Method for the prevention of malaria infection of humans by hepatocyte
growth factor antagonists
United States Patent: 7,670,631
Issued: March 2, 2010
Inventors: Mota; Maria M. (Lisboa,
PT), Rodriguez; Ana (Great Neck, NY), Giordano; Silvia (Turin, IT),
Rodrigues; Margarida Cunha (Parede, PT)
ALFAMA--Investigacao e Desenvolvimento de Produtos Farmac uticos, Lda.
(Porto Salvo, PT)
Appl. No.: 10/795,456
Filed: March 9, 2004
Covidien Pharmaceuticals Outsourcing
A method for the prevention of infection
of humans by plasmodium parasites is provided. The method consists of the
application of compounds that interfere with the infection of hepatocytes
by Plasmodium viax.
Description of the
SUMMARY OF THE INVENTION
This invention aids in fulfilling these needs in the art. The evasiveness
of malaria has made a definitive treatment difficult. Presented here is an
agent and a method capable of preventing the spread or acquisition of
malaria infection and of assisting in the prevention and treatment of such
More particularly, this invention provides a method for inhibiting the
activity of malaria in vivo, wherein the method comprises administering to
a human host an antimalarial agent, which is capable of exhibiting a
protective effect by preventing the initial replication of malaria
parasites in the liver of an infected host such as humans. The
antimalarial agent is comprised of at least one inhibitor of HGF activity,
and optionally, an antimalarial drug, such as primaquine. The antimalarial
agent is administered to the human in an amount sufficient to prevent or
at least inhibit infection of hepatocytes by malaria in vivo or to prevent
or at least inhibit replication or spread of a malaria parasites in vivo.
The present invention relates to the ability of hepatocytes to support the
growth of parasites that cause the human disease malaria. Plasmodium
parasites that cause human disease are Plasmodium falciparum, Plasmodium
vivax, Plasmodium malariae and Plasmodium ovale. More specifically the
invention reveals Met activation and downstream signals to be essential
for the establishment of plasmodium infection. It has previously been
known that Plasmodium sporozoites pass through several hepatocytes before
they are able to establish a vacuole in hepatocytes in which they divide.
It has not previously been known that the passage of sporozoites through
hepatocytes is associated with the production of a well known cytokine,
referred to as hepatocyte growth factor (HGF). HGF is known to be released
as an inactive, single chain protein. It is activated by proteolytic
cleavage that forms a disulfide bridge linked heterodimer. The heterodimer
binds to and activates the receptor protein tyrosine kinase Met. The
cytoplasmic domain of activated Met recruits a variety of proteins that
transmit signals through several distinct pathways. These signals result
in a variety of responses such as cell scattering, proliferation,
tubulogenesis and invasive growth. The present invention reveals a novel
Met mediated response of hepatocytes to HGF. Hepatocytes are rendered
permissive by HGF to the invasion by sporozoites in a manner that allows
their proliferation within a vacuole.
The present invention also provides a novel strategy for the prevention of
In preferred embodiments plasmodium infection of hepatocytes is prevented
by molecules which interfere with HGF production by wounded hepatocytes.
Also suitable for the prevention of infection are molecules, which
interfere with the proteolytic cleavage of HGF into its active form and
molecules which sequester HGF and thereby prevent it from binding to
hepatocytes via its receptor Met.
In another aspect, the invention reveals Met to be a target for drugs that
prevent malaria infection.
In a preferred embodiment of the invention, malaria infection is prevented
by molecules, which interfere with the binding of HGF to its receptor Met.
Such molecules are antibodies specific for HGF which block its binding
site for Met. Also in a preferred embodiment of the invention such
molecules are antibodies against Met, or fragments of such antibodies,
which block HGF binding but do not activate Met. In another embodiment of
the invention such molecules are oligonucleotides (aptamers) which bind to
Met but do not activate Met. In yet another embodiment of the invention
such molecules are HGF variants that interfere with Met activation by HGF.
Such variants include, but are not restricted to NR4.
In another aspect of the invention, plasmodium infection of hepatocytes is
prevented by drugs, which interfere with signal transduction by activated
Met. In a preferred embodiment of the invention such drugs are protein
tyrosine inhibitors. An example of such a drug is genistein.
In another preferred embodiment such drugs are selective inhibitors of the
protein tyrosine kinase Met. In preferred embodiments these inhibitors are
small molecular weight compounds and are administered by the oral route or
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "antimalarial agent" means a composition
comprising one or more inhibitors of HGF activity. The term "inhibitor of
HGF activity" means one or more compounds independently selected from HGF
receptor antagonists, inhibitors of HGF-mediated signal transduction, and
protein tyrosine kinase inhibitors. The inhibitor of HGF activity can be
employed alone or in combination with each other. The inhibitors of HGF
activity optionally can be combined with one or more known antimalarial
drugs to form the antimalarial agent of the invention.
The present invention relates to the invasion of hepatocytes by malaria
parasites. After transmission by a mosquito bite to a human host, malaria
sporozoites find their way to the liver where each sporozoite can give
rise to as many as 10,000 merozoites that are released into the blood. The
invasion of hepatocytes is an obligatory step of malaria infection.
