Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice

Article abstract of DOI:10.1002/anie.202015845

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages—erythrocytic, gametocyte, liver, and gamete activation stages—indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophyla

Full text of DOI:10.1002/anie.202015845

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 9279–9283
International Edition: doi.org/10.1002/anie.202015845
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Medicinal Chemistry Very Important Paper
Development of a Highly Selective Plasmodium falciparum
Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice
Wenhu Zhan+, Hao Zhang+, John Ginn+, Annie Leung, Yi J. Liu, Mayako Michino,
Akinori Toita, Rei Okamoto, Tzu-Tshin Wong, Toshihiro Imaeda, Ryoma Hara,
Takafumi Yukawa, Sevil Chelebieva, Patrick K. Tumwebaze, Maria Jose Lafuente-Monasterio,
Maria Santos Martinez-Martinez, Jeremie Vendome, Thijs Beuming, Kenjiro Sato,
Kazuyoshi Aso, Philip J. Rosenthal, Roland A. Cooper, Peter T. Meinke, Carl F. Nathan,
Laura A. Kirkman,* and Gang Lin*
Abstract: Plasmodium falciparum proteasome (Pf20S) inhib-
itors are active against Plasmodium at multiple stages—
erythrocytic, gametocyte, liver, and gamete activation
stages—indicating that selective Pf20S inhibitors possess the
potential to be therapeutic, prophylactic, and transmission-
blocking antimalarials. Starting from a reported compound, we
developed a noncovalent, macrocyclic peptide inhibitor of the
malarial proteasome with high species selectivity and improved
pharmacokinetic properties. The compound demonstrates
specific, time-dependent inhibition of the b5 subunit of the
Pf20S, kills artemisinin-sensitive and artemisinin-resistant
P. falciparum isolates in vitro and reduces parasitemia in
humanized, P. falciparum-infected mice.
mosquito stages, indicating that Pf20S inhibitors can poten-
tially have therapeutic, prophylactic and transmission-block-
ing indications.[2] Given that human hematopoietic cells,
plasma cells and plasmacytoid dendritic cells are highly
susceptible to proteasome inhibition,[3] proteasome inhibitors
for treatment of malaria must be highly selective for the
malarial proteasome over the three proteolytic subunits of the
human constitutive proteasome (c-20S) and the three pro-
teolytic subunits of the human immunoproteasome (i-20S) to
avoid toxicity and interference with immune responses.
We recently reported noncovalent Pf20S inhibitors that
showed marked antimalarial activity against multiple stages
of the parasitesꢀ life cycle.[2d,4] However, these inhibitors all
demonstrated fast clearance in vivo (Figure S1). To identify
a compound for use in mouse proof of concept studies, we
explored an alternative scaffold for Pf20S-selective inhibitors.
Bogyo and co-workers recently reported a macrocyclic pep-
tide, compound 1, that shows potent proteasome inhibition,
selectivity and effective antimalarial activity.[5] However,
compound 1 suffers from poor solubility and fast clearance
by liver microsomes (Table S1).
