Development of piperazine-based hydroxamic acid inhibitors against falcilysin, an essential malarial protease

Article abstract of DOI:10.1016/j.bmcl.2018.04.010

The human parasite Plasmodium falciparum kills an estimated 445,000 people a year, with the most fatalities occurring in African children. Previous studies identified falcilysin (FLN) as a malarial metalloprotease essential for parasite development in the

Full text of DOI:10.1016/j.bmcl.2018.04.010

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of piperazine-based hydroxamic acid inhibitors against
falcilysin, an essential malarial protease
Jeffrey P. Chance a,e, Hannah Fejzic a,e, Obiel Hernandez a, Eva S. Istvan b,c, Armann Andaya d,
Nikolay Maslov a, Ruby Aispuro a, Teodulo Crisanto a, Huyen Nguyen a, Brian Vidal a, Whitney Serrano a,
Bradley Kuwahara a, Corey Pugne Andanado a, Daniel E. Goldberg b,c, Jeremy P. Mallari a,
⇑
a Department of Chemistry and Biochemistry, California State University, San Bernardino, USA
b Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, Saint Louis, USA
c Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, USA
d Campus Mass Spectrometry Facilities, University of California, Davis, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The human parasite Plasmodium falciparum kills an estimated 445,000 people a year, with the most fatal-
ities occurring in African children. Previous studies identified falcilysin (FLN) as a malarial metallopro-
tease essential for parasite development in the human host. Despite its essentiality, the biological roles
of this protease are not well understood. Here we describe the optimization of a piperazine-based
hydroxamic acid scaffold to develop the first reported inhibitors of FLN. Inhibitors were tested against
cultured parasites, and parasiticidal activity correlated with potency against FLN. This suggests these
compounds kill P. falciparum by blocking FLN, and that FLN is a druggable target. These compounds rep-
resent an important step towards validating FLN as a therapeutic target and towards the development of
chemical tools to investigate the function of this protease.
Received 23 February 2018
Revised 3 April 2018
Accepted 4 April 2018
Available online xxxx
Keywords:
Falcilysin
Malaria
Protease inhibitors
Metalloprotease
Hydroxamic acid
Ó 2018 Elsevier Ltd. All rights reserved.
Malaria remains a serious threat to human health in many parts
of the world, and it is estimated that the disease kills nearly half a
million people annually.1 The causative agent of the most serious
form of human malaria is Plasmodium falciparum, a protozoan
parasite spread by the bite of an Anopheles mosquito vector. After
entering the human host at the site of the bite, the parasite transits
the host liver before entering the bloodstream to begin its intraery-
throcytic development. During this part of the life cycle, the para-
site invades host red blood cells (RBCs) and replicates within a
parasitophorous vacuole to produce 16–32 daughter merozoites.
Following replication, the host cell is lysed, and the merozoite
progeny reinvade new RBCs to begin another round of infection.
This part of the parasite life cycle gives rise to all clinical symptoms
of the disease, and despite recent management efforts malaria con-
tinues to be a severe burden on the health of subtropical regions
around the world. Emerging resistance against current clinical
drugs underscores the need for the identification and validation
of new therapeutic targets.
We are developing small molecule inhibitors as chemical tools
to investigate the function of falcilysin (FLN), a metalloprotease
essential for parasite development in the RBC. It is known that
FLN localizes to multiple subcellular compartments, and that FLN
carries out distinct roles based on its localization. It is thought that
FLN is required for protein import into the apicoplast2 (a relict
plastid involved in biosynthesis of fatty acids, heme, and iso-
prenoids), and for host cell hemoglobin degradation in the food
vacuole (an acidic organelle involved in catabolism of host cell pro-
teins).3 The majority of FLN appears to be distributed throughout
the cell, possibly in the ER or cytosol. Whether the protease func-
tions in either of these compartments, or if it is simply in transit
on the way to other organelles is not known. In addition, it is
unclear where FLN carries out its essential function (e.g. does FLN
carry its vital role in the apicoplast, the food vacuole, the cytosol,
or some combination of these), or what the nature of this function
is.
Abbreviations: RBC, Red blood cell; IPTG, Isopropyl b-D-1-thiogalactopyranoside;
FLN, Falcilysin; PMSF, Phenylmethylsulfonyl fluoride; TEA, Triethylamine; ER,
Endoplasmic reticulum; DMAP, 4-Dimethylaminopyridine.
⇑
Corresponding author at: California State University, Department of Chemistry
and Biochemistry, 5500 University Parkway, Chemical Sciences, Room 211, San
Bernardino, CA 92407, USA.
E-mail address: jeremy.mallari@csusb.edu (J.P. Mallari).
These authors contributed equally to this manuscript.
e
https://doi.org/10.1016/j.bmcl.2018.04.010
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chance J.P., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.010