Sporozoites can invade hepatocytes through disruption of the plasma
membranes followed by parasite migration through the cells or, like
intracellular bacteria and other parasites, through the formation of an
internalization vacuole around the invading pathogen. Initially
sporozoites pass through hepatocytes without forming an internalization
vacuole. Sporozoites enter hepatocytes by breaching their plasma
membranes, traverse the cytosol and leave the host cell which either dies
or succeeds to repair the membrane. The molecular mechanisms underlying
the passage of plasmodium through hepatocytes and the subsequent
establishment of a parasitophorus vacuole are poorly understood. Vacuole
formation by Plasmodium yoelii and Plasmodium falciparum, but not by the
rodent malaria parasite Plasmodium berghei, is dependent on CD81, a
tetraspin protein expressed by hepatocytes. CD81 is known to be a receptor
for hepatitis C virus but it does not appear to interact with any ligand
on the surface of sporozoites. Its role in hepatocyte invasion by certain
plasmodium species remains to be elucidated [Silvie et al., Nature
Medicine 9:93-96, (2003)]. Interestingly, sporozoites must traverse the
cytosol of several cells before invading a hepatocyte by formation of a
parasitophorous vacuole, which is indispensable for the differentiation
into the next infective stage [Mota et al., Science 291:440-42, 2001)].
This finding suggests that hepatocytes wounded by sporozoites release one
or more infection susceptibility inducing factors (ISIF) that render
neighbouring hepatocytes susceptible to infection. An important aspect of
the present invention is the discovery that a protein known as hepatocyte
growth factor serves as ISIF in malaria infections.
I. HGF and its Receptor Met
Hepatocyte growth factor was discovered as a mitogen for hepatocytes [Michalopolous
et al, Cancer Res., 44:441-4419 (1984); Rusasel et al J. Cell Physiol.,
119:183-192 (1984); Nakamura et al., Biochem. Biophys. Res. Comm.,
122:1450-1459 (1984)] and independently as a scatter factor that promotes
the dissociation of epithelial cells and vascular endothelial cells
[Stocker et al, Nature 327, 239-242 (1987)]. For simplicity the factor is
referred to as HGF. HGF was first purified from the serum of
hepatectomized rats [Nakamura et al., Biochem. Biophys. Res. Comm.,
122:1450-1459 (1984)] and subsequently from rat platelets [Nakamura et al.
Proc. Natl. Acad. Sci. USA, 83:6849-6493 (1986)] and from human plasma [Gohda
et al., J. Clin. Invest. 81:414-419 (1988)]. The cDNAs encoding rat HGF,
human HGF and a naturally occurring variant referred to as "delta5 HGF"
were cloned [Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973
(1989); Nakamura et al. Nature 342:440-443 (1989); Seki et al., Biochem.
Biophys. Res. Commun., 172:321-327 (1990); Tashiro et al., Proc. Natl.
Acad. Sci. USA, 87:3200-3204 (1990); Okajima et al., Eur. J. Biochem.,
193:375-381 (1990)]. Human HGF consists of an .alpha.-subunit of 440 amino
acids (M, 62 kDa) and a .beta.-subunit of 234 amino acids (M, 34 kDa). It
is produced as biologically inactive pro-HGF (728 aa) that is cleaved by
proteases between Arg494 and Val495 to form a disulfide-linked heterodimer.
The 62-kDa .alpha.-subunit contains an N-terminal hairpin domain (about 27
aa) followed by four canonical kringle domains, which are 80-aa
double-looped structures stabilized by three S-S bridges. The first
kringle domain binds to a protein tyrosine kinase receptor, Met, which is
described more in detail below. The hairpin loop and second kringle domain
binds membrane-associated heparan sulfate proteoglycans with low-affinity.
The 34 kDa .beta.-subunit contains a serine protease-like domain very
similar to that of the serine protease blood clotting factors but which
has no protease activity. HGF shows 38% overall sequence identity with
plasminogen and 45% identity with another cytokine known as macrophage
stimulating protein (MSP). HGF binds to a protein tyrosine kinase receptor
referred to as Met, while its close relative, MSP, binds to another
protein tyrosine kinase receptor known as Ron.
HGF is secreted as single chain Pro-HGF. This HGF precursor is bound to
proteoglycans that are associated with the extracellular matrix or with
cell surfaces in the vicinity of the producer cells. Activation of the
single chain precursor into the biologically active heterodimer by
proteolytic cleavage between Arg494 and Val495 is a tightly controlled
process [(for a review see Kataoka et al., Life XY 1:1036-1042 (2001)].
The enzymes first implicated in HGF activation were urokinase-type
plasminogen activator (uPA) and tissue-type plasminogen activator (tPA).
Subsequently three additional HGF activating enzymes have been identified,
namely coagulation Factor XIIa, membrane type serine protease-1 (MT-SP1)
also known as matriptase and HGF activator (HGFA). Each of these enzymes
is under the control of endogenous inhibitor proteins. HGFA is the most
effective HGF cleaving enzyme. Like HGF, HGFA is a heterodimer that is
generated from a single chain Pro-HGFA by cleavage following Arg407. One
of the HGFA cleaving enzymes is thrombin, an enzyme that is activated in
injured tissues through the coagulation cascade. Active HGFA heterodimers
are not inhibited by the major serum proteinase inhibitors, but are under
the control of two proteins, HGA inhibitor type I (HAI-1) and HGA
inhibitor type 2 (HAI-2), the latter being identical with placental
bikunin (PB). HAI-1 is upregulated in injured and regenerating tissues. It
is expressed at the cell surface where it binds and inhibits HGFA.