T
he spread of drug resistance threatens recent gains in
malaria control even as the current burden of disease remains
intolerable, with 228 million new cases and 405000 deaths,
primarily in children, estimated in 2018.[1] The limited range
of clinically validated antimalarial targets and the danger of
increasing resistance to current antimalarial regimens high-
light the need for novel antimalarial agents. Proteasomes of
pathogens have recently become targets of interest in tuber-
culosis, malaria, Chagas disease, leishmaniasis and schistoso-
miasis. The Plasmodium falciparum proteasome (Pf20S) has
served as a promising target for parasite-selective proteasome
inhibitors acting against erythrocytic, gametocyte, liver and
We conducted an extensive structure–activity relationship
(SAR) study to improve the pharmacokinetic properties of
this scaffold (Figure 1). The full SAR study will be described
in a separate paper. In brief, starting from compound 1, we
first explored substitution at P5 to reduce hydrogen bond
[*] Dr. W. Zhan,[+] Dr. H. Zhang,[+] Prof. Dr. C. F. Nathan, Prof. Dr. G. Lin
Department of Microbiology & Immunology, Weill Cornell Medicine
1300 York Ave, New York, NY 10065 (USA)
P. K. Tumwebaze
Infectious Diseases Research Collaboration, Kampala (Uganda)
Dr. M. J. Lafuente-Monasterio, Dr. M. S. Martinez-Martinez
Diseases of the Developing World (DDW), Tres Cantos Medicine
Development Campus, GlaxoSmithKline
E-mail: gal2005@med.cornell.edu
Dr. J. Ginn,[+] Dr. M. Michino, Dr. A. Toita, Dr. R. Okamoto, T.-T. Wong,
Dr. T. Imaeda, Dr. R. Hara, Dr. T. Yukawa, Dr. K. Sato, Dr. K. Aso,
Dr. P. T. Meinke
Tri-Institutional Therapeutics Discovery Institute
413 E 69th St, New York, NY 10065 (USA)
Severo Ochoa 2, 28760, Tres Cantos, Madrid (Spain)
Dr. J. Vendome, Dr. T. Beuming
Schrçdinger, Inc., New York, NY 10036 (USA)
Prof. Dr. P. J. Rosenthal
A. Leung, Y. J. Liu, Prof. Dr. L. A. Kirkman
Department of Medicine, Division of Infectious Diseases, Weill
Cornell Medicine
1300 York Ave, New York, NY 10065 (USA)
E-mail: lak9015@med.cornell.edu
Department of Medicine, University of California
San Francisco, CA 94143 (USA)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202015845.
S. Chelebieva, Prof. Dr. R. A. Cooper
Department of Natural Sciences and Mathematics, Dominican
University of California, San Rafael, CA 94901 (USA)
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Figure 2. Docking model of macrocyclic peptides in Pf20S. The struc-
ture of Pf20S b5-6 was obtained by homology modeling based on
yeast 20S b5-6 (3MG4), and then refined using molecular dynamics
simulations. The amide bonds of all compounds form 5 well-conserved
hydrogen bonds with the backbone of Ser21, Gly47 and Ala49 as well
as the side chain of Asp153. Additionally, the pyrrolidone in P5 of
compounds 2, 3 and TDI-8304 forms a hydrogen bond with Ser154,
and the morpholino oxygen in compound 3 and TDI-8304 form
a hydrogen bond with hydroxyl group of Ser157 in b6. For compound
3, the protonated form of the morpholine has been represented to
illustrate the possible salt bridge with Asp153, but the neutral form
might predominate at relevant pH (predicted pKa ꢀ6.6). Compound 2
is shown in yellow, compound 3 in purple and TDI-8304 in green.
Figure 1. SAR evolution from compound 1 to TDI-8304.
donor count and improve passive permeability, which yielded
compound 2. Compound 2 is highly potent against P.
falciparum strain Pf 3D7 (EC50 1 nM) and has activity against
c-20S (IC50 0.34 mM) and i-20S (IC50 > 34 mM) (Figure 1).
However, the poor solubility and mouse microsomal stability
of compound 2 needed additional optimization. Substitutions
at P1, P3, and the tether were structure-guided using a docking
workflow where macrocycles underwent extended conforma-
tion sampling followed by docking with a constraint to align
the core of the query molecule with that of a reference pose
model.[6] This pose model for the macrocycle series in Pf20S
was initially developed for Compound 2 based on the acyclic
analog bound in the yeast proteasome crystal structure (PDB:
3MG4) (Figure 2).[7] This docking workflow was implemented
into LiveDesign for automated docking pose generation. We
modified the P3 moiety to reduce LogD by replacing the
phenyl with a morpholino group, and further modified the P1
moiety to reduce a lability to hydrolysis by replacing 2,2,2-
trifluoroethylamino with cyclopropylmethyl, yielding com-
pound 3. Compound 3 showed a comparable EC50 against P
falciparum and demonstrated improved selectivity for c-20S
(Figure 1). This optimization also improved metabolic stabil-
ity and solubility (> 130 mgmLÀ1). Further improvements in
metabolic stability were realized by substitution at P1 with
a cyclopentyl moiety and replacement of the biaryl ether with
a mono phenyl alkyl chain. The final compound, TDI-8304,
was highly potent against Pf 3D7 with marked selectivity for
inhibition of Pf20S over human proteasomes (Figure 3A).
