human proteasome (Xꢀaxis) and P. falciparum
proteasome (Yꢀaxis) normalized to the control substrate containing a P1 leucine (shown in blue). The points above the dashed line
represent the P1 substrates with increased activity for the P. falciparum proteasome compared to the control substrate. The closer
the points are to Y axis from the diagonal line, the more specific the substrate is for the P. falciparum proteasome over the human
proteasome compared to the control substrate. (CꢀE) Plots as in (B) of the substrate processing rate for each of the P1, P2 and P3
positional scanning libraries containing nonꢀnatural amino acids compared to the template substrate MorꢀHfeꢀSer(Me)ꢀLeuꢀACC
(blue points). For each of the P1 (C), P2 (D), and P3 (E) positions, 45 – 50 nonꢀnatural amino acids were tested, while keeping the
other positions fixed with the corresponding amino acids in the template sequence as indicated. The RFU/s values of each substrate
for human and P. falciparum proteasome 20S were normalized to the values of a control MorꢀHfeꢀSer(Me)ꢀLeuꢀACC. All experiꢀ
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ments were performed at least in triplicate. (F) Structure of the optimal combination of residues found in the substrate profiling,
MorꢀHfeꢀSer(Me)ꢀThiꢀACC. Mor= morpholinoacetyl, Hfe= homophenylalanine, Ser(Me)= methylserine, Thi= 2ꢀthienylalanine,
RFU= relative fluorescence unit
Table 2. Potency and selectivity of VS inhibitors designed based on substrate screening data. EC50 values for inhibitors 11ꢀ17
in 72 h and 1 h treatment of P. falciparum W2 strain at ring stage and nonꢀconfluent HFFs.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
2 h EC50 (ꢀM)
1 h EC50 (ꢀM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
No.
Cap
R
3
R
2
R
1
Pf
HFF
SI
Pf
HFF
SI
0
.359
78.41
± 18.01
1
1
1
1
1
1
2
3
4
5
218
nd
nd
nd
± 0.042
0.021
13.96
± 3.58
1.013
± 0.169
652
482
> 250
> 250
> 250
> 250
± 0.005
0.018
8.54
± 2.00
0.33
± 0.047
±
0.005
0.008
0.002
9.80
± 2.96
0.137
± 0.095
1240
445
±
0.035
15.77
± 3.51
8.03
± 1.33
±
±
±
0.009
0.007
7.4
± 1.50
0.051
± 0.006
207
± 23.2
16
17
1138
667
4043
0.002
0.369
246
± 49.63
> 2.5
> 250
0.125
SI; selectivity index, nd; not determined
the Ser(Me) in our template sequence (Figure 2D). The P3
library demonstrated, as expected, that the S3 pocket of the
Plasmodium proteasome accommodates bulky and hydrophoꢀ
bic residues (Figure 2E). Interestingly, among the 47 amino
acids tested at P3, the Hfe in the template molecule proved to
be optimal in terms of overall activity and selectivity. Taken
together, these substrate data identify multiple nonꢀnatural
amino acids that could be used in each of the P1ꢀP3 positions
to optimize overall binding and selectivity for the parasite
proteasome with the sequence MorꢀHfeꢀSer(Me)ꢀThi being the
most optimal combination of residues (Figure 2F).
Conversion of optimal substrates into corresponding vi-
nyl sulfone inhibitors. We selected residues from the subꢀ
strate screening that showed the most promising specificity
and binding properties and used that information to build inꢀ
hibitors by replacement of the ACC reporter with the vinyl
sulfone electrophilic warhead (Compounds 11-17; Table 2).
Encouragingly, the profiles found in the substrate screening
translated into similar structureꢀactivity relationships (SAR)
for the vinyl sulfone inhibitors. Consistent with the fluorogenꢀ
ic substrate data, replacement of the P3 Hfe with a bulky but
nonꢀaromatic cyclohexylalanine (Cha; compound 11) reduced
potency. Inhibitors with 2ꢀaminobutyric acid (Abu; compound
Notably, we observed a significant increase in potency and
selectivity by incorporation of thiophene in the P1 position
(compound 14). The compound with 2ꢀfurylalanine at the P2
position displayed similar potency (compound 16, EC = 7
5
0
nM) to the analog containing the methylserine. However, citꢀ
rulline (Cit) substitution (compound 17) resulted in loss of
activity probably due to poor permeability of the compound.
Assessment of lead compound liabilities. Given that
epoxyketoneꢀbased inhibitors have been shown to have short
3
1ꢀ34
half lives in vivo due to hydrolysis of the reactive epoxide
,
we were concerned about the overall stability of the vinyl sulꢀ
foneꢀbased inhibitors. Therefore, we performed metabolite
profiling studies on compound 7. Compound 7 exhibited good
aqueous solubility (198 ꢀM) and rat hepatocyte stability (t ;
1
/2
75 min). Yet, it is predicted that the compound would have
short halfꢀlife and be rapidly cleared in vivo based on its staꢀ
bility in human liver microsomes (Table S1). The formation of
metabolites of compound 7 was liver microsomeꢀ and
NADPHꢀdependent and the compound was stable to hydrolyꢀ
sis in human and mouse plasma (Figure S4). In fact, > 85% of
the compound remained stable in plasma after 6 h incubation.
The tentative identity of the metabolites was deduced by comꢀ
parison of the product ion spectra to that of the parent comꢀ
pound. The microsomal metabolites of inhibitor 7 were mainly
derived from oxidative reactions on the morpholine cap and
homophenylalanine side chain as well as some breakage of the
1
2) and propargylglycine (Pra; compound 13) at the P1 posiꢀ
tion exhibited comparable activities (EC = 21 nM and 18 nM,
5
0
respectively) and retained similar selectivity as the leucine P1.
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morpholine ring and hydrolysis of the peptide backbone beꢀ
tween Hfe and Ser(Me) (Figure S5ꢀ6). These findings suggest
that the vinyl sulfone and P1/P2 residues remain unmodified
and that the most significant liabilities are related to the P3
Hfe and morpholino capping group.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Potency and selectivity of VS inhibitors designed to optimize capping group and solubility. EC50 values for inhibitors
8ꢀ30 in 72 h and 1 h treatment of P. falciparum W2 strain at ring stage and nonꢀconfluent HFFs.