Cytokines such as IL-1.beta. induce shedding of the HGFA/HAI-1 complex by
TNF-.alpha. converting enzyme (TACE) and the TACE-like metalloproteinases
of the ADAM (a disintegrin and metalloproteinase) family of proteins.
After shedding HGFA dissociates from HAI-1 and is then able to activate
HGF. Thus, HAI-1 is not only an inhibitor but also a specific acceptor of
mature HGFA, acting as a reservoir of this enzyme on the cell surface.
HAI-1 is described in U.S. Pat. No. 6,465,622B2, published in Oct. 15,
2002, wherein it is claimed for its use as control factor for HGF and HGFA.
The HGF receptor Met was originally discovered as a component of an
oncogenic fusion protein that was generated in a carcinogen treated
sarcoma cell line [Cooper et al., Nature, 311:29-33 (1984)]. In normal
cells the primary Met transcript produces a 150 kDa polypeptide that is
glycosylated and then cleaved to form a S-S linked heterodimer. HGF and
its receptor Met is subject of U.S. Pat. No. 5,648,273, published in July
15, which claims the use of the ligand-receptor for the diagnosis of
proliferative disorders and diseases such as hepatitis and
The Met heterodimer consists of a .beta.-subunit, that is highly
glycosylated and entirely extracellular and a .alpha.-subunit with a large
extracellular region and an intracellular tyrosine kinase domain. Met is a
member of a superfamily of receptor tyrosine kinases (RTKs). The
superfamily is divided into at least 19 families including the Her family
(EGFR, Her 2, Her 3, Her 4), the insulin receptor family (insulin
receptor, IGF-1R, insulin-related receptor), the PDGF receptor family (PGFRa
and b, CSF-R, kit, Flk2), the Flk family (Flk-1, Flt-1, Flk-4), the FGF
receptor family (FGF-R1, 2, 3, and 4) and others. Met and its close
relative Ron form a distinct family of receptors for the ligands HGF and
macrophage stimulating protein (MSP), respectively.
Upon HGF binding, c-Met undergoes autophosphorylation of specific tyrosine
residues. While phosphorylation of Tyr1234 and Tyr1235 located within the
activation loop of the tyrosine kinase domain activates the intrinsic
kinase activity of c-Met, phosphorylation of Tyr1349 and Tyr1356 in the
C-terminus generates a multisubstrate docking site for signal transducing
proteins such as phosphotidylinositol 3-kinase (PI3K), phospholipase
C-.gamma. PLC-.gamma.), src, Stat3, Grb2 and the Grb2 associated docking
protein Gab1. Grb2 also interacts with Met through the adaptor protein Shc.
Grb2 recruits the Ras nucleotide exchange protein SOS which activates the
Ras-MAPK signaling pathway. Thus, the docking of signal transducers to the
activated Met receptor initiate signaling through a variety of pathways.
The c-terminal 26 amino acids of Met provide not only docking sites for
signal transducers, but also regulate the enzymatic activity of Met. A
mutation (M1250T) in the kinase domain bypasses the regulatory role of the
C-terminal amino acids [Gual et al., Oncogene 20:5493-502 (2001)].
A variety of responses to HGF have been described in different Met
expressing target cells. These responses include proliferation, programmed
cell death, dissociation of cells, mutual repulsion, movement of cells
through the extracellular matrix and branching morphogenesis. During
embryogenesis interactions between HGF producing, mesenchymal cells and
Met expressing, epithelial cells appear to be involved in the formation of
a neuronal tissues. HGF gene knock out mice as well as Met gene knock out
mice exhibit defects in the development of the placenta, liver and muscles
and die between E13.5 and 15.5 [(Schmidt et al., Nature 373:699-702
(1995); Uehara et al., Nature 373:702-705 (1995); Bladt et al., Nature
376:768-771 (1995)]. In adult life, HGF-Met interactions are involved in
wound healing, angiogenesis, and tissue regeneration. Not surprisingly Met
activation by HGF has been implicated in the growth, invasion and
metastasis of tumors. The biology of HGF and of its receptor Met is well
described in several review articles [Maulik et al., Cytokine & Growth
Factor Reviews, 13: 41-59 (2002)] and in numerous publications referenced
Based on their biological properties, both HGF and HGF antagonists have
been proposed to be useful for the treatment of a variety of diseases. The
production of HGF and its therapeutic applications have been claimed in
several patents. HGF has been isolated from blood on the basis of its high
affinity for heparin (U.S. Pat. No. 5,004,805 published Apr. 2, 1991).
Pegylation of HGF prolongs its clearance, reduces the dose required, and
is thought to ameliorate side effects of HGF therapy (U.S. Pat. No.
5,977,310, published in Nov. 2, 1999). HGF levels may be increased by HGF
degradation inhibiting polysaccharides such as heparin, hyaluronic acid,
dextran, dextran sulfate, heparan sulfate, dermatan sulfate, keratan
sulfate, chodroitin, or chondroitin sulfate (U.S. Pat. No. 5,736,506,
published in Apr. 17, 1998). A HGF activating protease has also been
claimed (U.S. Pat. No. 5,677,164, published in Oct. 14, 1997).