To determine the inhibition modality of TDI-8304, we
monitored the hydrolysis of the proteasome substrate suc-
LLVY-AMC by Pf20S in the presence of 0.5 mM b2-specific
inhibitor WLW-VS and TDI-8304 at indicated concentrations
(Figure 3B). TDI-8304 demonstrated time-dependent inhib-
ition with an induced-fit mechanism.[8] The hyperbolic
correlation of kobs as a function of TDI-8304 concentrations
was fit to equation kobs = k6 + (k5/ (1 + (Kiapp/ [I]) and the
values of Kiapp 1007 nM, Ki*app 89.6 nM and t1/2 14.4-minute
Figure 3. TDI-8304 selectively inhibits Pf20S. A) Dose-dependent inhib-
ition of chymotryptic b5 subunits of Pf20S, human c-20S and i-20S.
B) Time courses of inhibition of Pf20S b5 by TDI-8304 at indicated
concentrations (nM). C) Plot of kobs values obtained from the time
course curves with varied TDI-8304 concentrations yields
KIapp =1007 nM, Ki*=89.6 nM and koff =0.0008 SÀ1. D) TDI-8304 spe-
cifically inhibited Pf20S b5 but not b2 or b1, from being labeled by
MV151, either in the absence of a Pf20S b2 specific inhibitor WLW-VS
(top) or in the presence of WLW-VS (bottom).
half-life for the Pf20S:TDI-8304 complex were calculated by
equation Ki* = Ki/ (1 + k5/ k6) (Figure 3C). TDI-8304 dose-
dependently blocked labelling of Pf20S b5, but not of Pf20S
b1 or b2 (Figure 3D), by MV151, suggesting that TDI-8304 is
a highly specific inhibitor of Pf20S b5.
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Figure 4. TDI-8304 inhibits growth of erythrocytic stage P. falciparum. A) TDI-8304 potently inhibited Pf3D7 growth (open black circles) but is not
cytotoxic for HepG2 human hepatoma cells (solid black circles); B) TDI-8304 inhibits growth of Pf Dd2 and two proteasome inhibitor-resistant
strains, Pf Dd2b6A117D and Pf Dd2b5A49S. C) TDI-8304 at 1 mM led to accumulation of polyubiquitinated proteins after treatment of
P. falciparum schizonts for 6 hours. D) EC50 values of TDI-8304 against P. falciparum strains with various drug resistance profiles; E) Ex vivo
activity of TDI-8304 against 38 clinical isolates from Uganda in years 2018 and 2019. The geometric mean EC50 was 18 nM. Each black spot
represents a single P. falciparum isolate. Blue and green spots represent Dd2 and 3D7 laboratory controls, respectively. F) In vitro synergy of TDI-
8304 and DHA against artemisinin-sensitive P. falciparum Dd2 Cam3.IRev and artemisinin-resistant Cam3.IR539T. Data were average of two
independent experiments and each in duplicates. FIC=fractional inhibitory concentration.
