Polyamine Pathway as Drug Target against Malaria

Sammanfattning: 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.