Polyamine Pathway as Drug Target against Malaria
(2015) In Lund University Faculty of Medicine Doctoral Dissertation Series- Abstract
- Malaria, caused by the protozoan parasite Plasmodium falciparum is responsible for
about 600.000 death cases every year. Mainly affected are populations of subtropical
countries in Africa and the largest groups of victims are children below the age of 5
years. The fast evolving drug resistances of Plasmodium against the the most pow-
erful antimalarials threatens the successful containment of this disease in the future.
Therefore new, cheap and powerful antimalarial are urgently needed. A better under-
standing of the parasite’s unique molecular biology would help to identify new drug
targets and could predict resistances. This thesis describes aspects of drug design
... (More) - Malaria, caused by the protozoan parasite Plasmodium falciparum is responsible for
about 600.000 death cases every year. Mainly affected are populations of subtropical
countries in Africa and the largest groups of victims are children below the age of 5
years. The fast evolving drug resistances of Plasmodium against the the most pow-
erful antimalarials threatens the successful containment of this disease in the future.
Therefore new, cheap and powerful antimalarial are urgently needed. A better under-
standing of the parasite’s unique molecular biology would help to identify new drug
targets and could predict resistances. This thesis describes aspects of drug design
and the parasite’s unique feature of sequence insertions within conserved proteins by
studies on two enzymes of the polyamine pathway that are suggested drug targets.
These enzymes are S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine
synthase (SpdS) from Plasmodium falciparum.
The first part of this work describes the heterologous expression and biochemi-
cal characterization of Pf AdoMetDC. The enzyme contains a 150 amino acid long
Plasmodium specific insert domain, compared to its homologs. This domain is
known to interact with an ornithine decarboxylase domain (ODC) in the native
Pf AdoMetDC/ODC bifunctional enzyme. Using several biochemical and biophysical
techniques including limited proteolysis, CPMG-NMR, UV-CD and ab-initio SAXS
modeling it is shown that the quaternary structure, like that of the mammalian ho-
mologs, is a dimer. Furthermore comparison of SAXS models from Pf AdoMetDC
with and without the insert shows the positions of the insert domain. All together the
results give new insights into the structural biology Pf AdoMetDC/ODC complex and
demonstrate that the 150 amino acid insert domain mainly adopts a three-dimensional
structure.
The second part includes studies on Pf SpdS with the focus on inhibitor design.
Several structures of the enzyme with various potential inhibitors (described earlier
for homologous SpdS or newly discovered by virtual screening and rational design ap-
proach) bound are presented. Using enzyme activity assays and isothermal titration
calorimetry (ITC) the binding and inhibition of Pf SpdS by potential inhibitors is in-
vestigated. It is demonstrated that there is discrepancy between binding and inhibition
potency. Predicted inhibitors can bind to the enzyme in vitro without inhibiting the
enzyme activity. A sequential binding process, suggested earlier by crystallographic
data, is supported by the binding data, and is proposed to explain the discrepancies
between ligand-binding affinity and inhibition. The present findings may explain the
limited success of previous efforts at structure-based inhibitor design for Pf SpdS, and
they may be relevant for other drug targets that follow a sequential binding process. (Less) - Abstract (Swedish)
- Popular Abstract in English
An algae that has lost its ability to do photosynthesis over the last million years and
started to feed on human blood causes about 600.000 death every year, a number
equals about 8 times Lund’s present population. This evolved algae named Plasmod-
ium falciparum causes Malaria, a tropical disease mainly affecting economically weak
regions in Africa. Unlike bacteria or viruses, Plasmodium is an eukaryotic organism
as humans are, but it lives as a single cell. This parasite has a very complex biology
that is poorly understood today but it is known to evolve rapidly. Its fast adaptation
to the environment is problematic for the fight... (More) - Popular Abstract in English
An algae that has lost its ability to do photosynthesis over the last million years and
started to feed on human blood causes about 600.000 death every year, a number
equals about 8 times Lund’s present population. This evolved algae named Plasmod-
ium falciparum causes Malaria, a tropical disease mainly affecting economically weak
regions in Africa. Unlike bacteria or viruses, Plasmodium is an eukaryotic organism
as humans are, but it lives as a single cell. This parasite has a very complex biology
that is poorly understood today but it is known to evolve rapidly. Its fast adaptation
to the environment is problematic for the fight against Malaria, since it gains resis-
tances against effective antimalarials very fast. In 2014 reports of resistances against
the most powerful antimalaria drug today, artemisinin, are a big warning sign that we
might lose the fight against the disease if new drugs are not found soon.
But how can we find new drugs? In the last century most drugs against malaria
were extracts or isolated compounds from plants. Research developments in the last
decades allows the design of drugs against a specific target of the pathogen. These
targets are mainly enzymes, the working horses of every cell that are running the
metabolism. Enzymes catalyze specific reactions in metabolic pathways and are large
polymers of up to thousands of amino acids. They also have a three dimensional
structure that is required for their function and the structure can be visualized by
x-ray crystallography, but it requires the growth of protein crystals which is a difficult
process in some cases. Knowing the structure, molecules can be designed that bind to
that enzyme and potentially inhibit it and eventually kill the parasite.
Two plasmodial enzymes and potential drug targets S-adenosylmethionine decar-
boxylase (AdoMetDC) and spermidine synthase (SpdS) that are involved in the syn-
thesis of small molecules called polyamines that are important for cell growth of the
parasite are the subjects of this thesis work. AdoMetDC has several features that are
typical for malarial proteins and are associated with the organism’s fast evolution.