TDI-8304 is nontoxic to HepG2 cells (Figure 4A) and
potent not only against Pf 3D7 (Figure 4A) but also against
the P. falciparum lab strain Dd2 and two Dd2-derived strains
that are resistant to our previous noncovalent proteasome
inhibitors PKS21004 and TDI4258 by virtue of mutation
Pf20Sb6A117D in the adjacent b6 subunit (which changes the
conformation of S3 and S4 binding pockets of the b5 active
site) and mutation Pf20Sb5A49S in the active site of the b5
catalytic domain, respectively.[2d,4] The EC50 values of TDI-
8304 against Dd2 and Dd2b5A49S were comparable, suggest-
ing that its binding mode, which is stabilized by its macro-
cyclic tether structure, is unaffected by the mutation. How-
ever, an ꢀ 18-fold reduction in potency against Dd2b6A117D
(Figure 4B) suggests that the conformational changes caused
by the b6A117D mutation affect the binding of TDI8304
more than the conformational changes caused by the b5A49S
mutation. Supporting on-target whole-cell activity, a 6-hour
exposure to TDI-8304 led to accumulation of polyubiquiti-
nated proteins in Pf trophozoites (Figure 4C). TDI-8304
showed comparable activity against Pf strains HB3 (chlor-
oquine-sensitive), 3663 (ART-sensitive) and 4884 (ART-
resistant)[9] (Figure 4D), indicating no apparent cross-resist-
ance.
between sensitivities to TDI-8304 and chloroquine resistance
among the clinical isolates.
Several studies have established that proteasome inhib-
itors are synergistic with ARTs.[2a,b,d] To investigate the
synergy of TDI-8304 with ART, we tested TDI-8304 in
combination with dihydroartemisinin (DHA) using a modi-
fied ring-stage survival assay (RSA) with both artemisinin-
sensitive Pf Cam3.IRev and artemisinin-resistant Pf
Cam3.IR539T in a multidrug-resistant Dd2 strain background.
We exposed early (1- to 3-h) ring-stage parasites to a 3-h pulse
of DHA and continuous TDI-8304 for further 69 h, as
described.[2d] Parasitemia was measured at 72 h post initial
exposure. Isobologram analyses clearly demonstrated syner-
gistic killing of artemisinin-sensitive (Figure 4F Left) and
artemisinin-resistant (Figure 4F right) parasites by the com-
bination of TDI-8304 and DHA.
TDI-8304 is metabolically stable upon incubation with
human, mouse and rat microsomes and cell-permeable (Fig-
ure 5A). It inhibited by less than 10% a panel of CYP
enzymes, and showed no cytotoxicity to HepG2 cells. TDI-
8304 further showed no inhibition of the hERG (Ether-ꢁ-go-
go) channel, implying low risk of cardiac arrhythmia through
action on this ion channel. TDI-8304 is rapidly cleared when
administrated to mice by i.v. (Figure 5B) or p.o. routes
(Figure S1), but shows prolonged clearance when adminis-
trated s.c. (Figure 5B). This allowed twice-daily dosing (b.i.d)
to afford sufficient exposure for efficacy experiments in mice.
We tested TDI-8304 in a humanized mouse model of Pf
TDI-8304 was further tested for ex vivo activity against
fresh falciparum isolates from 38 malaria patients in Uganda.
The EC50 values ranged from 5–30 nM, with a mean of 18 nM,
in agreement with the results from P. falciparum laboratory
strains (Figure 4E). There was no observed association
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Figure 5. Pharmacokinetic properties of TDI-8304 and its efficacy in humanized, P. falciparum-infected mice. A) PK properties of TDI-8304. B) In
vivo PK of TDI-8304 administrated i.v. (1 mgkgÀ1) or s.c. (100 mgkgÀ1). C) Parasitemia reduction of P. falciparum Pf3D70087/N9 in NOD-SCID IL-2R-
null mice transfused with human erythrocytes. TDI-8304 was given twice-daily by s.c. (100 mgkgÀ1) on days 3 through 6. Two mice were used in
each group, each serially sampled on days 3 to 7. Percentage parasitemia was calculated by acquiring a minimum number of 500 parasitized
erythrocytes.
