Malaria is one of the major public health problems in the world. Approximately 500 million people are afflicted and almost 3 million people die from the disease each year. Of the four causative species Plasmodium falciparum is the most lethal. Due to the rapid spread of parasite resistance there is an urgent need for new antimalarial drugs with novel mechanisms of action. Several promising targets for drug intervention have been revealed. This thesis addresses the parasitic aspartic proteases termed plasmepsins (Plm), which are considered crucial to the hemoglobin catabolism essential for parasite survival. The overall aim was to identify inhibitors of the P. falciparum Plm I, II, and IV. More specific objectives were to attain activity against P. falciparum in infected erythrocytes and selectivity versus the most homologous human aspartic protease cathepsin D (Cat D). To guide the design process the linear interaction energy (LIE) method was employed in combination with molecular dynamics. Initial investigations of the stereochemical requirements for inhibition resulted in identification of an L-mannitol derived scaffold encompassing a 1,2-dihydroxyethylene transition state isostere with affinity for Plm II. Further modifications of this scaffold provided inhibitors of all three target plasmepsins (Plm I, II, and IV). Apart from the stereochemical analysis three major kinds of manipulation were explored: a) P1/P1′ and P2/P2′ side chain alterations, b) replacement of amide bonds by diacylhydrazine, 1,3,4-oxadiazole, and 1,2,4-triazole, and c) macrocyclization. Several inhibitors of Plm I and II with Ki values below 10 nM were discovered and one Plm IV selective inhibitor comprising two oxadiazole rings was found which represents the most potent non-peptide Plm IV inhibitor (Ki = 35 nM) reported to date. Some of the identified plasmepsin inhibitors demonstrated significant activity against P. falciparum in infected erythrocytes and all inhibitors showed a considerable selectivity for the plasmepsins over the human Cat D.

Today, the demand for speed in drug discovery is constantly increasing, particularly in the iterative processes of hit validation and expansion and lead optimization. Irradiation with microwaves (MWs) has been applied in the area of organic synthesis to accelerate chemical reactions and to facilitate the generation of new chemical entities since 1986. In the work presented in this thesis, the use of MW-mediated heating has been expanded to address three fields of drug discovery, namely hit expansion, chemical library generation and genomics. In the first project, potential inhibitors of malaria aspartic proteases were designed and synthesized, partly by MW-assisted organic chemistry, and evaluated with regard to their inhibitory efficacy on five malaria aspartic proteases and their selectivity over two human aspartic proteases. The synthetic work included the development of fast and convenient methods of MW-assisted formation of thiazolidines and epoxy esters. Some of the resulting structures proved to be efficacious inhibitors of the aspartic protease that degrades haemoglobin in all four malaria parasites infecting man. No inhibitor affected the human aspartic proteases. Expedient, two-step, single-operation synthetic routes to heterocycles of medicinal interest were developed in the second and third projects. In the former, the use of a versatile synthon, Ph3PCCO, provided α,β-unsaturated lactones, lactams and amides within 5–10 minutes. In the latter project, saturated lactams were formed from amines and lactones in 35 minutes, in the absence of strong additives. These two MW-mediated protocols allowed the reduction of the reaction time from several hours or days to minutes. In the fourth project, a fully automated MW-assisted protocol for the important enzyme-catalysed polymerase chain reaction (PCR) was established. In addition, the PCR reaction could be performed in unusually large volumes, 2.5 mL and 15 mL, with yields corresponding to those from conventional PCR. Good amplification rates suggested that the thermophilic enzyme, Taq polymerase, was not affected by the MW radiation.

Enolate formation by proton abstraction from an sp3-hybridized carbon atom situated next to a carbonyl or carboxylate group is an abundant process in nature. Since the corresponding nonenzymatic process in water is slow and unfavorable due to high intrinsic free energy barriers and high substrate pKa s, enzymes catalyzing such reaction steps must overcome both kinetic and thermodynamic obstacles. Computer simulations were used to study enolate formation catalyzed by glyoxalase I (GlxI) and 3-oxo-Δ5-steroid isomerase (KSI). The results, which reproduce experimental kinetic data, indicate that for both enzymes the free energy barrier reduction originates mainly from the balancing of substrate and catalytic base pKas. This was found to be accomplished primarily by electrostatic interactions. The results also suggest that the remaining barrier reduction can be explained by the lower reorganization energy in the preorganized enzyme compared to the solution reaction. Moreover, it seems that quantum effects, arising from zero-point vibrations and proton tunnelling, do not contribute significantly to the barrier reduction in GlxI. For KSI, the formation of a low-barrier hydrogen bond between the enzyme and the enolate, which is suggested to stabilize the enolate, was investigated and found unlikely. The low pKa of the catalytic base in the nonpolar active site of KSI may possibly be explained by the presence of a water molecule not detected by experiments. The hemoglobin-degrading aspartic proteases plasmepsinI and plasmepsin II from Plasmodium falciparum have emerged as putative drug targets against malaria. A series of C2- symmetric compounds with a 1,2-dihydroxyethylene scaffold were investigated for plasmepsin affinity, using computer simulations and enzyme inhibition assays. The calculations correctly predicted the stereochemical preferences of the scaffold and the effect of chemical modifications. Calculated absolute binding free energies reproduced experimental data well. As these inhibitors have down to subnanomolar inhibition constants of the plasmepsins and no measurable affinity to human cathepsin D, they constitute promising lead compounds for further drug development.