| Principal Investigator : Ram A Vishwakarma
Project Associates
/ Assistants Ph D Students Collaborators The
project aims at chemical synthesis and biosynthesis of GPI cell surface
molecules (Lipophosphoglycan, Proteophosphoglycans, and free GPIs) of Leishmania
parasite, and design and synthesis of the structural and functional mimics
and inhibitors of GPI biosynthetic pathway. The GPIs are expressed abundantly
on the cell surface of Leishmania, Trypanosoma and Plasmodium
species, and have been implicated in the infectivity, transmission and
intracellular survival of the parasites. The parasitic GPIs have distinct
structural features, compared to mammalian protein GPI-anchor, and are
interesting targets for chemical synthesis, biosynthesis and molecular design.
The objectives include, (i) design and synthesis of GPI anchor, LPG structural
domains (PI, GPI, conserved glycan-core, variable phosphoglycan repeats and
terminating cap), and GPI analogues, (ii) synthesis of labeled biosynthetic
precursors and their incorporation in the parasite to elucidate the steps
involved in the LPG/GPI assembly, (iii) design and synthesis of structural and
functional mimics, and mechanism based inhibitors of GPI biosynthetic pathway,
and (iv) role of parasitic GPIs and their synthetic analogues in PKC and
PI-3-kinase mediated events in macrophages. Lipophosphoglycan,
the major cell-surface molecule unique to Leishmania species,
represents one the most complex carbohydrates present in Nature posing
substantial challenge for organic synthesis and glycobiology. LPG is a
multifunctional virulence-factor critical for infectivity and survival of
parasite, and its remarkable function is manifested in severe inhibition (in
nM range) of PKC dependent signaling of host macrophages. Structure
of
LPG consists of four distinct domains: alkyl-lyso-GPI anchor, conserved
glycan core with internal galactofuranose residue, variable phosphoglycan
repeats, and neutral oligomannose cap.
In
the last couple of years we have reported syntheses of three individual
structural domains of LPG viz. the GPI anchor, tetrasaccharide cap and
phosphoglycan repeats, and synthesis of the last formidable target i.e.
conserved hexasaccharide core has been achieved this year. This has brought us
fairly close to completing first chemical synthesis of full-length LPG of L.
donovani. The synthesis of suitably functionalised glycan core (see the
following structure) containing internal galactofuranose residue was achieved
through a convergent route that required three key building blocks; (a)
Gal-1,6a-Gal-1,3a-Galf
intermediate (Segment-A) with a suitable protecting group at the non-reducing
(6-OH) end, (b) bifunctional Man-1,3a-Man
segment-B with suitably blocked 3-OH of distal mannose and (c) the
glucosamnine-inositiol acceptor (segment C). The trisaccharide A was
synthesized from following three intermediates, allyl-2,3,4-tri-O-benzyl-a-D-galactoside,
allyl-2,3,4-tri-O-benzyl-6-O-PMB-a-D-galactoside,
and 4,6-isopropylidene-2-O-benzoyl-D-galactonolactone, which themselves were
prepared in multi-step syntheses. The key reaction of this synthesis was the
construction of Galf
residue and reduction of galactonolactone to convert this trisaccharide into
appropriate donor. The segment-B was prepared from central intermediate
allyl-3-PMB-2,4,6-tri-O-benzyl-a-D-mannoside
which served as donor (allyl removal and activation) as well as acceptor (PMB
removal). This molecule was obtained by selective PMB protection of 3-OH, and
benzylation of allyl-a-D-mannoside.
Synthetic approach followed for preparation of Gln-Ino (segment C) was
essentially the same as reported last year, except that an efficient
deacetylation and selective 3,6-di-O-benzoylation of masked glucosamine
residue was applied. For the construction of full-length glycan core, the
trichloroacerimidate donor B was first coupled to 4-OH of the
glucosamine-inositol intermediate C to get required tetrasaccharide. Now the
3-OPMB from distal mannose was removed and coupled with Gal1,6aGal1,3aGalf
intermediate to obtain the target glycan core. With conserved glycan-core with
non-reducing end 6-OPMB correctly placed, we are poised to achieve first
chemical synthesis of full-length LPG by bringing PG domain at the
non-reducing end and glycerolipid at the 1-OH of inositol (required PG and
lipid intermediate already available from our efforts in preceding years).
This
year we have devised a new and efficient solid-phase methodology to construct
linear Leishmania phosphoglycans (upto 3 PG repeats) based on our
design and application of a novel cis-allyloxy-phosphoryl solid-phase
linker that enabled the selective cleavage of first anomeric-phosphodiester
linkage, using organometallic catalysis, without affecting any of the other
internal anomeric-phosphodiesters of growing PG chain on the solid support.