1
72 h EC50 (ꢀM)
1 h EC50 (ꢀM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
No.
Cap
R
3
R
2
R
1
Pf
HFF
SI
Pf
HFF
SI
0
.034
13.63
± 2.63
0.384
± 0.071
18
19
20
21
22
402
> 250
±
0.006
0
.009
1.7
± 0.26
0.155
± 0.060
54.3
± 11.2
187
nd
349
nd
± 0.0059
nd
nd
nd
nd
0.013
2.75
± 0.50
0.251
± 0.027
95.3
± 15.3
212
nd
379
nd
±
0.004
nd
nd
nd
nd
0
.003
2.22
± 0.077
0.048
± 0.009
23
24
716
346
281
493
1016
1561
2559
11.43
> 250
±
0.0007
0.002
0.727
± 0.092
0.012
± 0.003
28.73
± 5.65
2355
906
±
±
0.0004
0.004
0.985
± 0.093
0.021
± 0.004
18.66
± 2.95
25
26
27
28
29
0.0007
0.007
3.45
± 0.30
0.042
± 0.001
113
± 23.3
2690
± 0.002
0.072
± 0.008
73.39
± 7.21
> 2.5
> 250
> 250
> 250
0.019
29.67
± 4.45
0.648
± 0.099
±
±
±
0.003
0.009
23.13
± 3.53
1.065
± 0.242
0.001
0.005
0.061
± 0.008
0.121
± 0.032
42.45
± 7.7
30
SI; selectivity index, nd; not determined
Because a major metabolite of our lead molecule is a preꢀ
351
0.001
fore, finding ways to block oxidative metabolism on Hfe may
be important for retaining in vivo activity.
sumed hydroxylation on the Hfe sidechain, we synthesized
two vinyl sulfones containing P3 homotyrosine residues. Theꢀ
se analogs inhibited the P. falciparum β2 and β5 subunits
Optimization of pharmacological properties of lead mol-
ecules. Given that major metabolism occurred on the morphoꢀ
line ring of compound 7, we decided to make further modifiꢀ
cations to replace the capping group with molecules that might
improve stability and retain overall potency and selectivity of
the lead molecules. We evaluated several fiveꢀmembered aroꢀ
(
Figure S7) but were found to be inactive at killing parasites in
cellular assays suggesting a loss in cell permeability that reꢀ
sulted from addition of the hydroxyl group on the Hfe. Thereꢀ
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pound against parasite growth. Comparing the potency and
matic or nonaromatic heterocycles as replacements for the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
morpholine cap based on prior use of these groups to generate
Oprozomib (PRꢀ047), a secondꢀgeneration orally bioavailable
selectivity index measured for each compound against parasite
and human cell cultures (Figure 3C) to the competition labelꢀ
ing results for the human proteasome (Figure 3B), we found
that even highly potent inhibition (nM) of the human β5 subuꢀ
nit did not result in host cell toxicity, consistent with reports
that selective inhibition of host β5 activity is well tolerated in
1
8
human proteasome inhibitor . Within the same core Hfeꢀ
Ser(Me)ꢀLeuꢀvs scaffold, compounds with 2ꢀMeꢀ5ꢀthiazole, 5ꢀ
Meꢀ3ꢀisoxazole, and 1ꢀEtꢀ3ꢀMeꢀ5ꢀpyrazole rings showed poꢀ
tent activities in parasite killing (Table 3). The most selective
of the three compounds was 18 but it was less selective than
the original compound 7 with the morpholine cap. We then
used these three selected capping groups on the optimized
sequence, HfeꢀSer(Me)ꢀThiꢀvs. Again, the 2ꢀMeꢀ5ꢀthiazole
cap showed a good selectivity profile (compound 23, SI= 716)
and was the most potent compound yet described for killing P.
37ꢀ38
nonꢀtransformed mammalian cells . However, compounds
that had potent inhibition of the β5 activity combined with
even weak inhibition of the β2 subunit lost virtually all selecꢀ
tivity. This is most clearly illustrated when comparing comꢀ
pounds 28 and 30, for which both compounds had similar poꢀ
tencies for parasite killing, yet compound 30 was more than
100 times less selective than compound 28. The competition
labeling results confirmed that both have similar potencies for
the human β5 subunit yet compound 30 showed weak cross
reactivity with the human β2 subunit when used at high dose.
This apparently minor cross reactivity, therefore leads to a
dramatic loss of selectivity due to the highly toxic effects of
inhibiting both the β5 and β2 subunits in the human cells.
The immunoproteasome (i20S), which is a modified form of
the constitutively active 20S proteasome core particle, has
subtle differences in proteolytic activity and substrate preferꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
falciparum (EC = 3 nM).
50
For further inhibitor design, we also considered physicoꢀ
chemical properties such as molecular weight (MW), lipoꢀ
philicity (cLogP), hydrogen bond donors/acceptors
(HBDs/HBAs) and topological polar surface area (tPSA) (Taꢀ
ble S2). Inhibitor 23, although highly selective and exceptionꢀ
ally potent, has overall poor aqueous solubility (Table S1).
Also, the P1 thiophene can be converted to reactive metaboꢀ
35
lites such as Sꢀoxides and thiophene epoxides . Therefore, we
synthesized two analogs with homoserine (Hse) at the P2 posiꢀ
tion to help enhance solubility and substituted the P1 thienylꢀ
alanine by either leucine or norvaline. Compounds 28 and 29
both have low nanomolar activity at killing parasite in the 72 h
treatment with exceptionally low toxicity against HFFs resultꢀ
ing in greatly enhanced selectivity ratios (Table 3). While
compound 29 with norvaline at the P1 position decreased parꢀ
asite survival after 72 h treatment, it was only active in the
micromolar concentration range when used for a 1 h pulse
treatment, suggesting that it may have reduced cellular uptake
compared to compound 28. The selectivity profile of the most
optimal inhibitors was also confirmed with a screen against P.
falciparum Dd2 and HepG2 (Table S3). These data confirmed
similar selectivity ratios as those observed for HFF cells but
showed an even greater specificity of compound 28, which
had no toxicity to HepG2 cells even after 72 hour treatment
with doses as high as 10 ꢀM, supporting this as a promising
lead molecule for further development.