infection[10] with s.c. administration at 100 mgkgÀ1 b.i.d., on
days 3–6 post infection and monitoring parasitemia from day
Conflict of interest
3 until day 7 (Figure 5C). TDI-8304 reduced the parasitemia
by a factor of 2 log10 in one mouse and cleared the
parasitemia in another mouse. Pharmacokinetics of TDI-
8304 from the infected SCID mice during the course of the
assay were also determined (Table S2), and the data showed
that the whole blood concentration of TDI-8304 was con-
stantly greater than the EC90 (29 nM) of TDI-8304 in both
mice, except for one data point at the 24 h time-point in
mouse 2 (Figure S2), in agreement with the observed
reduction of parasitemia in infected mice.
In conclusion, we developed a preclinical and species-
selective P. falciparum proteasome inhibitor with marked
anti-malarial activity in a humanized mouse model of P.
falciparum infection. To our knowledge, this is the first
noncovalent Pf20S inhibitor shown to have anti-malaria
activity in Pf infected mice.
The authors declare no conflict of interest.
Keywords: inhibitors · malaria · proteasome · selectivity ·
therapeutics
[1] WHO, World Health Organization, Geneva: World Health
Organization, 2018.
[2] a) C. Dogovski, S. C. Xie, G. Burgio, J. Bridgford, S. Mok, J. M.
McCaw, K. Chotivanich, S. Kenny, N. Gnadig, J. Straimer, Z.
Bozdech, D. A. Fidock, J. A. Simpson, A. M. Dondorp, S. Foote,
N. Klonis, L. Tilley, PLoS Biol. 2015, 13, e1002132; b) H. Li, A. J.
OꢀDonoghue, W. A. van der Linden, S. C. Xie, E. Yoo, I. T. Foe,
L. Tilley, C. S. Craik, P. C. da Fonseca, M. Bogyo, Nature 2016,
530, 233; c) M. Zhang, C. Wang, T. D. Otto, J. Oberstaller, X.
Liao, S. R. Adapa, K. Udenze, I. F. Bronner, D. Casandra, M.
Mayho, J. Brown, S. Li, J. Swanson, J. C. Rayner, R. H. Y. Jiang,
J. H. Adams, Science 2018, 360, eaap7847; d) L. A. Kirkman, W.
Zhan, J. Visone, A. Dziedziech, P. K. Singh, H. Fan, X. Tong, I.
Bruzual, R. Hara, M. Kawasaki, T. Imaeda, R. Okamoto, K.
Sato, M. Michino, E. F. Alvaro, L. F. Guiang, L. Sanz, D. J. Mota,
K. Govindasamy, R. Wang, Y. Ling, P. K. Tumwebaze, G.
Sukenick, L. Shi, J. Vendome, P. Bhanot, P. J. Rosenthal, K.
Aso, M. A. Foley, R. A. Cooper, B. Kafsack, J. S. Doggett, C. F.
Nathan, G. Lin, Proc. Natl. Acad. Sci. USA 2018, 115, E6863 –
E6870.
[3] a) M. Hirai, N. Kadowaki, T. Kitawaki, H. Fujita, A. Takaori-
Kondo, R. Fukui, K. Miyake, T. Maeda, S. Kamihira, Y. Miyachi,
T. Uchiyama, Blood 2011, 117, 500; b) K. Neubert, S. Meister, K.
Moser, F. Weisel, D. Maseda, K. Amann, C. Wiethe, T. H.
Winkler, J. R. Kalden, R. A. Manz, R. E. Voll, Nat. Med. 2008,
14, 748; c) R. de Luna Almeida Santos, L. Bai, P. K. Singh, N.
Murakami, H. Fan, W. Zhan, Y. Zhu, X. Jiang, K. Zhang, J. P.
Assker, C. F. Nathan, H. Li, J. Azzi, G. Lin, Nat. Commun. 2017,
8, 1692.
