Polyamine Pathway as Drug Target against Malaria

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.