39ꢀ40
ences . Inhibition of immunoproteasomes can diminish
immune responses against infection, therefore, we sought to
profile our new inhibitors against human immunoproteasome
as well. We used the broadꢀspectrum activityꢀbased probe that
specifically targets all active subunits of the constitutive proꢀ
41
teasome and immunoproteasome . Although β1c, β1i, β5c,
and β5i subunits cannot be clearly resolved on SDSꢀPAGE,
we were still able to assess the inhibition of compounds for
i20S and compare that to h20S and Pf20S inhibitions (Figure
S8). Compounds 3, 11, and 30 that showed crossꢀreactivity
towards the human β2c were also found to inhibit β2i at high
concentrations (50 ꢀM), while compounds 7, 14, 23, and 28
showed virtually no inhibition of this immunoproteasome subꢀ
unit. To more effectively monitor inhibition of the β5i subunit,
we used the β5iꢀselective fluorogenic substrate AcꢀANWꢀ
42
AMC . Compounds 23 and 30 exhibited subꢀmicromolar IC
50
values, while the others showed only weak micromolar activiꢀ
ty (Table S4). Overall, the selectivity of compounds was the
same for β5i as β5c suggesting that enhanced crossꢀreactivity
for the immunoproteasome is not likely to be a significant
problem for our lead molecules.
Identification of determinants that drive selectivity and
potency. Having identified a number of lead molecules with
enhanced parasiteꢀspecific killing, we wanted to better underꢀ
stand the determinants that drive the potency and selectivity.
We therefore performed direct binding studies on purified
proteasomes from both parasites and the human host using
A dominant role for the electrophile in the selectivity of
lead molecules. The primary electrophiles used in the clinicalꢀ
ly approved human proteasome inhibitors bortezomib and
carfilzomib are a boronic acid and an epoxyketone, respectiveꢀ
ly. Both of these electrophiles are substantially more reactive
towards the active site threonine hydroxyl compared to the
vinyl sulfone electrophile. This is because the vinyl sulfone is
generally designed to target cysteine nucleophiles by way of a
Michael addition reaction. Therefore, we reasoned that use of
a highly reactive ‘warhead’ on the inhibitor might reduce the
overall selectivity of our lead molecules that derive selectivity
through binding elements at the P1ꢀP3 positions on the peptide
scaffold. We replaced the vinyl sulfone group of the lead
compound 28 with the epoxyketone and boronic acid electroꢀ
philes. Interestingly, the epoxyketone derivative 33 exhibited
potent inhibition of β2 and β5 subunits of the P. falciparum
proteasome, while the boronic acid derivative 34 inhibited all
three catalytic subunits. In fact, the inhibitory activities of
1
3, 36
activityꢀbased probes
(Figure 3B). This approach allows
direct assessment of compound binding to each of the active β
subunits of the proteasome through the quantification of residꢀ
ual protein labeling after compound treatment. The resulting
gel labeling data can then be used to calculate IC50 values for
inhibitor binding at each subunit. Using this approach, we
confirmed that potency for killing parasites tracked with the
ability of the compounds to bind and inhibit both the β2 and
β5 subunit of the parasite proteasome, consistent with our
36
earlier studies . Thus, compounds 3, 23, 28 and 30 all were
potent inhibitors of parasite growth (low nM EC50 values; Taꢀ
bles 1ꢀ3), which corresponded to low micromolar IC50 values
in the competition labeling studies for both the β2 and β5 subꢀ
units. Compound 11, on the other hand, showed comparable
β2 potency and reduced potency for the β5 subunit, with only
partial inhibition of β5 even at 50 ꢀM concentrations. This
lack of β5 activity resulted in overall poor activity of the comꢀ
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these analogs for the β2 subunit of the parasite proteasome
were greater than that of the parent vinyl sulfone 28. However,
analogs 33 and 34 also inhibited both β5 and β2 subunits of
the human proteasome, which became more apparent when
enzymes were treated with inhibitors for longer times (Figure
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Lead optimization of Plasmodium-selective proteasome inhibitors. (A) Structures of inhibitors designed based on
the substrate screening data with enhanced selectivity and potency for the parasite proteasome. (B) Inhibition of purified P. falcipa-
rum and human 20S as assessed by competitive activityꢀbased probe labeling. Purified 20S proteasomes were incubated with each
inhibitor at the indicated final concentrations for 1 h or 18 h followed by labeling of residual proteasome activity using the activityꢀ
based probes. The individual subunits of the proteasome (β1, β2 and β5) are indicated on the gel. The IC50 values were calculated
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for each inhibitor for the P. falciparum and human 20S proteasomes by quantification of labeled proteins using Image J and then
normalizing to mockꢀtreated control. (C) A stacked bar plot of EC50 values (logarithmic scale) of each inhibitor in 72 h treatment of
P. falciparum W2 strain at ring stage (black bar) and nonꢀconfluent HFFs (white bar) with error bars, s.d. (upper panel), and selecꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tivity index (comparing the P. falciparum activity versus HFF activity, HFF EC50 / P. falciparum EC , bottom panel) for selected
50
inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Choice of electrophile is important for obtaining optimal selectivity of proteasome inhibitors. (A) Structures of
compounds 28, 33, and 34 and inhibition of purified P. falciparum and human proteasome 20S as assessed by competitive activityꢀ
based probe labeling. Purified 20S proteasomes were incubated with each inhibitor for indicated times followed by labeling of reꢀ
sidual proteasome activity using the activityꢀbased probes. The IC50 values were calculated for each inhibitor for the P. falciparum
and human 20S proteasomes by quantifying labeled proteins using Image J and normalizing them to mockꢀtreated control. (B) Inhiꢀ
bition of compounds 28, 33, and 34 for processing of the fluorogenic substrates, SucꢀLLVYꢀAMC (β5) and BocꢀLRRꢀAMC (β2)
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by human proteasome 20S. The inhibitors were incubated with h20S for 1 h followed by the addition of each fluorogenic substrate.