Acknowledgements
This work is supported by NIH grants R01AI143714 (G.L.),
R21AI123794 (G.L. and L.A.K.), AI139179 (P.J.R. and
R.A.C.), and T37MD003407 (S.C.); The Brockman Founda-
tion (L.A.K.); Department of Medicine, Weill Cornell
Medicine Seed fund (L.A.K.); Tri-Institutional Therapeutics
Discovery Institute; Weill Cornell Medicine Matching Fund
(G.L.); Milstein Program in Chemical Biology and Transla-
tional Medicine; and Medicines for Malaria Venture RD/15/
0001 (P.J.R. and R.A.C.). We gratefully acknowledge in-kind
support of the Tri-Institutional Therapeutics Discovery Insti-
tute (TDI), a 501(c)(3) organization. TDI receives financial
support from TDIꢀs owners (Memorial Sloan Kettering
Cancer Center, The Rockefeller University and Weill Cornell
Medicine), Takeda Pharmaceutical Company, and generous
contributions from Mr. Lewis Sanders, Mr. Howard Milstein
and other philanthropic sources. We thank Drs. Stacia Karg-
man and Leigh Baxt at TDI for their constructive suggestions.
The Department of Microbiology and Immunology is sup-
ported by the William Randolph Hearst Foundation.
[4] W. Zhan, J. Visone, T. Ouellette, J. C. Harris, R. Wang, H. Zhang,
P. K. Singh, J. Ginn, G. Sukenick, T. T. Wong, J. I. Okoro, R. M.
Scales, P. K. Tumwebaze, P. J. Rosenthal, B. F. C. Kafsack, R. A.
Cooper, P. T. Meinke, L. A. Kirkman, G. Lin, J. Med. Chem.
2019, 62, 6137.
[5] H. Li, C. Tsu, C. Blackburn, G. Li, P. Hales, L. Dick, M. Bogyo, J.
Am. Chem. Soc. 2014, 136, 13562.
[6] D. Sindhikara, S. A. Spronk, T. Day, K. Borrelli, D. L. Cheney,
S. L. Posy, J. Chem. Inf. Model. 2017, 57, 1881.
9282 www.angewandte.org
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[7] C. Blackburn, K. M. Gigstad, P. Hales, K. Garcia, M. Jones, F. J.
Benoit-Vical, O. Mercereau-Puijalon, D. Menard, Nature 2014,
505, 50.
[10] I. Angulo-Barturen, M. B. Jimenez-Diaz, T. Mulet, J. Rullas, E.
Herreros, S. Ferrer, E. Jimenez, A. Mendoza, J. Regadera, P. J.
Rosenthal, I. Bathurst, D. L. Pompliano, F. Gomez de las Heras,
D. Gargallo-Viola, PLoS One 2008, 3, e2252.
Bruzzese, C. Barrett, J. X. Liu, T. A. Soucy, D. S. Sappal, N.
Bump, E. J. Olhava, P. Fleming, L. R. Dick, C. Tsu, M. D.
Sintchak, J. L. Blank, Biochem. J. 2010, 430, 461.
[8] R. A. Copeland, Evaluation of enzyme inhibitors in drug
discovery: A guide for medicinal chemists and pharmacologists,
2nd ed., Wiley, Hoboken, 2013.
[9] F. Ariey, B. Witkowski, C. Amaratunga, J. Beghain, A. C.
Langlois, N. Khim, S. Kim, V. Duru, C. Bouchier, L. Ma, P. Lim,
R. Leang, S. Duong, S. Sreng, S. Suon, C. M. Chuor, D. M. Bout,
S. Menard, W. O. Rogers, B. Genton, T. Fandeur, O. Miotto, P.
Ringwald, J. Le Bras, A. Berry, J. C. Barale, R. M. Fairhurst, F.
Manuscript received: November 27, 2020
Revised manuscript received: December 29, 2020
Accepted manuscript online: January 12, 2021
Version of record online: March 11, 2021
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