2
J.P. Chance et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Scheme 1. Inhibitor synthesis route. Conditions: (i) di-tert-butyl dicarbonate, NaOH, 1,4-dioxane:H2O (2:1), 0 °C ? 20 °C, 12–24 h; aryl sulfonyl chloride, TEA, 20 °C, 12 h; (ii)
Thionyl chloride, MeOH, 20 °C, 12–24 h; (iii) alkyl halides or acid halides, DMAP (cat), TEA, 1,4-dioxane, 20 °C, 4–48 h; (iv) NH2OH, MeOH, 20 °C, 6 h.
Table 1
Activity of inhibitor series against FLN and cultured P. falciparum.
X
R
IC50 vs FLN (mM)
IC50 vs. P. falciparum (mM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H-
H-
H-
H-
H-
H-
H-
H-
3-hydroxypropyl-
butyl-
benzoyl-
>50
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
30
40
7
20
3-F-benzoyl
4-F-benzoyl
2-Br-benzoyl
4-Br-benzoyl
3,4-Cl2-benzoyl
4-phenyl-benzoyl
phenylsulfonyl-
benzoyl-
2-Br-benzoyl
3-Br-benzoyl
4-Br-benzoyl
3,4-Cl2-benzoyl
4-phenyl-benzoyl
propanoyl-
>50
>50
7
13
5
1
3
1
2
H-
8
4j
4k
4l
MeO-
MeO-
MeO-
MeO-
MeO-
MeO-
MeO-
Br-
Br-
Br-
Br-
Br-
>50
>50
8
2
3
5
1
1
4m
4n
4o
4p
4q
4r
4s
4t
10
14
6
10
5
2
>50
>10
>10
>10
>10
1.4
benzoyl-
35
31
16
1.5
9
9
3
0.2
3-F-benzoyl
4-F-benzoyl
4-phenyl-benzoyl
4u
0.1
These remaining gaps in our understanding of FLN biology are
largely due to challenges in generating loss-of-function mutants
(i.e. parasites in which FLN is inactive or absent), a result of the par-
asite’s well-known resistance to genetic manipulation. To address
this need, we are working towards the development of potent and
selective chemical inhibitors of FLN. Such inhibitors could be used
to block FLN activity in cultured parasites in lieu of genetic disrup-
tion and provide valuable insights into the biological roles of this
essential protein. These compounds would also help to evaluate
the potential of FLN as a therapeutic target. To date, no inhibitors
against FLN have been reported. Here we describe the development
of a panel of FLN inhibitors using a piperazine-based hydroxamic
acid scaffold. This scaffold has several known advantages over pep-
tidyl inhibitors, including good membrane permeability and an
ability to target multiple binding surfaces of a protease active site.4
These properties make this scaffold a promising platform for the
development of selective, cell-permeable FLN inhibitors.
Previous studies of this scaffold against a human orthologue
(MMP-3) indicate similar compounds are competitive inhibitors
which utilize the hydroxamic acid moiety to coordinate the cat-
alytic zinc atom in the protease active site, while the N1 and N4
substituents target subsites in the active site cleft involved in sub-
strate recognition.4 We began exploring structural requirements
for FLN inhibition by synthesizing (Scheme 1) compounds with
aryl sulfonyl groups at the N1 position and a variety of functional
groups at the N4 position (Table 1).
We initially focused on probing SAR at N4 with various func-
tional groups. Introduction of aryl sulfonyl or smaller alkyl or
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J.P. Chance et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
site cleft. It is notable that the N1 position also contributes to inhi-
bitor potency, and relatively small changes in the X substituent can
result in a >5-fold change in potency (i.e. 4u vs. 4p). Future studies
will focus on further optimization of the N1 and N4 aryl groups.
Inhibitors were then screened against cultured 3D7 P. falci-
parum at 1 and 10 mM. Compounds with parasiticidal activity at
1 mM were further tested in a dose response assay to determine
EC50 values. These experiments identified 4 inhibitors with weak
(<50% parasite killing) parasiticidal activity at 10 mM, as well as 2
compounds (4p and 4u) with low micromolar potency against cul-
tured parasites. It is notable that both compounds are active
against FLN, and the most potent FLN inhibitor (4u) is also the
most potent antimalarial (see Fig. 1b). Together, these data indicate
FLN may be a promising point for chemotherapeutic intervention,
and these inhibitors represent an important step towards the
development of selective chemical tools to further probe the
biological functions of this protease. As similar inhibitors have
been shown to target human MMP-3,4 future studies will focus
on optimizing specificity for the parasite and FLN relative to
human cell lines and metalloproteases.
Fig. 1a. Dose response of lead compound 4u against FLN.
Acknowledgements
The authors would like to thank Edwin Chen for helpful discussions
on protein expression and purification. The authors would also like
to thank the CSUSB Institute for Child Development and Family
Relations for supported writing time.
Funding
This work was supported by the CSUSB Office of Research and
the California State University CSUPERB New Investigator Grant
program.
Fig. 1b. Dose response of lead compound 4u against cultured P. falciparum.
A. Supplementary data
amide groups at N4 did not yield active inhibitors (4a, 4b, 4j, 4q).
However, installation of a phenyl ring through an amide linkage
produced compounds (4c, 4r) with modest activity against the
metalloprotease target (IC50 ꢀ 30–40 mM). We expanded on this
observation by testing a range of ring substitution patterns and
found that introduction of smaller fluoro groups at the 3 or 4 posi-
tions of the phenyl group generally did not improve activity
against FLN, while larger halogen substituents (3-Br, 4-Br, or 3,4-
Cl2) led to consistent increases in potency (3–10-fold). These data
indicate that steric bulk at N4 is important driver of FLN inhibition,
and we further tested this by introducing a second phenyl ring (4-
Ph). This approach yielded the most potent inhibitor of the series
(4u, IC50 = 1.4 mM, see Fig. 1a), indicating that the N4 substituent
targets a large binding site or possibly multiple sites in the active
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.04.010.
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Please cite this article in press as: Chance J.P., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.010