The AMC molecules released by residual proteasomal activity were measured (excitation at 380 nm, emission at 460 nm) and the
slope over the linear range of the reaction was used for the calculation of IC50 values using Prism. (C) A stacked bar plot of EC50
values (logarithmic scale) for the vinyl sulfone (VS)ꢀ, epoxyketone (EK)ꢀ and boronic acid (BA) derivatives 28, 33 and 34 in 72 h
treatment of P. falciparum W2 strain at ring stage (black bar) and nonꢀconfluent HFFs (white bar) with error bars, s.d. (left) and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
selectivity index (comparing the P. falciparum activity to HFF activity, HFF EC50 / P. falciparum EC , right).
50
4
A). This coꢀinhibition of both subunits resulted in toxicity
and a complete loss of selectivity of the compounds (Figure
C and Table S5). The inhibitory activities of these analogs
(PK) to evaluate its overall stability and oral uptake in rats.
We therefore performed standard PK analysis following IV
and PO administrations to SpragueꢀDawley rats (Figure 5C)
and found that after IV administration at 50 mg/kg, plasma
concentrations of 28 reached a Cmax of 20 ꢀM (12017 ng/mL)
with a halfꢀlife (t ) of 0.49 h (Table S7). Following oral
4
for human proteasome β2 and β5 subunits were also conꢀ
firmed using fluorogenic substrate assays (Figure 4B). The
boronate 34 showed potent inhibition of the human proꢀ
teasome activity which was not reflected in the competition
labeling experiments due to the fact that its reversible inhibiꢀ
tion mechanism made it less effective at competing for the
labeling by the irreversible activityꢀbased probe. The loss of
potency observed in parasite killing with the boronic acid deꢀ
rivative 34 despite strong inhibition of the P. falciparum proꢀ
teasome in lysates also suggests poor cell permeability of the
compound as another potential liability. Taken together, these
results demonstrate that optimization of selectivity using pepꢀ
tide backbone changes requires the use of a suitable electroꢀ
phile such as the vinyl sulfone to prevent potency being driven
mainly by the reactivity of the electrophile.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
/2
A
Cam3.II K13 WT
Cam3.II K13 C580Y
a
c
e
g
i
b
d
f
1.5
1.5
1.0
0.5
1.0
0.5
0.0
0.0
0.0 0.5 1.0 1.5
0.0 0.5 1.0 1.5
FIC50 DHA
FIC50 DHA
1.5
1.5
1.0
0.5
0.0
1.0
0.5
0.0
0.0 0.5 1.0 1.5
0.0 0.5 1.0 1.5
The P. falciparum-specific proteasome inhibitors en-
hance parasite killing by ART in both sensitive and re-
sistant parasites. Recent data suggest that artemisininꢀ
resistant (ARTꢀR) parasites are vitally dependent on proꢀ
teasomal degradation of ARTꢀalkylated and polyꢀubiquitinated
FIC50 DHA
FIC50 DHA
1.5
1.5
1.0
0.5
0.0
1.0
0.5
0.0
14, 43ꢀ44
parasite proteins
. In support of this finding, DHA and
0.0 0.5 1.0 1.5
0.0 0.5 1.0 1.5
13
FIC50 DHA
FIC50 DHA
1.5
WLWꢀvs are highly synergistic in killing parasites in culture .
Therefore, we tested our new P. falciparumꢀspecific proꢀ
teasome inhibitors 3, 7, 23, and 28 on artemisininꢀsensitive
ARTꢀS) parasites originated from Cambodia (Cam3.II) harꢀ
boring a wildꢀtype version of Kelch 13 (Cam3.II WT K13) as
h
1.5
1.0
1.0
0.5
(
0
.5
.0
0
0.0
C580Y
0.0 0.5 1.0 1.5
0.0 0.5 1.0 1.5
well parasites isogenic at the K13 locus (Cam3.II K13
)
3
FIC50 DHA
FIC50 DHA
1.5
that rendered them ARTꢀR . All of the tested inhibitors were
potent on both ARTꢀS and ARTꢀR parasites, with killing acꢀ
tivities in the nanomolar range. When parasites were exposed
to combinations of the compounds and DHA (dihydroartemisꢀ
inin) at the early ring stage of the intraerythrocytic cycle, we
observed strong synergism in both ARTꢀS and ARTꢀR paraꢀ
sites (Figure 5A). In particular, the inhibitor 28 exhibited the
strongest synergism among the compounds tested. These reꢀ
sults confirm that Plasmodiumꢀspecific proteasome inhibitors
potentiate ART action and can be used to eliminate ARTꢀR
parasites.
j
1.5
1.0
0.5
1.0
0.5
0.0
0.0
0.0 0.5 1.0 1.5
0.0 0.5 1.0 1.5
FIC50 DHA
FIC50 DHA
B
15
C
102
IV (50 mg/kg)
PO (100 mg/kg)
1
10
1
0
1
00
The lead vinyl sulfone reduces parasite growth in vivo
without host toxicity. Encouraged by the selective parasite
killing and synergism with DHA in our culture model, we
proceeded to test the efficacy of the new analogs compared to
the original WLLꢀvs lead molecule in the Plasmodium berghei
mouse model. In this model of human malaria infection, mice
are infected and total parasite burden in blood is measured
1 -
0
1
5
0
10-2
10-3
0
2
4
6
8
Vehicle WLL-vs
3
7
28
time (h)
45
over time by simple blood smear analysis . We infected CD1
Figure 5. The lead vinyl sulfone enhances parasite killing
by ART and reduces parasite growth in vivo. (A) Isoboloꢀ
grams depicting P. falciparum in vitro cultureꢀbased fractional
IC50 (FIC ) values in the presence of (a, b) dihydroartemisinin
DHA) and inhibitor 3, (c, d) DHA and 7, (e, f) DHA and 23,
g, h) DHA and 28 or (i, j) DHA and WLLꢀvs. The dashed line
represents additive interactions. Concave shapes (sum of FIC50
values <1) indicate synergistic effects between compounds.
