Malaria is a disease that causes more than 1 million deaths per year world wide and more than 400 million clinical cases. Due to the acquired resistance of Plasmodium falciparum to the drugs used to control the infection, searching for new anti-malaria drugs is necessary in modern days. Recent studies have shown that the parasite synthesizes fatty acids using a fatty acid synthase type II (FAS-II) instead of a type-I fatty acid synthase (FAS-I) that is present in other eukaryotes. Plasmodium falciparum enoyl reductase (PfENR) is responsible for the last step of fatty acid biosynthesis in the parasite. This enzyme is located within the apicoplast, a plastid-like organelle that is responsible for several important metabolic pathways, including fatty acid biosynthesis. It is known that triclosan is an inhibitor of ENR in bacteria and we and others have shown that it is also effective against ENR in apicomplexan organisms such as P. falciparum. However triclosan cannot be used to treat malaria in humans because it has metabolic liability (glucoronidation) which limits its inhibitory potency. We have used X-ray crystallography and a Structural Activity Relationship (SAR) strategy to design and cocrystallize a tertiary complex of PfENR with NAD+ and triclosan derivatives to improve their properties as drugs to treat malaria. More than five hundred triclosan derivatives were synthesized, and their in vitro and in vivo inhibitory activity evaluated. Furthermore, structural studies were made of their affinity to interact with residues in PfENR active sites, as well as with the cofactor NAD+. A total of six PfENR-NAD+-triclosan analog/complexes structures were determined. Analogs which had replacements of chloride groups at position 5 of ring A and 4' of ring B were determined, allowing the structural analysis of the binding of these triclosan analogs to PfENR. In addition, the urea derivatives (modification at position 1) as well as phenylsulphonamides (modification at position 2') have shown to be more potent inhibitors than triclosan in the in vivo assay. The analysis of the inhibitory properties and the structure of these analogs bound to PfENR will provide novel compounds in the search for new anti-malarial drugs.

Tuberculosis and malaria are among the most deadly infectious diseases in the world. The prevalence in regions without well-established public health causes economical and financial burdens for both society and patients. There is an urgent need to find effective treatments due to the emergence of drug-resistant strains. The aim of the studies reported here was to gain knowledge from the protein structures that can lead to the elimination of these pathogens. In these studies, protein crystallography is the main method used to solve protein structure. Based on the protein structure, we used different methods to characterize the protein function of three lipid-binding proteins (LprG, LprA, and gp232), and to identify potent inhibitors against Plasmodium falciparum enoyl-ACP reductase (PfENR), a drug target protein involved in central lipid metabolism. To characterize the function of two lipid-binding proteins (LprG and LprA), liquid chromatography-mass spectrometry (LC-MS) was used to analyze the ligand extract. In the study of tail fiber protein from mycobacteriophage, we used protein sequence alignment to identify gp232 as a major tail fiber protein, which potentially binds to lipids on the cellular surface of mycobacteria. A pull-down assay and imaging methods (fluorescence microscopy and electron microscopy) were conducted to confirm the function of gp232. In the structural study of PfENR, the structure-activity relationships method was used to find potent inhibitors against PfENR, which would show stronger inhibition than the known inhibitor triclosan. The triclosan-like analogs with modification at the 5-position revealed a new binding site in PfENR that has great potential for improving the potency of inhibition. We found that two inhibitors containing the core structure of piperidine and tetrahydroquinoline reached this new binding site and were 10-fold more potent than triclosan. The structural study of PfENR provides structural insights into the inhibitor-binding site that can lead to the discovery of new drugs. The comprehensive knowledge that we gained from the structural studies of these lipid-binding proteins provide new information that could lead to a greater understanding of pathogen physiology or guide the discovery of effective treatments to eliminate the pathogens.