Applications of HGF therapy include the treatment of arterial occlusive
disease (U.S. Pat. No. 6,133,231, published in Oct. 17, 2000), the
occlusive disease (U.S. Pat. No. 6,133,231, published in Oct. 17, 2000),
the treatment of inflammatory bowel diseases (U.S. Pat. No. 6,319,899B1,
published in Nov. 20, 2001), the enhancement of re-surfacing of blood
vessels traumatized or damaged, for instance by vascular surgery or
angioplasty (U.S. Pat. No. 6,133,234, published in Oct. 17, 2000). HGF has
also been claimed to ameliorate side effects caused by commonly used
immunosuppressants (U.S. Pat. No. 5,776,464, published in Jul. 7, 1998).
Finally, the topical application of HGF gene containing vectors to blood
vessels or other target organs has been described for a variety of
therapeutic purposes (U.S. Pat. No. 6,248,722B1, published in Jun. 19,
2001). Met and downstream signal transduction pathways have long been
regarded as attractive targets for cancer therapy. First, studies with
tumor cell lines and tumor models in animals have shown that Met plays an
important role in the invasive growth and in metastasis of cancer cells.
Second, Met gene amplification has been observed in liver metastasis of
colorectal carcinomas. Third, Met is overexpressed in several types of
human tumors such as thyroid and pancreatic carcinomas. Fourth, germ line
mutations in the Met gene are found in hereditary, papillary renal
carcinoma, and somatic Met gene mutations are found in sporadic papillary
The present invention identifies a previously unknown function of the Met
receptor, the inhibition of which represents a novel therapeutic
application of Met antagonists. Signaling through Met renders hepatocytes
permissive to productive invasion by malaria sporozoites. Met signaling is
essential for the entry of sporozoites into hepatocytes via the formation
of an internalization vacuole and/or the proliferation of sporozoites
within vacuoles that are formed by the plasma membranes of hepatocytes.
The discovery of this function of Met is the basis of a novel approach to
the prevention of malaria infection. A further embodiment of the present
invention is the use of compounds that prevent the establishment of a
malaria infection by interfering with HGF mediated activation of Met or
signaling events downstream of Met, that are involved in rendering
hepatocytes permissive to the infection by malaria parasites. Several Met
antagonists have been described in the literature and some have been
claimed in patents for applications in the treatment of diseases that are
caused at least in part by the excessive or aberrant function of Met. The
previously claimed indications of Met antagonists are the treatment of
malignant tumors. The potential application of Met antagonists for the
treatment of infectious disease, and in particular of infections by
malaria parasites becomes apparent through the present invention. The
claim of the present invention is the use of Met antagonist for the
prevention of human infections by malaria parasites. Known HGF antagonists
are described in the following sections.
II. HGF Receptor Antagonists
A. HGF Variants
Various forms of HGF--both occurring naturally and generated by genetic
manipulation of HGF encoding cDNA antagonize some or all Met functions.
Uncleaved pro-HGF binds to but cannot activate Met. Several HGF isoforms
are generated by differential splicing of primary HGF transcripts. These
include NK1 (consisting of an N domain and the first kringle domain of HGF)
and NK2 (consisting of an N domain and the first two kringle domains of
HGF). Two additional variants discovered in the macaque endometrium and
placenta, namely dNK1 and dNK2 are similar to the NK1 and NK2 isoforms,
except that they encode proteins with a five amino acid deletion in the
first kringle domain [Lindsey and Brenner, Mol Human Reprod. 8:81-87
(2002)]. NK1, and NK2 bind with high affinity to the HGF receptor Met and
have been reported to act as HGF antagonists [Lokker and P. J. Godowski,
J. Biol. Chem. 268: 17145-17150 (1993); Chan et al., Science 254:1382-1387
(1991)]. However, subsequent studies have shown that these HGF variants
may act either as partial HGF agonists or as HGF antagonists depending on
the cell context, the presence or absence of heparin, and the HGF function
analyzed. In vivo studies with mice overexpressing transgenic HGF, NK1,
NK2, HGF+NK1, or HGF+NK2 have revealed potential in vivo functions of HGF
isoforms. Transgenic expression of HGF has a variety of phenotypic
consequences such as enhanced liver growth, progressive glomerulosclerosis,
disruption of olfactory mucosa, aberrant localization of muscle cells in
the central nervous system and of melanocytes in the dermis and epidermis,
precocious mammary lobuloalveolar development and susceptibility to tumor
induction. Transgenic expression of NK1 produces a similar phenotype,
while transgenic expression of NK2 exhibits none of the HGF and NK1
induced phenotypic characteristics. In HGF+NK2 bitransgenic mice NK2
antagonizes the pathological consequences of HGF overexpression and
downregulates the subcutaneous growth of transplanted, Met expressing
tumor cells. However, transgenic overexpression of NK2 promotes metastasis
of these same tumor cells. Thus, NK2 antagonizes many of the responses to
HGF, but shares with HGF the ability to dissociate (scatter) cells, a
response that facilitates metastasis [Otsuka et al., Molecular and
Cellular Biology 20:2055-2065 (2000)].
NK4, another HGF variant, is generated by a single cut digestion of HGF
with elastase. NK4 contains the N terminal hairpin structure and four
kringle domains. In contrast to NK1 and NK2, NK4 is a pure HGF antagonist
[Date et al., FEBS Letters 420:1-6 (1997)]. Like the isolated HGF a chain,
NK4 binds to Met but does not induce its autophosphorylation unless an
isolated HGF .beta.-chain is added. Because of its ability to antagonize
HGF, administration of the NK4 protein or NK4 gene transfer [Hirao et al.,
Cancer Gene Ther 9:700-7 (2002); Maehara et al., Clin Exp Metastasis
19:417-26 (2002)] is being evaluated as a novel approach to the treatment
of Met expressing cancers. Single chain HGF variants similar to NK4, which
have been engineered to be resistant against proteolytic cleavage are
described in U.S. Pat. No. 5,879,910, published in Mar. 9, 1999, and in
U.S. Pat. No. 5,580,963 published in Dec. 3, 1996.