B) Reduction of parasite burden after in vivo inhibitor treatꢀ
mice with P. berghei and treated with compounds at 50 mg/kg
once daily for 4 consecutive days of intravenous injections.
We observed almost complete reduction of parasite burden on
day 4 (3: 99% and 28: 96% reduction of parasites, Figure 5B)
suggesting that the new analogs are highly effective at killing
parasites in vivo at levels comparable to WLLꢀvs (98% reducꢀ
tion). Given the overall high specificity ratio and low toxicity
for compound 28, we selected it for further pharmacokinetic
50
(
(
(
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Journal of the American Chemical Society
with a SAR investigation of the corresponding vinyl sulfone
ment on day 4. CD1 mice were infected with P. berghei and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
2
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6
treated with vehicle (n= 5) or 50 mg/kg compounds WLLꢀvs,
3 (n= 3), 7 and 28 (n= 5) once daily for 4 consecutive days
starting 2 h after infection. Parasitemia was monitored daily by
Giemsaꢀstained thin blood smears. (C) Plasma concentrationꢀ
time profiles of inhibitor 28 after intravenous (50 mg/kg) and
oral (100 mg/kg) administration to SpragueꢀDawley rats (n=
inhibitors, we identified bulky and hydrophobic P3 residues
that are preferred by the parasite enzyme. Steric and conforꢀ
mational constraint appear to be key determinants of the speciꢀ
ficity as narrower binding pockets of human β2 reduce the
accessibility of ligands. Furthermore, our data show that toxꢀ
icity of compounds towards host cells is associated specificalꢀ
ly with an increase in potency and inhibition of the β2 subunit
of the human enzyme. We also found that the P1 side chain
can be exploited to enhance selectivity using small aliphatic
and aromatic residues, exemplified by 2ꢀthienylalanine. Our
data further support the finding that the P1 residue directs the
specificity for the β2 subunit of the parasite proteasome beꢀ
cause only this subunit is compatible with bulky side chains
such as Trp and Hfe at the P1 position. However, it is imꢀ
portant to retain potency for the β5 subunit as β2ꢀselective
inhibitors are not effective at killing parasites on their own.
Recently, Kirkman and coworkers also demonstrated a synerꢀ
gistic effect between β2 and β5 inhibition of the P. falciparum
proteasome. Their β5ꢀselective inhibitor did not yield a signifꢀ
icant reduction in parasitemia in vivo on its own, but when
combined with the β2ꢀselective inhibitor WLWꢀvs, reduced
parasitemia by 95% . We also found that while the P1 and P3
positions are the major determinants of selectivity, the P2 and
capping group also influence the selectivity subtly, especially
when combined with specific P3 and P1 variants. In fact, the
substitution of the morpholinoacetyl cap with a 2ꢀMeꢀ5ꢀ
thiazole ring led to an increase in β5 inhibition. To provide a
structural understanding of the specificity determinants, a
study of highꢀresolution electron microscopy structure of the
P. falciparum proteasome with our new potent and selective
inhibitor bound in multiple active sites is in progress.
6
), error bars, s.d.
administration (100 mg/kg), the compound displayed some
plasma exposure with a Cmax of 1 ꢀM and an area under the
curve (AUC) of 1294 hr×ng/mL. The overall oral bioavailabilꢀ
ity (F) was 2.6%, which while a promising sign that the comꢀ
pound could make it into blood upon oral dosing, suggests that
additional formulation and compound optimization likely will
be required to achieve therapeutically relevant doses of the
drug by oral delivery.
0
1
2
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9
0
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0
DISCUSSION
The proteasome has proven to be a viable target in humans
4
6ꢀ50
for the treatment of multiple myeloma . This multimeric
complex has recently emerged as a viable target in Plasmodi-
um falciparum, the causative agent of malaria, due to its cenꢀ
52
51
tral role in parasite growth, replication and transmission . Its
potential as an antiꢀmalarial target has recently been further
strengthened by the fact that a functional proteasome is reꢀ
quired for P. falciparum resistance to ART derivatives, which
are the core components of global firstꢀline ACTs. Therefore,
inhibitors of the Plasmodium proteasome have the potential to
block infection, replication, transmission and possibly the
dissemination of ART resistance. However, to be viable as a
therapy, compounds must be highly selective to avoid toxicity
resulting from inhibition of the host proteasome. We recently
reported on peptide vinyl sulfones that are capable of selective
inhibition of the parasite enzyme. However, these lead moleꢀ
cules still suffer from some level of reactivity towards the
human enzyme and also contain significant liabilities in terms
of their pharmacological properties. Here we report the use of
a combination of substrate screening and rational design based
on the prior highꢀresolution EM structure to optimize both the
selectivity and physicochemical properties to provide a next
generation of inhibitors of the Plasmodium proteasome. Using
these new compounds, we were able to define the elements of
specificity of compounds for the parasite enzyme over the
human counterpart, which resulted in a lead molecule with
high potency and exceptional selectivity that can clear paraꢀ
sites in mouse models of infection. Furthermore, the structural
and SAR data presented here provide a roadmap for further
optimization of compounds for advancement into clinical triꢀ
als.
Most peptideꢀinspired proteasome inhibitors derive their
subunit selectivity from the amino acid sidechains that engage
the S pockets of the enzyme active sites. Understanding the
structural differences between the parasite and human proꢀ
teasomes through highꢀresolution electron microscopy in our
previous study facilitated the discovery of specific nonꢀnatural
amino acids that could be used to greatly enhance the specificꢀ
ity of inhibitors for the parasite enzyme over the human host
enzyme. Incorporation of nonꢀnatural amino acids into a subꢀ
strate library design allowed us to generate 140 fluorogenic
peptides (45 – 50 peptides/subꢀlibrary), which were then asꢀ
sayed for specificity in a simple and rapid format. Coupled
We have found that the vinyl sulfone is an ideal reactive
electrophile for development of parasiteꢀselective inhibitors.
