This thesis entitled “Mechanism of Anticancer and Antimalarial Action of
a Modulator of Heat Shock Proteins” describes the successful elucidation of the
mechanism of anticancer and antimalarial action of 15-Deoxyspergualin (DSG).
DSG, a relatively well known immunosuppressant and antitumor molecule has
been demonstrated to kill the malaria parasite in vitro and in vivo (Midorikawa et
al., 1997; Midorikawa et al., 1998). A highly polar molecule, DSG binds the
carboxy terminal “EEVD” motif of heat shock proteins, Hsp70 and Hsp90,
enhances the ATPase activity of Hsp70 (Nadler et al., 1992; Nadler et al., 1998),
and modulates several seemingly unrelated cellular processes. DSG has also been
demonstrated to inhibit protein synthesis and polyamine synthesis in cells
(Kawada et al., 2002; Hibasami et al., 1991), and previously speculated to inhibit
malaria parasite growth by inhibiting polyamine synthesis. The grim situation
with regard to malaria infection and mortality, principally an offshoot of the
emergence of chloroquine resistant strains of the causative agent of malaria -
Plasmodium falciparum, calls for intense efforts towards developing efficacious
antimalarial agents with few side effects. DSG, having been used already in graft
rejection cases in man and demonstrated to potently inhibit malaria in mice
(Midorikawa et al., 1997), offers promise in this regard. It was, therefore, of
interest to solve the mystery of its mechanism of antimalarial action.
Chapter 1 surveys literature related to DSG mechanism of action and
presents the thesis objective. Chapter 1 also gives an overview of heat shock
proteins and their role in cancer, and the biology of the malaria parasite
(Plasmodium falciparum), the working of the principal metabolic pathways
existing in it, and a description of processes related to the intriguing, relict plastid present in apicomplexans. The metabolic processes previously speculated to be targeted by DSG, and those later found to be involved in DSG mechanism of action – polyamine synthesis and transport, protein synthesis and apicoplast
processes are dealt with in more detail. Though DSG has been speculated to kill
the malaria parasite by inhibiting polyamine synthesis, that DSG could clear
malaria infection in Plasmodium berghei infected mice did not corroborate with
the observation that inhibitors of polyamine biosynthesis are incapable of
inhibiting the malaria parasite in vivo probably because the parasites make do with
polyamines salvaged from the host (Assaraf et al., 1984; Bitonti et al., 1987). On
the other hand, DSG is known to bind heat shock proteins, and inhibit protein
synthesis, and heat shock proteins are speculated to be involved in the activation
of HRI (heme regulated inhibitor), a type of eIF2á kinase that phosphorylates the
eukaryotic initiation factor, eIF2á in conditions of heme deficiency or other
cellular stress. eIF2á phosphorylation leads to stalling of protein synthesis. It
seemed likely that if HRI is activated upon sequestration of heat shock proteins by
DSG, it would culminate in protein synthesis inhibition and ultimately, cell death.
With the intention to investigate this line of thought, the PlasmodB
database was mined for proteins essential to the existence of heme dependent
protein synthesis in Plasmodium falciparum. Two Hsp70 proteins from
Plasmodium falciparum, one with the carboxy terminal “EEVD” motif implicated
in DSG binding, and one without, and an Hsp70 interacting protein were cloned
and expressed in their recombinant form in Escherichia coli. The preliminary
characterization of these heat shock proteins described in Chapter 2 revealed that
they were functionally active. DSG did not inhibit either the chaperone activity of
the Hsp70s or the interaction of Hsp70 with Hip, but stimulated their ATPase
activity as anticipated.
Chapter 3 gives a complete picture of the mechanism of protein synthesis
inhibition by DSG in the standard protein synthesis system – reticulocyte lysate.
The experiments carried out revealed that DSG inhibits protein synthesis precisely
through the mechanism envisaged, i.e. through phosphorylation of HRI following
sequestration of Hsp70. Experiments involving exogenous addition of heat shock
protein to in vitro translation reactions confirmed this hypothesis. Moreover, DSG
inhibited protein synthesis in cancer cells in vivo, too, and HRI knockdown cells
were not affected by DSG. Interestingly, the Hsp70 levels in various cancer cell
lines inversely correlated with the inhibitory activity of DSG, and modulation of
Hsp70 levels through standard methods altered DSG inhibition of protein
synthesis in these cells. It was thus confirmed that DSG did indeed inhibit
mammalian cells through the pathway envisaged. Its previously reported
antitumor property is probably through this outlined mechanism of interference
with protein regulation.
In the malaria parasite, too, DSG inhibited protein synthesis through eIF2
alpha phosphorylation following Hsp70 sequestration as outlined in Chapter 4.
However, while the concentration of DSG required for inhibition of malaria
parasite growth was in the nanomolar range, high micromolar concentrations of
DSG were required to effect protein synthesis inhibition in the malaria parasite,
indicating that yet another target for DSG existed in the malaria parasite.
With protein synthesis no longer a candidate target of DSG, I looked into
the previously implicated polyamine synthesis pathway. In the event of DSG
inhibiting polyamine transport in addition to polyamine biosynthesis, it would be
expected to clear malaria infection in vivo contrary to other inhibitors of
polyamine biosynthesis. In Chapter 5, evidence for the polyamine synthesis
pathway in the malaria parasite is provided. Experiments involving incorporation
of radiolabeled precursors in the malaria parasite and in mammalian cells,
however, revealed that only high micromolar concentrations of DSG inhibit
polyamine synthesis. Polyamine transport was also studied in considerable detail
in malaria parasite infected red blood cells. Though infected red blood cells
demonstrated different kinetic parameters, implying that new polyamine
transporters were employed by the parasite on the red blood cell upon infection,
DSG did not potently inhibit polyamine transport, either.
The mystery of the target of DSG in the malaria parasite was, however, close to solution, when the growth inhibition of the malaria parasite by DSG was studied carefully. DSG invoked “delayed death” – a phenomenon wherein death is invoked only one cycle after incubation with the inhibitor. “Delayed death” is
typical of inhibitors that target apicoplast processes (Fichera and Roos, 1997).
DSG did not inhibit either fatty acid synthesis or prokaryotic protein synthesis –
processes that occur in the apicoplast, but effected a decrease in the amount of
nucleus encoded proteins that are targeted to the apicoplast, suggesting that it
inhibited the trafficking of nucleus encoded proteins to the apicoplast. Confocal
microscopy of parasites transfected with GFP fusion protein confirmed these
findings, and is described in Chapter 6.
The thesis ends with a summary of the findings in Chapter 7. Apicoplast
processes have always been considered to harbor immense potential in the
development of antimalarial agents, thanks to the absence of an equivalent
organelle and hence pathways, in the human host. Trafficking of nucleus encoded
proteins to the apicoplast has remained unexplored however. The work done in
this thesis not only serves to demystify DSG with regard to its mechanism of
action, but also paves the way for further studies in this area of intracellular
trafficking, which could help in the development of more efficacious antimalarial
agents. It also adds a new dimension to previous work conducted with regard to
the anticancer action of DSG.
Appendix 1 revolves around inhibitors which target various apicoplast
processes. Apicoplast processes have been conventionally linked to the intriguing
but unfortunate (with respect to clinical application) “delayed death”. Results
presented in this section demonstrate that not all apicoplast processes invoke
“delayed death”. Inhibition of apicoplast processes such as fatty acid biosynthesis
and heme synthesis evoke rapid death. Inhibitors designed to target these
processes could, therefore, be highly efficacious.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/326 |
| Date | 06 1900 |
| Creators | Ramya, T N C |
| Contributors | Surolia, Avadhesha |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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