B. Soluble Met receptors
A soluble form of Met is released from cultured endothelial cells, smooth
muscle cells, and various tumor cell lines. The soluble receptor is
thought to counteract the activation of cell surface associated Met by HGF.
Met-IgG fusion proteins have been generated which retain the ability to
bind HGF with high affinity and thus are able to neutralize HGF activity.
Angiostatin, an inhibitor of angiogenesis, is a fragment of plasminogen
that contains 3-4 kringles domains. The anti-angiogenic effects of
angiostatin are thought to be based on its ability to inhibit ATPase on
the endothelial cell surface, and to interfere with integrin functions and
with pericelluar proteolysis. Recent research indicates that the anti-angiogenic
activity of angiostatin is at least in part due to its ability to
neutralize the effects of HGF [Wajih and Sane, prepublished online in
Blood, Oct. 24, (2002)].
Angiostatin, which has 47% sequence homology with HGF, binds to Met and
prevents HGF mediated signaling in endothelial cells and smooth muscle
cells. It inhibits the proliferation of these cells in response to HGF but
not in response to other growth factors such as vascular endothelial cell
growth factor (VEGF) or basic fibroblast growth factor (BFGF), which act
through protein tyrosine kinase receptors other than Met. Thus angiostatin
functions as a selective Met antagonist.
D. Anti-HGF Receptor Antibodies
While some anti-Met antibodies are receptor agonists others block ligand
mediated receptor activation. Met blocking monoclonal antibodies and
various derivatives of such antibodies have been developed by the company
Genentech and are described in U.S. Pat. No. 6,468,529 B1 (published in
Oct. 22, 2002), U.S. Pat. No. 6,214,344B1 (published in Apr. 10, 2001),
U.S. Pat. No. 6,207,152B1 (published in May 1996) and of U.S. Pat. No.
5,686,292 (published in June 1995). These antibodies or derivatives of
such antibodies are claimed to be useful for the treatment of cancer.
E. Met Selective Aptamers
Single stranded oligonucleotides with random sequences can form a large
variety of structures. Oligonucleotides which bind to a particular target
can be selected from large random oligonucleotide libraries by a method
known as the SELEX process. Oligonucleotide ligands that selectively bind
to Met and block ligand mediated Met activation have been identified by
the company Gilead using the SELEX method. These HGF antagonists are
described in U.S. Pat. No. 6,344,321 B1 (published in Feb. 2, 2002), in
U.S. Pat. No. 5,843,653 (published in June 1995) and in U.S. Pat. No.
5,475,096 (published in June 1991).
III. Inhibitors of HGF-Mediated Signal Transduction
A. Met c-Tail Peptide
Modeling of the cytoplasmic domain of Met suggests that the c-terminal
tail gets into contact with the catalytic pocket and thereby acts as an
intramolecular modulator of the receptor. Bardelli et al designed peptides
that correspond to sequences in the c-tail of Met. The peptides were
rendered cell-permeable by extending them with sequences corresponding to
internalization mediating sequences of the Antennapedia homeodomain. A Met
tail peptide blocked ligand induced autophosphorylation as well as
downstream Met signaling. The peptide also blocked signal transduction by
Ron, a close relative of Met, but did not affect signaling by EGF, PDFG or
VEGF through other protein tyrosine kinase receptors. Thus, the Met c-tail
peptide is a selective Met/Ron antagonist.
B. Grb2 Antagonists
SH2 domains recognize phosphotyrosine residues (Tyr-P) with additional
secondary binding interactions within two or three amino acids C-proximal
to the Tyr-P residue. Differences in residues adjacent to Tyr-P generate
differential affinity toward SH2 domain subfamilies. Thus, SH2 domains of
particular sets of signal transducers can be selectively blocked by Tyr-P
containing tripeptides. Inhibitors of SH2 domain interactions with
phosphorylated tyrosine are described in U.S. Pat. No. 5,922,697,
published in Jul. 13, 1999. Compounds in which the Tyr-P residue is
replaced by phosphonomethyl phenylalanine or related structures, are
resistant to degradation phosphatases. A variety of other modifications of
the peptides increase the affinity for particular SH2 domains or increase
the ability of the compounds to pass through plasma membranes to reach
their intracellular targets [Yao et al J. Med. Chem., 42:25-35 (1999)].
Tripeptide based inhibitors of the Grb2 SH2 domain have been reported to
block HGF mediated cell motility, matrix invasion, and branching
morphogenesis. These same inhibitors have only a minor effect on HGF
mediated cell proliferation. Inhibitors with particularly high affinity
for the SH2 domain of Grb2 are described in U.S. Pat. No. 6,254,742B1,
published in Jun. 12, 2001 as compounds that are useful for the treatment
of cancer, metastasis, psoriasis as well as allergic, autoimmune, viral
and cardiovascular diseases.
