Kerri-Lynn Conrad '11 and Chinagozi Ugwu '10
protease(HAP) is one of 10 plasmepsins (PMs) identified in the genome
responsible for the most widespread form of malaria. Plasmepsins are
aspartic acid proteases that catalyze the hydrolysis of peptide bonds.
HAP is one of 4 PMs residing in the food vacuole of the parasite (5).
falciparum uses host erythrocyte
hemoglobin as a major nutrient source. The
degrades host erythrocyte
by means of a specialized structure called a cytostome,
which spans between the membranes of the erythrocyte and the
parasite cytoplasm. Hemoglobin is packaged into vesicles (3), which
are transported from the cytostome to the food vacuole, where it is
active site of HAP, containing catalytic aspartate and histidine
has been studied as a potential target for novel antimalarial therapy
By inhibiting catalytic residues in the active site, the protease
activity of HAP would be inhibited. Knockout of HAP's protease function
could potentially preserve hemoglobin of erythrocytes infected with Plasmodium
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II. General Structure and Active Site
is a dimer composed of 2 identical monomers, A
Together, the 2
monomers form a large substrate binding cleft,
can bind an individual substrate.
Although HAP exists as a dimer, there is no proof, to
date, that dimerization is required for enzymatic activity.
amino and carboxy
terminal domains of the HAP chains are assembled into characteristic
6-stranded interdomain β-sheets
interactions between the 2 monomers have a profound impact on the
conformation of a functionally important loop, the "flap" residues
"flap" remains in
an open conformation in the apoenzyme.
active site of HAP is located in a large formed by the amino
carboxy terminal domains of each monomer.
are the two catalytic residues located in the active site of each
monomer of the protein.
contribute directly to HAP's
unusual feature of unliganded HAP is the presence of a Zn
active site of each monomer the enzyme, as well as 4 other Zn ions
on the surface
of the HAP dimer.
ion in the active site is
coordinated by the side chains of His32
from one HAP
from the other monomer, and a H2O
presence of a metal ion in the active sites of aspartic proteases
not been observed before. Although the coordination of the Zn ion in
the active site of HAP resembles the active site of known
metalloproteases, it is not clear whether HAP functions as a
metalloprotease toward synthetic peptides. If HAP does function as a
metalloprotease, it may promote nucleophilic attack on the carbonyl
carbon of the protein by the oxygen atom of the aspartic acid residue,
or the water molecule.
Pepstatin A Binding
A is a potent
inhibitor of aspartyl proteases. It is a hexa-peptide containing the
unusual amino acid statine, having the sequence
bonds with His32
in the active site of HAP. Pepstatin
A has 2 hydroxyl groups on
side chain residues. They are
located 2 residues from one another. The residue closer to the amino
end of Pepstatin
A participates in a hydrogen
bond with His32
residue closer to the carboxy end of Pepstatin
A participates in a
hydrogen bond with Asp215
Because the nucleophilic O and N atoms of Asp215
respectively, are stabalized by strong hydrogen bonds
A hydroxyl residues, Asp215
can no longer
participate in catalysis of peptide bonds via nucleophilic attack at
carbonyl residues of peptide bonds.
with HAP in the active site, the "flap"
at residues 70-83
shifts to a "closed" conformation
The change in
conformation is most dramatic in the Lys76 residue,
shifts 6.9 angstroms from the open position in the apoenzyme. The Lys76
residue participates in a hydrogen bond with the carbonyl oxygen at the
carboxy terminus of an alanine residue of the inhibitor
This is the
only bond the inhibitor forms to the flap, due to the orientation of
the carboxy terminus when the flap "closes" upon Pepstatin
A cannot form bonds to the
flap upon binding to HAP, the primary means of inhibition of HAP
protease function is by hydrogen bonding to the catalytic His32
residues in the active site.
inhibitor, a family of
inhibitors aimed as anti-HIV agents targeting the retroviral protease. KNI-10006
a potent inhibitor of HAP, with an IC50=0.69
HAP is drastically
different than that of Pepstatin
A. In the KNI-10006-HAP
complex, the hydroxyl
group at the central part of
the inhibitor points away
residues in the active site.
