Polyamine Pathway as Drug Target against Malaria

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

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.

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 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.

• author: Sprenger, Janina
• supervisor: Lo Persson; Jannette Carey; Salam Al-Karadaghi
• opponent: Professor Schramm, Vern, Albert Einstein College of Medicine, New York City, USA
• organization: Biochemistry and Structural Biology; Biogenic Amines (research group)
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Pharmacology and Toxicology
• 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
• 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