Swapping of the vinyl sulfone on our highly selective lead
molecule for an epoxyketone or boronic acid shows that the
high degree of reactivity of these electrophiles effectively neꢀ
gates all the specificity gained by design of an optimal peptide
sequence. The epoxyketone and boronic acid groups also have
additional liabilities that may prevent their effective use as
antiꢀmalarial agents. Human proteasome inhibitors such as
carfilzomib and oprozomib are rapidly metabolized and
31ꢀ34
cleared via epoxide hydrolysis within minutes
and the boꢀ
ronic acid is a highly efficient inhibitor for diverse enzymes
that use nucleophilic hydroxyl residues. Our in vitro metaboꢀ
lism study with compound 7 demonstrated that the vinyl sulꢀ
fone is stable in microsomes. Given that the vinyl sulfone
functions by irreversible inhibition, it is possible that our lead
compounds could induce prolonged pharmacological effects in
spite of rapid clearance. Furthermore, the vinyl sulfone has
been shown to have a limited spectrum of possible target, all
of which are cysteine protease such as the cathepsins. This
small family of potential off targets are not particularly probꢀ
lematic as they can be inhibited without causing shortꢀterm
53ꢀ54
toxic effects
.
The drug exposure of our optimized Plasmodiumꢀspecific
proteasome inhibitor 28 following oral administration in a rat
PK study is promising although the overall oral bioavailability
needs to be further improved. Previous studies have demonꢀ
strated that toxicity of irreversible proteasome inhibitors correꢀ
31, 55
lates with the Cmax values rather than the AUC
. The fact
that compound 28 showed a low peak plasma concentration
therefore might not be problematic, especially if the comꢀ
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Journal of the American Chemical Society
Page 12 of 16
pound is used in combination with ART where complete inhiꢀ the compound. Compounds were diluted for doseꢀresponse
1
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9
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6
bition of the proteasome is not required to enhance ARTꢀ
mediated parasite killing.
concentrations in media and added to the cells for either 1 h or
72 h. Cell viability was measured using the CellTiterꢀBlue
Assay (Promega) as per the manufacturer’s instructions. Valꢀ
ues in Tables 1ꢀ3 and S3 are the average n = 9 cell cultures
from three independent experiments of triplicates for HFF
treatment.
Inhibitor 28 was able to reduce parasite growth in vivo. Givꢀ
en the large therapeutic window and no noticeable toxic effect
or other signs of sickness in the mice, we conclude that the Pfꢀ
specific proteasome inhibitor 28 could be used to attenuate
parasite growth without appreciable toxicity to the host. More
importantly, the strong synergy displayed between the proꢀ
teasome inhibitors and ART derivatives in both ARTꢀsensitive
and ꢀresistant lines suggests that highly selective Pf proꢀ
teasome inhibitors may be most beneficial when used in comꢀ
bination with ART for malaria treatment. Furthermore, beꢀ
cause the proteasome activity is critical for the onset of a deꢀ
layed response to ART, even lowꢀlevel inhibition of the paraꢀ
site proteasome (i.e. at levels not sufficient to kill parasites
through proteasome inhibition alone) may be sufficient to enꢀ
hance ART killing and prevent the onset of resistance. Thereꢀ
fore, the compounds described here already have the potential
to be used as highly safe adjunct therapies for the effective
treatment of complicated cases of malaria.
Activity-based probe labelling of mammalian and P. fal-
36
ciparum proteasome. BMV037 was used at a final concenꢀ
tration of 10 ꢀM to label P. falciparum proteasome activity,
41
and MV151 was used at a final concentration of 2 ꢀM to
0
1
2
3
4
5
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7
8
9
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0
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3
4
5
6
7
8
9
0
label mammalian proteasome activity. Purified proteasomes
(human or P. falciparum, 10 nM) were incubated with each
inhibitor for 1 h at 37°C before addition of BMV037 or
MV151 for a further 2 h at 37°C. Samples were denatured by
addition of SDS sample buffer, boiled briefly and run on a
12% SDS PAGE. Gels were scanned on the Cy5 channel (for
BMV037) or the Cy3 channel (for MV151) on a Typhoon
Scanner (GE Healthcare).
Assessment of DHA synergism against ART-sensitive
and -resistant P. falciparum. Tightly synchronized 0ꢀ3 h
postꢀinvasion rings were exposed to combinations of the drugs
(
with fixed ratios of 1:0, 4:1, 2:1, 1:1, 1:2, 1:4 and 0:1 of 16×
EXPERIMENTAL SECTION
IC50 drug stocks) for 3 h. Drugs were then removed with three
washes, and the wells were incubated with fresh drugꢀfree
media. Parasitemias were determined following an additional
69 h of culture. Data were collected from two separate biologꢀ
ical repeats (indicated as different colors), each with technical
replicates.
Fluorogenic substrate assay. Substrate assays were perꢀ
formed in black 96 or 384ꢀwell Costar plates. For these assays,
purified human (purchased from Boston Biochem) or P. falci-
3
8
parum 20S proteasomes were activated using human PA28α.
Each enzyme was added to a final concentration of 2 nM and
the reaction proceeded in the presence of substrates (100 ꢀM)
for 1 h at 37°C. The fluorescent signal (RFU, relative floꢀ
rescence unit) of ACC was measured at 380 nm (excitation)
and 460 nm (emission). The slope over the linear range of the
reaction (RFU/s) was determined for each of the enzymes. All
experiments were performed at least in triplicate.
In vivo assessment. CD1 mice were infected by intraperiꢀ
7
toneal (IP) injection with 10 P. berghei ANK strain MRAꢀ
868 parasites isolated from an infected mouse on Day 0.
WLLꢀvs, 3, 7, and 28 (50 mg/kg), or vehicle (10% DMSO,
40% PEGꢀ400, 10% (2ꢀhydroxypropyl)ꢀβꢀcyclodextrin in
PBS) was administered by intravenous injection for 4 consecuꢀ
tive days. For each drug dose, three to five mice were used.
Due to the technical challenge of 4 consecutive days of tail
vein injections, some doses were administered as retroꢀorbital
injection when required. Treatment with 50 mg/kg compounds
for all mice was administered as tail vein injection (day 0 and
3) and retroꢀorbital injections (day 1 and 2). During drug
treatment, the health of the mice was assessed daily by observꢀ
ing their physical appearances and activities of mice. Paraꢀ
sitemia was monitored daily by Geimsaꢀstained thin blood
smears obtained from the tail vein and quantified by light miꢀ
croscope counting, containing at least 1000 red blood cells.