C. Inducers of Gab1 Phosphorylation
Phosphorylation of serine/threonine residues of the Grb2 associated binder
1 (Gab1) by PKC-.alpha. and PKC-.beta.1 provides a mechanism for the
downregulation of Met signals. Inhibition of serine/threonine phosphatases
PP1 and PP2A by okadaic acid results in the activation of serine/threonine
kinases such as PKCs, and in the hyperphosphorylation of the serine/threonine
residues of gab1. The concomitant hypophosphorylation of tyrosine residues
prevents Gab1 from recruiting PI 3 kinase to Met [Gual et al., Oncogene
D. Dominant Negative Src Variants
Src binds via its SH2 domain to phosphorylated tyrosine residues of ligand
activated Met. The mutant receptor MET M1268T binds src constitutively and
NIH3T3 cells expressing the mutant receptor gene form tumors in nude mice.
Transfection of dominant negative src constructs into these cells was
reported to retard their growth, and to downregulate the phosphorylation
of the focal adhesion kinase (FAK) and of paxicillin, but had no effect on
Grb2 binding or PLC-.gamma. phosphorylation [Nakaigawa et al., Oncogene
E. PI3K Inhibitors
The binding of PI3K to Met is unusual in that it does not involve the
canonical motif YXXM but a novel motif YVXV. Although the novel motif has
low affinity for the N- and C-terminal SH2 domains of the p85 subunit of
PI3K, two closely spaced YVXV motifs in the c-tail of Met represent a
docking site for PI3K. The binding is inhibited by synthetic
phosphopeptides. The PI3K-mediated signal appears to be essential for HGF
induced cell scattering (cytoskeletal reorganization, loss of
intercellular junction, cell migration) and morphogenesis. Wortmannin, an
inhibitor of PI3K, inhibits Met induced branching of renal cells on a
collagen matrix. PI3K signals appear not to be essential for cell
transformation, but do contribute to metastasis.
F. NFkB Inhibitors
In liver cells HGF stimulates NF-kappaB DNA binding and transcriptional
activation via the canonical IkappaB phosphorylation-degradation cycle and
via the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated
protein kinase cascades. Studies with NFkB inhibitors indicate that HGF
induced NFkB activation is required for proliferation and tubulogenesis,
but not for scattering nor for the antiapoptotic function of HGF [(Muller
et al., Mol Cell Biol 22:1060-72, (2002)].
G. Inhibitors of Small GTP-Binding Proteins
Inhibition of Ras interferes with the spreading, actin reorganization, and
scattering of epithelial cells. Dominant negative Rac abolishes HGF
induced spreading and actin reorganization in non-small cell lung cancer
cells. Microinjection of Rho inhibits HGF induced spreading and scattering
but not motility.
H. Hsp90 Antagonists
The chaperone Hsp90 stabilizes many proteins involved in signal
transduction. The chaperone appears to be required for the stability and
function of a variety of mutated or aberrantly expressed signaling
proteins that promote the growth and/or survival of cancer cells. Hsp90
client proteins include mutated p53, Bcr-Abl, src, Raf-1, Akt, ErbB2 and
hypoxia-inducible factor 1.alpha. (HIF-1.alpha.). The benzoquinone
ansamycin compounds geldanamycin and herbimycin and the structurally
unrelated radicicol block the N-terminal nucleotide binding pocket of
HsP90 and cause the degradation of Hsp90 client proteins, many of which
are involved in tumor progression. One Hsp90 inhibitor,
17-allylaminogeldanamycin (17AAG), is currently in phase I clinical trial,
and a novel oxime derivative of radicicol (KF58333) is in preclinical
evaluation [(Soga et al., Cancer Chemother Pharmacol 48: 435-45, (2001)].
Recent research has shown that Met is a Hsp90 client that is particularly
sensitive to geldanamycin or related compounds. At nanomolar
concentrations, geldanamycins downregulates Met protein expression,
inhibit HGF-mediated cell motility and invasion and revert the transformed
phenotype of cells expressing HGF and Met or constitutively activated Met
mutants. Signaling pathways downstream of Met appear to be even more
sensitive to Hsp90 inhibitors. Geldanamycins inhibited HGF-mediated
plasmin activation at femtomolar concentrations which is nine orders of
magnitude below their growth inhibitory concentrations. Interestingly,
radicicol has been reported to be moderately active against Plasmodium
berghei in mice [Tanaka et al., J. Antibiot. 51:153-60 (1998)]. However,
this activity is likely not related to Met inhibition [Tanaka et al., J
Antibiot 10:880-8 (1999).
IV. Protein Tyrosine Kinase Inhibitors
The reversible phosphorylation of tyrosine residues on proteins is an
important mechanism of signal transduction. A large variety of natural and
synthetic compounds are known to be tyrosine kinase inhibitors. Almost all
of these inhibitors block protein kinases by blocking the ATP pocket of
the enzymes. Therefore, many have a broad spectrum of activity not only
against tyrosine kinases but also against serine/threonine kinases and/or
other ATP utilizing proteins.
1. General Protein Kinase Inhibitors
The Indrocarbazole K252a was first isolated from the culture broth of
Actinomadura and later from Nocardiopsis in a screen for antagonists of
Ca2+-mediated signaling. K252a inhibits serine/threonine protein kinases
such as various isoforms of protein kinase C (PKCs), cAMP and cGMP
dependent kinases as well as protein tyrosine kinases, in particular those
of the Trk and Met families. K252a inhibits Met mediated signals at
nanomolar concentrations. The compound inhibits Met autophosphorylation
and prevents activation of its downstream effectors MAPKinase and Akt. It
prevents HGF-mediated scattering in MLP-29 cells, reduces Met-driven
proliferation in GTL-16 gastric carcinoma cells, and reverses Met mediated
transformation of NIH3T3 fibroblasts. K252a and related compounds are
promising leads of drugs that may be used against Trk and Met driven
cancers [Morotti et al., Oncogene 21:4885-4893, (2002)]. Conceivably,
K525a may serve as a lead in the development of Met specific inhibitors.