This enzyme is about 50 % longer than the equivalent in other organisms due to so
called ’amino acid insertions’. In this thesis a variety of biochemical and biophysical
studies provide new insights into possible evolution and structure of these insertions
and the AdoMetDC enzyme itself that might apply for other plasmodial enzymes. The
second enzyme, SpdS has a known crystal structure and the design of inhibitors that
are specific for this enzyme is one focus of the present work. Conventional methods
to find new inhibitors using computational screenings have a very low success rate,
but studies on the mechanism presented here on how this enzyme binds its ligands
provide a new strategy to find potential drugs. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/5276391
- author
- Sprenger, Janina LU
- supervisor
-
- Lo Persson LU
- Jannette Carey LU
- Salam Al-Karadaghi LU
- opponent
-
- Professor Schramm, Vern, Albert Einstein College of Medicine, New York City, USA
- organization
- publishing date
- 2015
- type
- Thesis
- publication status
- published
- subject
- keywords
- Amino acid insertions, Crystallography, polyamine pathway, drug design, enzyme inhibition, ITC, ligand binding, malaria, Plasmodium falciparum, S-Adenosylmethionine decarboxylase, spermidine synthase.
- categories
- Higher Education
- in
- Lund University Faculty of Medicine Doctoral Dissertation Series
- pages
- 206 pages
- publisher
- Biogenic Amines
- defense location
- Belfragesalen, BMC D15, Klinikgatan 32, Lund
- defense date
- 2015-05-11 10:00:00
- ISSN
- 1652-8220
- ISBN
- 978-91-7619-125-5
- language
- English
- LU publication?
- yes
- id
- c27d2610-48df-45aa-814c-60dce0cdc61c (old id 5276391)
- date added to LUP
- 2016-04-01 14:37:45
- date last changed
- 2019-05-22 05:53:01
@phdthesis{c27d2610-48df-45aa-814c-60dce0cdc61c, abstract = {{Malaria, caused by the protozoan parasite Plasmodium falciparum is responsible for<br/><br> about 600.000 death cases every year. Mainly affected are populations of subtropical<br/><br> countries in Africa and the largest groups of victims are children below the age of 5<br/><br> years. The fast evolving drug resistances of Plasmodium against the the most pow-<br/><br> erful antimalarials threatens the successful containment of this disease in the future.<br/><br> Therefore new, cheap and powerful antimalarial are urgently needed. A better under-<br/><br> standing of the parasite’s unique molecular biology would help to identify new drug<br/><br> targets and could predict resistances. This thesis describes aspects of drug design<br/><br> and the parasite’s unique feature of sequence insertions within conserved proteins by<br/><br> studies on two enzymes of the polyamine pathway that are suggested drug targets.<br/><br> These enzymes are S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine<br/><br> synthase (SpdS) from Plasmodium falciparum.<br/><br> The first part of this work describes the heterologous expression and biochemi-<br/><br> cal characterization of Pf AdoMetDC. The enzyme contains a 150 amino acid long<br/><br> Plasmodium specific insert domain, compared to its homologs. This domain is<br/><br> known to interact with an ornithine decarboxylase domain (ODC) in the native<br/><br> Pf AdoMetDC/ODC bifunctional enzyme. Using several biochemical and biophysical<br/><br> techniques including limited proteolysis, CPMG-NMR, UV-CD and ab-initio SAXS<br/><br> modeling it is shown that the quaternary structure, like that of the mammalian ho-<br/><br> mologs, is a dimer. Furthermore comparison of SAXS models from Pf AdoMetDC<br/><br> with and without the insert shows the positions of the insert domain. All together the<br/><br> results give new insights into the structural biology Pf AdoMetDC/ODC complex and<br/><br> demonstrate that the 150 amino acid insert domain mainly adopts a three-dimensional<br/><br> structure.<br/><br> The second part includes studies on Pf SpdS with the focus on inhibitor design.<br/><br> Several structures of the enzyme with various potential inhibitors (described earlier<br/><br> for homologous SpdS or newly discovered by virtual screening and rational design ap-<br/><br> proach) bound are presented. Using enzyme activity assays and isothermal titration<br/><br> calorimetry (ITC) the binding and inhibition of Pf SpdS by potential inhibitors is in-<br/><br> vestigated. It is demonstrated that there is discrepancy between binding and inhibition<br/><br> potency. Predicted inhibitors can bind to the enzyme in vitro without inhibiting the<br/><br> enzyme activity. A sequential binding process, suggested earlier by crystallographic<br/><br> data, is supported by the binding data, and is proposed to explain the discrepancies<br/><br> between ligand-binding affinity and inhibition. The present findings may explain the<br/><br> limited success of previous efforts at structure-based inhibitor design for Pf SpdS, and<br/><br> they may be relevant for other drug targets that follow a sequential binding process.}}, author = {{Sprenger, Janina}}, isbn = {{978-91-7619-125-5}}, issn = {{1652-8220}}, keywords = {{Amino acid insertions; Crystallography; polyamine pathway; drug design; enzyme inhibition; ITC; ligand binding; malaria; Plasmodium falciparum; S-Adenosylmethionine decarboxylase; spermidine synthase.}}, language = {{eng}}, publisher = {{Biogenic Amines}}, school = {{Lund University}}, series = {{Lund University Faculty of Medicine Doctoral Dissertation Series}}, title = {{Polyamine Pathway as Drug Target against Malaria}}, year = {{2015}}, }