Synthesis of fluorogenic substrate library. The preparaꢀ
tion of ACC and FmocꢀACCꢀresin was carried out as deꢀ
P. falciparum replication assays. P. falciparum W2 culꢀ
tures were maintained, synchronized, and lysed as previously
38
described . P. falciparum W2 was obtained originally from
the Malaria Research and Reference Reagent Resource Center.
Parasite cultures were grown in human erythrocytes (no blood
type discrimination) purchased from the Stanford Blood Cenꢀ
ter (approved by Stanford University). Ring stage P. falcipa-
rum culture at 2% parasitemia and 1% hematocrit was added
to 96ꢀwell plates spotted with compounds. The P. falciparum
culture was incubated with each compound for either 1 h or 72
h. After compound treatment for 1 h, the inhibitor was reꢀ
moved, and the culture was washed three times in media, beꢀ
fore fresh media was added for a further 71 h of growth. After
incubation, the culture was fixed in a final concentration of
1% paraformaldehyde (in PBS) for 30 min at room temperaꢀ
ture. The nuclei stain YOYOꢀ1 was then added to a final conꢀ
centration of 50 nM and incubated overnight at 4°C. Parasite
replication was monitored by observation of a YOYOꢀ1ꢀ
positive population (infected) and YOYOꢀ1ꢀnegative populaꢀ
tion (uninfected) using a BD Accuri C6 automated flow cyꢀ
tometer. Values in Tables 1ꢀ3 and S3 are the average; n = 6
parasite cultures from two independent experiments with tripꢀ
licates.
3
0
scribed previously . The substitution level of the resin (0.5
mmol/g) was determined by a spectrophotometric Fmoc quanꢀ
titative assay. The synthesis of the library was performed usꢀ
ing a Syro II 96ꢀtip synthesis module (Biotage). To prepare the
P1 library, each of 45 of single Fmocꢀamino acids 10 eq (50
ꢀmol) with 10 eq of HATU (50 ꢀmol), 10 eq of collidine (50
ꢀmol) in DMF (180 ꢀL/tip) was added to the same tips as
ACCꢀresin (10 mg, 5 ꢀmol). The reaction was carried out for
2
4 h. Then, resin was washed five times with DMF and the
reaction was repeated using above reagents. After washing
with DMF, Fmoc protecting group was removed using 20%
piperidine (v/v) in DMF. For fixed positions, 5 eq of single
amino acid (FmocꢀSer(Me)ꢀOH at P2 and FmocꢀHfeꢀOH at
P3) was used. Reactions were carried out with 5 eq of HATU
and 5 eq of collidine in DMF for 3 h. Nꢀterminus capping was
Cell viability assay in mammalian cells. HFFs used in all
experiments were nonꢀtransformed primary cells (obtained
from J. Boothroyd) derived from human foreskin tissues and
tested for Mycoplasma contamination. HFFs were plated at
2
,500 cells (nonꢀconfluent) per well <24 h before addition of
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Journal of the American Chemical Society
compound (10 ꢀM) was incubated at 37°C in a solution conꢀ
carried out using 5 eq of morpholinꢀ4ꢀylꢀacetic acid with 5 eq
1
2
3
4
5
6
7
8
9
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5
6
of HATU and 5 eq of collidine in DMF for 3 h. The resin was
washed with DMF and then with CH Cl . The substrates were
cleaved from the resin by treating with a solution of trifluoroaꢀ
taining 1 mg/mL microsomes (MLM, male mouse CD1, Lot
No. 1510043, Xenotech; HLM; mixed gender, Lot No.
1410013 Xenotech), magnesium chloride (5 mM) and
NADPH (1 mM) in potassium phosphate buffer (100 mM, pH
7.4) for 60 min while shaking. The samples were then preꢀ
pared by iceꢀcold acetonitrile precipitation, centrifuged and
filtered for LCꢀMS/MS analysis. Controls containing all the
sample constituents (not incubated), and in which NADPH,
microsomes or the test compound were individually excluded
were also prepared and handled similarly to the test sample.
Propranolol (10 ꢀM) was incubated concomitantly as a posiꢀ
tive control.
2
2
cetic acid : triisopropylsilane : H O (95 : 2.5 : 2.5, 200 ꢀL/tip)
for 1 h, and the collected material was precipitated with cold
diethyl ether (Et O, 2 mL). After precipitation, the mixture
was centrifuged and washed with Et O (1 mL). After centrifuꢀ
2
2
2
gation, a white precipitate was dissolved in acetonitrile/H O
2
and lyophilized. The final products were dissolved in DMSO
to a concentration of 20 mM and used without further purificaꢀ
tion. For the P2, and P3 libraries, FmocꢀLeuꢀOH bound to
ACCꢀresin was prepared. The resin was dried and then split
into the tips (5 ꢀmol/tip, 50 tips per each library). In the same
manner as described above, P2 and P3 libraries were syntheꢀ
sized by coupling 50 of single Fmocꢀamino acid residues to P2
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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2
3
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5
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8
9
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2
3
4
5
6
7
8
9
0
Metabolite identification. Metabolites were identified by
comparison of the chromatograms of the incubated samples to
those at T0 using Lightsight 2.3. The tentative identity of the
metabolites was deduced by comparison of the product ion
spectra of the [M+H] ions of the metabolites with that of the
parent compound using Analyst 1.6.
Rat pharmacokinetics. SpragueꢀDawley rats, weighing apꢀ
proximately 235 to 265 g were obtained and used for all of the
pharmacokinetic (PK) studies. Animals were allowed to acꢀ
climate for a minimum of 3 days before experimentation and
allowed food and water ad libitum. Compound 28 was formuꢀ
lated in 10% DMSO, 40% PEGꢀ400, 10% (2ꢀhydroxypropyl)ꢀ
βꢀcyclodextrin in PBS (pH 7.4). For intravenous bolus adminꢀ
istration, five animals received a single dose of 28 via retroꢀ
orbital injection at 50 mg/kg. For oral administration, six rats
received 28 via oral gavage at 100 mg/kg. Blood samples were
collected at 0.25, 0.5, 1, 2, 4, 8 h postdose and processed to
plasma. Compound concentrations were determined by LCꢀ
MS/MS.