2. Inhibitors with Selectivity for Protein Tyrosine Kinases
Several classes of compounds are known protein tyrosine kinase inhibitors.
Several such compounds have been isolated from plants or microorganisms
and have been extensively used for research purposes. The best known are
genistein, lavendustin A, tyrphostin 47, herbimycin, staurosporin and
radicicol. Herbimycin A is a benzoquinoid ansamycin antibiotic that
inhibits a broad spectrum of protein tyrosine kinases by covalently
interacting with their kinase domains. Staurosporin is an indole carbazole
antibiotic which inhibits a broad spectrum of kinases including scr family
members, and serine/threonine kinases. More recently a large number of
protein tyrosine kinase inhibitors have been synthesized and are claimed
in several patent applications. 1) bis monocyclic, bicyclic or
heterocyclic aryl compounds (PCT WO 92/20642); 2) vinylene-azaindole
derivatives (PCT WWO 94/14808); 3) 1-cyclopropyl-4-pyridyl-quinolines
(U.S. Pat. No. 5,330,992). 4) styryl compounds (U.S. Pat. No. 5,217,999);
2) styryl-substituted pridyl compounds (U.S. Pat. No. 5,302,606); 5)
quinazoline derivatives (EP Application No. 0 566 266A1 and U.S. Pat. No.
6,103,728); 6) selenoindoles and selenides (PCT WO 94/03427); 7) tricyclic
polyhydroxylic compounds (PCT WO 92/21660); 8) benzylphosphonic acid
compounds (PCT WO 91/15495); 9) tyrphostin like compounds (U.S. Pat. No.
6,225,346B1); 10) thienyl compounds (U.S. Pat. No. 5,886,195). 11)
benzodiazepine based compounds with some selectivity for src and FGF-r
tyrosine kinases (U.S. Pat. No. 6,100,254, published in Aug. 8, 2000).
Tyrosine kinase inhibitors from various classes are claimed for the
treatment of cancers that are driven by tyrosine kinases such as Met as
well as HER2, EGFR, IGFR, PDGFR, src and KDR/FLK-1. None of the known
tyrosine kinase inhibitors are selective for Met. However, it is
conceivable that a Met specific inhibitor can be developed in the future.
This optimism is based on the fact that several compounds have been
synthesized which inhibit a limited set of protein tyrosine kinases one of
which is approved for cancer therapy and several of which are in clinical
development. These compounds include: 1) The pyrazole pyrimidine PP1 shows
selectivity for lck and src kinases over ZAP-70, JAK2 and EGF receptor
kinases. 2) STI-571 (GLEEVEC.RTM.) inhibits all forms of abl, PDGF
receptor, and c-kit tyrosine kinases. 3) ZD1839 is a synthetic
anilinoquinazoline with some selectivity for the EGF receptor. 4) OSI-774
is another orally active quinazoline derivative with some selectivity for
the EGF receptor. 5) 4-anilinoquinazoline derivatives show selectivity for
the VEGF-R (U.S. Pat. No. 6,291,455B1, published in Sep. 18, 2001). 6)
SU101 shows selectivity for the PDGF receptor, but its antiproliferative
effects are in part due to an ring-opened metabolite which inhibits
dihydro-orotate dehydrogenase, a mitochondrial enzyme crucial to
pyrimidine biosynthesis. 7) Aryl and heteroaryl quinazoline compounds show
selectivity for CSF-R (U.S. Pat. No. RE 37,650 E, published in Apr. 9,
2002.8) SU 5416, a VEGF receptor (Flk1/KDR) antagonist was designed on the
basis of crystallographic studies of the indolin-2-one pharmacophore and
the FGF receptor tyrosine kinase domain. 9) Bis mono- and bicyclic aryl
and hetero aryl compounds show selectivity for EGFR and PDGFR (U.S. Pat.
No. 5,409,930). 10) Piceatannol (3,4,3,5V-tetrahydroxy-trans-stilbene)
shows selectivity for syk and lck, but also inhibits serine/threonine
kinases and ATPase. 11) Several compounds that are based on
benzodiazepines show some selectivity for the non-receptor tyrosine kinase
src and for the FGF-R tyrosine kinase receptor family. These examples show
that compounds with selectivity for one or a few tyrosine kinases can be
V. Anti-Malarial Effects of Protein Kinase Inhibitors
Like plants the related apicomplexan parasites such as plasmodium appear
not to produce protein tyrosine kinases. A few reports suggest that
protein tyrosine phosphorylation occurs in plasmodium (see section A
below). However homology searches have failed to detect any sequences
related to the known protein tyrosine kinase families. Therefore it is
conceivable that antimalarial effects of protein tyrosine kinase
inhibitors are due to the inhibition of the enzymes that are produced by
the human host. A variety of quinazoline derivatives have been reported to
have antimalarial activity. These compounds include
2,4-diamino-6(3,4-dichlorobenzylamine quinazoline (PAM1392 [Thompson et
al. Exp. Parasitol 25:32-49, 1969)],
[Schmidt and Rossan, Am. J. Trop. Med. Hyg. 28:781-92, (1979)], several
other 2,4-diamine-6-substituted quinazoline derivatives Elslager and
colleagues [Elsager et al., J. Med. Chem. 21:1059-70, (1978)] and by
Chinese scientists [Gy et al., Xao Xue Bao 19:108-18, (1984), Yao et al.,
Yao Xue Bao 19:76-8, (1984)]. The antimalarial activity of
2,4-diamino-5-methyl-693,4,5-trimethoxyanilinomethyl) quinazoline salts is
described in U.S. Pat. No. 4,376,858, published in Mar. 15, 183. One
possible mode of action of quinazoline derivatives against plasmodium are
inhibition is the inhibition of tyrosine kinases (U.S. Pat. No. 6,103,728,
published in Aug. 15, 2000).