flap is in an
conformation in the KNI-10006-HAP
complex, creating a "flap pocket" with an abundance of
hydrophobic residues. KNI-10006
with the hydrophobic residues
70-83, including Val71,
entrance of the flap pocket is
unique to HAP. KN1-10006
hydrophobic interactions with
It is believed that Phe111
HAP as opposed to
proteases such as PMII and PMIV plays a role in HAP's ligand
Implications for Anti-Malarial Drug Design
KNI-10006 have been identified as potent inhibitory drugs to target the
falciparum HAP proteins. These
compounds are particularly
exciting for the
development of anti-malarial drugs because they are adaptive inhibitors
of the P.
falciparum aspartyl protease
The Pepstatin A peptide greatly
reduces the rate of erythrocyte degradation by inhibiting the activity
of the P.
falciparum digestive vacuole.
Pepstatin A particularly
reduces the activity
of the four most lethal P.
I, II, IV and HAP (6). This
binds to the active site of the HAP enzyme and blocks P.
protein from degrading the hemoglobin cells.
KNI-10006 does not bind
to the active site of the HAP protein unlike most other anti-malarial
This compound inhibits the four most virulent malarial plasmepsins at
concentrations (3,6). However the potency with which KNI-10006 peptide
the parasitic plasmepsins II and IV is much greater than all the other
concentration 50 (IC50)
values for plasmepsins
II and IV with the
KNI-10006 compound is 39 nM and 15 nM respectively (6). The KNI-10006
problem in drug design in that it inhibits human aspartic protease.
KNI-10006 only inhibits human aspartyl protease pepsin at several
magnitude less that it inhibits P.
As a consequence of
KNI-10006 cell/vacuole membrane impermeability, the capacity to which
growth in infected erythrocyte cultures with P.
low (4,6). Experimentation is ongoing to develop a more permeable
Xiao, H., Parr, C. L., Kiso, Y., A, Gustchina., Yada, R. Y., A.
Wlodawer. 2009.Crystal Structures of the Histo-Aspartic Protease (HAP)
from Plasmodium falciparum.
K., Kimura, T., Tsuchiya, Y., Kamiya, M., Ruben, A. J., Freire, E.,
Hayashia, Y., Kisoa, Y. 2007. Additional interaction of
allophenylnorstatine-containing tripeptidomimetics with malarial
aspartic protease plasmepsin II. Bioorganic & Medicinal
Chemistry Letters. 17:3048–3052.
A., Hidaka, K., Kimura, T., Hayashi, Y., Nezami, A., Ernesto, F., Kiso,
Y. 2004. Search
Substrate-Based Inhibitors Fitting the S2’
Space of Malarial Aspartic Protease Plasmepsin II. J. Peptide
A., Kimura, T., Hidaka, K., Kiso, A., Liu, J., Kiso, Y. Goldberg, D. E., Freire,
E. 2003. High-Affinity Inhibition of a Family of Plasmodium
falciparum Proteases by a
Designed Adaptive Inhibitor.
Biochemistry. 42: 8459-8464.
- Nguyen, J.T., Hamada, Y., Kimura, T.,
Y. 2008. Design
of Potent Aspartic Protease Inhibitors to Treat Various Diseases. Arch.
Pharm. Chem. Life Sci. 341: 523 – 535.
K. M., Marzahn, M. R., Gutiérrez-de-Terán, H.,
Åqvist, J., Dunn,
B. M., Larhed, M. 2009. α-Substituted norstatines as the
transition-state mimic in inhibitors of multiple digestive
proteases. Bioorganic & Medicinal Chemistry.
A.M., Lee, A.Y., Gulnik, S.V., Majer, P., Collins,
J., Bhat, T.N., Collins, P.J., Cachau,
R.E., Luker, K.E., Gluzman, I.Y., Francis, S.E., Oksman, A., Goldberg,
D.E., Erickson, J.W. 1996. Structure and Inhibition of Plasmepsin II, a
Hemoglobin-Degrading Enzyme from Plasmodium
Proc. Natl. Acad. Sci. 93: 10034-10039.
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