Enzyme inhibitors and structure-aided drug-design

Tropical diseases are by far the leading infectious killer of the world. It is estimated that almost 50% of the world’s population are at risk and each year about 500 million people directly suffer from these diseases. Only malaria, according to WHO, kills over 1 million people a year, that is about 3000 a day, (http://www.rbm.who.int). The limited repertoire of available drugs, which are of relatively low potency and/or of high toxicity, together with the development of resistance to these drugs, currently constitute a major problem. Unfortunately development of new drugs have been largely neglected by pharmaceutical industry.

Among the biochemical pathways which have been considered as a potential target for drugs against malaria, leishmaniasis, trypanosoma (sleeping sickness) and onchocerciasis (river blindness) is the polyamine biosynthesis pathway. The key enzymes involved in the synthesis of these compounds are ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SPDS). Several inhibitors that have originally been developed against human enzymes (for the treatment of cancer) were shown to have an effect on parasites (Heby et al., 2007).

We have solved the crystal structure of ODC in complex with the one of the most potent inhibitors, 1-aminooxy-3-aminopropane (APA) and studied the effect of this inhibitor on ODC from Leishmania donovani, the causative agent of leishmaniasis (Tamu Dufe et al., 2007). We have also studies the enzyme SPDS from C. elegans, a model organisms used in the study of parasitic nematodes (Tamu Dufe et al., 2005), and from Plasmodium falciparum, the causative agent of malaria (Tamu Dufe et al., 2007). We have determined the structure of SPDS in complex with substrate and two different inhibitors. These complexes open up the way for the design of better inhibitors of these enzymes, which hopefully can be developed to be drug candidates (Jacobsson et al., 2008).

Dihydroorotate dehydrogenase (DHODH) (EC 1.3.99.11) is a key enzyme in nucleotide synthesis. It catalyzes the fourth committed step in the de novo biosynthesis of pyrimidines. In this step of the synthesis, (S)-dihydroorotate (DHO) is stereospecifically oxidized to orotate, while the prosthetic flavin (FMN) group is reduced. Inhibition of DHODH leads to reduced levels of essential pyrimidine precursors, among which is UMP, a critical component of RNA and DNA synthesis. Most organisms are able to both synthesize and salvage pyrimidine bases; however, rapidly proliferating human cells such as activated T-lymphocytes and cancer cells are dependent on de novo nucleotide synthesis to meet their growth requirements. The suggestion that DHODH inhibitors may be used as antiproliferative agents in cancer therapy and in the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections.tropical parasitic infections is based on these findings.

In collaboration with Active Biotech AB and Saromics AB, in a drug design project we solved the structure of DHODH in complex with two inhibitors: a brequinar analogue and a novel inhibitor (a fenamic acid derivative), as well as the first structure of the enzyme to be characterized without any bound inhibitor (Walse et al., 2008). Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone, undergo changes in conformation upon inhibitor binding. Using our data, the loop regions Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/co-factor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.