(
using FmocꢀHfeꢀOH at P3) and respectively P3 position (usꢀ
+
ing FmocꢀSer(Me)ꢀOH at P2).
Analog synthesis. The synthesis of compounds was divided
into two parts, the amino acidꢀvs part (P1ꢀvs) and the peptide
part that contains the cap–P3–P2 moieties. Each of these two
parts was synthesized separately according to a previously
13
established procedure . The synthesis was completed by atꢀ
taching the P1ꢀvs moiety to the desired dipeptide moiety (Capꢀ
P3ꢀP2ꢀOH) using simple HBTU/HOBt coupling to afford the
desired analogs. The crude compounds were purified on RPꢀ
HPLC to afford pure final products.
Kinetic solubility. The compounds were added to 96ꢀwell
plates in duplicate, from their respective 10 mM stocks. A
MiVac sample evaporator was then used to dry down the
DMSO under vacuum. Buffer at pH 2 and pH 7.4 was then
added to the driedꢀdown wells. The plates were then incubated
at 25°C for 24 h with shaking. The plates were then centriꢀ
fuged and 30 ꢀL of samples was carefully transferred to an
analysis plate. Finally, 270 ꢀL of acetonitrile containing 100
nM carbamazepine as internal standard was added to the analꢀ
ysis plate. Six calibration standards and five quality control
samples were prepared by adding DMSO after the dryꢀdown
step to give final concentrations between 5 – 250 ꢀM and were
processed similarly. The positive controls hydrocortisone and
reserpine were prepared similarly, but with a 3ꢀpoint calibraꢀ
tion curve between 10 – 220 ꢀM.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Substrate assay data of the P. falciparum and human proteasome
2
0S using a focused library of fluorogenic substrates, additional
data of inhibition for activityꢀbased probe labeling of P. falcipa-
rum and human constitutive and immuneꢀproteasome 20S, metabꢀ
olite identification of compound 7, kinetic solubility, Log D, rat
hepatocyte, human liver microsomal stability, and calculated
physicochemical properties of analogues, in vivo efficacy of
selected lead compounds, cell viability data of P. falciparum Dd2
strain and HepG2 cells for selected inhibitors, inhibition for
cleavage processing of the fluorogenic substrates of human proꢀ
teasome 20S, in vivo PK parameters of compound 28, detailed
experimental procedures including synthesis, characterization
Microsomal stability. Metabolic stability was performed in
duplicate in a 96ꢀwell microtiter plate. The test compounds (1
ꢀ
M) were incubated individually in mouse, rat and pooled
human liver microsomes (0.4 mg/mL) at 37°C for predeterꢀ
mined time points, in the presence or absence of the cofactor
NADPH (1 mM). Reactions were quenched by adding 300 ꢀL
of ice cold acetonitrile containing internal standard (carbamꢀ
azepine, 0.0236 ꢀg/mL). Test compounds in the supernatant
were analyzed by means of LCꢀMS/MS (Agilent Rapid Resoꢀ
lution HPLC, AB SCIEX 4000 QTRAP MS). Metabolite
searches were not conducted during the metabolic stability
assay.
In vitro incubations with mouse and human plasma. The
test compound (10 ꢀM) was incubated at 37°C in human or
mouse plasma for 6 h while shaking. The samples were then
prepared by iceꢀcold acetonitrile precipitation, centrifuged and
filtered for LCꢀMS/MS analysis. Vinpocetine and Procaine (10
AUTHOR INFORMATION
Corresponding Author
* Matthew Bogyo – mbogyo@stanford.edu
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
ꢀ
M) were incubated concomitantly as positive controls.
In vitro incubations with mouse liver microsomes
(MLM) and human liver microsomes (HLM). The test
ACKNOWLEDGMENT
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 16
We thank Warren Olifant and Nesia Barnes for guidance on soluꢀ
bility and metabolic stability assays. We thank Christopher Kirk
Winzeler, E. A.; Gerwick, W. H.; Ottilie, S., J. Med. Chem. 2017, 60,
721ꢀ6732.
18) Zhou, H. J.; Aujay, M. A.; Bennett, M. K.; Dajee, M.; Demo, S. D.;
Fang, Y.; Ho, M. N.; Jiang, J.; Kirk, C. J.; Laidig, G. J.; Lewis, E. R.; Lu,
Y.; Muchamuel, T.; Parlati, F.; Ring, E.; Shenk, K. D.; Shields, J.;
Shwonek, P. J.; Stanton, T.; Sun, C. M.; Sylvain, C.; Woo, T. M.; Yang,
J., J. Med. Chem. 2009, 52, 3028ꢀ3038.
(19) Thornberry, N. A.; Rano, T. A.; Peterson, E. P.; Rasper, D. M.;
Timkey, T.; GarciaꢀCalvo, M.; Houtzager, V. M.; Nordstrom, P. A.; Roy,
S.; Vaillancourt, J. P.; Chapman, K. T.; Nicholson, D. W., J. Biol. Chem.
6
(
1
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9
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(Kezar Life Sciences) for helpful discussion. We thank Jeremy
Burrows and Stephen Brand (Medicines for Malaria Venture) for
advice and help with in vitro DMPK studies. We also thank Ellen
Yeh for access to the BD Accuri flow cytometer. Funding for this
work was provided in part by R21 AI127581 (to M.B. and
D.A.F.).
1
997, 272, 17907ꢀ17911.
(20) Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.; Ellman, J. A.;
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(
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Table of Contents (TOC)
OH
Human
proteasome
P. falciparum
proteasome
N
O
O
S
H
H
N
N
S
N
H
O
O
O
β1
β2
β1
β5
β2
β5
P. falciparum W2 EC50 (
HFF EC50 ( M)
Selectivity Index
ꢀ
M) = 0.019
= 29.67
= 1561
ꢀ
0
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9
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