A) Inhibition of Plasmodium Protein Kinases
1) Dluzeski and Garda reported that several protein kinase inhibitors (staurosporin,
genistein, methyl 2,5-dihydroxycinnamate, tyrphostin B44 and B46,
lavendustin A and RO3) inhibited the erythrocytic cycle of plasmodium
falciparum [Dluzewski and Garda, Experientia 52:621-623, (1996)]. With the
exception of staurosporin, a strong serine/threonine kinase inhibitor,
these compounds preferentially inhibit protein tyrosine kinases. These
inhibitors prevented the development of the parasites within erythrocytes
and/or invasion. Because of the broad spectrum of activities of these
inhibitors it is not clear whether inhibition of a protein tyrosine kinase
played any role in the observed effects, nor is it clear whether the
target proteins were derived from the erythrocytes or from the parasites.
2) While screening artemisinin like compounds from microorganisms, Tanaka
and colleagues identified seven fungal metabolites with antimalarial
activity. One of these compounds, radicicol, a broad spectrum protein
kinase inhibitor was moderately active against Plasmodium berghei in mice
[Tanaka et al., J. Antibiot 51:153-60, (1998)].
3) More recently Sharma reported that a membrane bound PTK activity was
increased during maturation from the ring stage to the trophozoite stage.
Inhibition of the PTK activity by chloroquine was proposed to represent
one possible mechanism of action of this drug against the parasite [Sharma
and Mishra, Indian J. Biochem. Biophys. 36:299-304 (1999); Sharma, Indian
J. Exp. Biol. 38:1222-6 (2000)].
B) Inhibition of Human Protein Tyrosine Kinases
A variety of pathogenic effects of plasmodium are mediated by protein
tyrosine kinases of the human host and thus can be inhibited by protein
tyrosine kinase inhibitors. Several examples have been reported in the
1) Adhesion of infected erythrocytes to vascular endothelium involves the
binding of P. falciparum membrane protein 1 (PfEMP1) to CD36 that is
expressed by endothelial cells of the host. A signal mediated by CD36 is
essential for adhesion. The pyrazolopyrimidine PP1, a selective inhibitor
of src and lck kinases, inhibits this signal and prevents adhesion [Yipp
et al., Blood online, (2002)].
2) CD36 and CD36 mediated, protein kinase dependent signals are also
involved in the nonopsonic clearance of P. falciparum infected
erythrocytes by monocytes and macrophages. Both genistein and selective
ERK and p38 MAPK inhibitors (PD98059 and SB203580, respectively) reduced
the uptake of infected erythrocytes to almost the same extent as CD36
blockade [McGilvray et al., Blood 96:3231-40, (2000)].
3) Glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium
falciparum. Malarial GPI induces rapid onset tyrosine phosphorylation of
multiple intracellular substrates within 1 min of addition to cells. These
signals are involved in the upregulation of parasite adherence and in the
induction of nitric oxide (NO) release by macrophages and endothelial
cells. Both adherence and NO release are prevented by the tyrosine kinase
antagonists tyrphostin and genistein [Tachado et al., J Immunol
156:1897-1907, (1996); Schofield et al., J. Immunol. 156:1886-96].
In previous work the protein tyrosine kinase receptor Met has not been
implicated in malaria infections. The present invention identifies the
protein tyrosine kinase Met as a crucial mediator of hepatocyte
susceptibility to infection by malaria sporozoites.
VI. HGF Related Anti-Malarials
A. Sulfated Polysaccharides
As mentioned above, HGF levels may be increased by HGF degradation
inhibiting polysaccharides including dextran sulfate, heparan sulfate,
dermatan sulfate, keratan sulfate, chondroitin, or chondroitin sulfate.
The combination of sulfated polysaccharides, such as sulfated curdlan,
dextrin sulfate, chondroitin sulfate, heparin, carageenan) with quinine
for the treatment of malaria is described in U.S. Pat. No. 5,780,452,
published in Jul. 14, 1998. The proposed strategy is based on the ability
of sulfated polysaccharides to inhibit the invasion of human erythrocytes
by malarial parasites. The present invention raises concerns regarding
this strategy, since sulfated polysaccharides may increase HGF levels by
inhibiting its degradation, a fact described in U.S. Pat. No. 5,736,506,
published in Apr. 17, 1998. Therefore sulfated polysaccharides are
excluded from the antimalarial agent of this invention.
Claim 1 of 5 Claims
1. A method of inhibiting malaria
infection, wherein the method comprises administering genistein to a human
in need thereof in an amount sufficient to inhibit infection of the human
by malaria parasites.
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