This strategy was further extended to construct larger phosphoglycans (19-22
repeats) in a one-pot synthesis by polycondensation of a single key
bifunctional H-phosphonate intermediate. Although this polycondensation
approach yielded heterogeneous mixture of 19-22 repeats, this allowed us to
initiate biophysical experiments using CD spectroscopy, and preliminary result
indicated helical nature of these phosphoglycans in solution. Another
interesting result this year was the observation of dual selectivity in
functionalisation of deprotected lactal (intermediate used in synthesis of
linear PGs of L. donovani) enabling regioselective PMB protection of
3-OH (among 6 free hydroxyls) followed by TBDMS protection of 6-OH of the Gal
residue. This intermediate turned out to be extremely important and opened up
the possibility of construction of branched phosphoglycans of Leishmania
major. This opportunity was exploited and taken to logical conclusion to
make desired branched phosphoglycans. In
Leishmania, the biosynthetic assembly of PG repeats occurs inside Golgi
(after the pre-assembled GPI core is translocated from ER to Golgi) and
involves a set of initiating and elongating Man-1a-PO4-transferases,
unique to Leishmania and capable of transferring intact man-1a-phosphate
(and not just the Man) from GDP-Man. Furthermore, presence of GDP-Man
transporter (GMP antipoter) specific to Leishmania Golgi membranes
makes the PG assembly even more interesting. Our interest has been to design
the mechanism based inhibitors of MPTs and GDP-Man transporter activities.
Last year we established synthetic routes for novel b-lactam
analogues of PG repeat, and several monofluoro-, difluoro- and chiral antipode
(with L-Man) of GDP-Man. These molecules have been biologically evaluated this
year using L.donovani membrane system and purified Golgi vesicles. This
has led to identification of two novel inhibitors (mM
range) of eMPT enzyme involved in PG assembly. Also one of analogue
demonstrated interesting property against GDP-Man tansporter of Golgi
vesicles. Among
the prominent biological activity displayed by the major Leishmania
GPIs (LPG and GIPLs) is the inhibition of macrophage functions such as PKC
dependent signaling pathway. The bioactivity of Leishmania GPIs is in
contrast to Trypanosoma brucei and Plasmodium falciparum GPIs,
which activate the macrophage functions. We have utilized some of the
synthetic material available to us for addressing this kind of biological
questions pertaining to Leishmania GPIs. To address the question as to
which structural domain of Leishmania GPIs is responsible for dramatic
down-regulation of PKC dependent transient c-fos expression, we have
chemically synthesized defined alkylacylglycerolipids domain of corresponding
GPIs (LPG and GIPLs of L.donovani), and evaluated them for inhibition
of PKC and c-fos gene expression in macrophages. The results unequivocally
demonstrated that the unusual lipid domain of Leishmania GPIs is
primarily responsible for inhibition of PKC dependent transient c-fos
gene expression (parallel experiments using synthetic PG and cap domains of
LPG did not show any appreciable inhibition). This
year we have prepared two new fluorescent (NBD) labeled GPI anchors and
started using them in our membrane experiments using synthetic GUVs. In
addition to our primary focus on Leishmania LPG, last year we initiated
synthetic efforts towards chemical synthesis of the GPI anchor of P.
falciparum to address questions related to glycobiology and immunology of
these so-called malaria toxins (with DC Gowda). This year we have made
substantial progress in synthesis of malarial GPI including the construction
of top tetra-mannose domain. This fragment is now being coupled to a carrier
protein for testing as synthetic vaccine. Under
the NMILTI program of CSIR (with A Mukhopadhyay and SK Basu), last year we
reported design strategy and synthesis of new drug conjugates of
antitubercular drug rifampicin with poly-G and fucoidin for scavenger receptor
mediated drug delivery into the macrophages. This year two new
rifampicin-fucoidin conjugates and a fluorescent (NBD) labeled analogue were
synthesized and used for macrophage experiments. In another project on natural products from medicinal plants, a novel anticancer drug lead was identified (with Arun Balakrishnan) which selectively inhibited telomerase, and activated c-myc and caspases leading to apoptosis in cancer cells. Publications Original
peer-reviewed articles 1.
Chawla M and Vishwakarma RA (2003) Alkylacylglycerolipid domain of Glycosyl
phosphatidylinositol (GPI) molecules of Leishmania is responsible for
inhibition of protein kinase C mediated c-fos gene expression in macrophages. J
Lipid Res 44:594-600. 2.
Arya R, Mehra A, Bhattacharya S, Vishwakarma RA and Bhattacharya A
(2003) Biosynthesis of Entamoeba histolytica proteophosphoglycan in-vitro. Mol
Biochem Parasitol 126:1-8. 3.
Giridharan P, Somasundaram ST, Perumal K, Vishwakarma RA, Karthikeyan
NP, Velmurugan R and Balakrishnan A (2002) Novel substituted
methylenedioxylignan suppresses proliferation of cancer cells by inhibiting
telomerase and activation of c-myc and caspases leading to apoptosis. Brit
J Cancer 87:98-105. Patents 1.
Jain DC, Bhakuni RS, Saxena S, Kumar S and Vishwakarma RA (2002) Preparation
of arteethers from dihydroartemisinin. US patent application no.
2000-535513, granted in Feb 2002 (US 6346631). 2.
Jain DC, Bhakuni RS, Saxena S, Kumar S and Vishwakarma RA (2001) Preparation
of arteethers. British patent application no. 2000-7261, granted in Sep
2001 (GB 2360517). 3.
Balakrishnan A, Vishwakarma RA and Ramakrishnan V (2001) Lignan compounds with
antiproliferative properties. PCT WO patent application no. 2000-IN 63,
granted in Dec 2001 (PCT-WO 2001096589). |
