Falcipain project focusing on cysteine proteases

The falcipain project dates back to the late 1980s, when scientists at the University of California, San Francisco (UCSF) identified a role for cysteine proteases in the breakdown of haemoglobin by malaria parasites. During the erythrocytic stage of their life cycle, which is the stage responsible for the disease in humans, malaria parasites live within red blood cells. The parasites take up haemoglobin from these cells, and break down this protein to provide amino acids for their growth. The UCSF group discovered that, when parasites were treated with inhibitors of cysteine proteases, haemoglobin breakdown was blocked and they failed to develop. Notably, after treatment with cysteine protease inhibitors, the parasite food vacuole, the organelle in which haemoglobin breakdown takes place, becomes swollen and filled with undegraded haemoglobin. This abnormality serves as a “signature” for effective inhibition of parasite cysteine proteases.

Subsequent work identified a family of cysteine proteases of P. falciparum, known as falcipains. Falcipain -2 and falcipain -3 appear to be the key enzymes required for haemoglobin break-down. Studies of these enzymes have included:

• the production of large quantities in bacteria for inhibitor screening, biochemical characterization and structure determination;
• stage-specific localization to the food vacuole of trophozoites (falcipain-2) and schizonts (falcipain -3);
• disruption of protease genes to characterize their functions as haemoglobinases;
• characterization of the specific biochemical roles of unique falcipain domains and determination of protease structures

In summary, molecular studies confirm critical roles for falcipain-2 and falcipain-3 in erythrocytic malaria parasites, suggesting that these are appropriate tar-gets for antimalarial chemotherapy.

A number of classes of proteases probably contribute to the process of haemoglobin hydrolysis. Most notably, in addition to the falcipains, a family of plasmpesin aspartic proteases has also been shown to break down haemoglobin and localize to the food vacuole. Interestingly, cysteine and aspartic protease inhibitors demonstrate synergistic antimalarial activity, suggesting that they act cooperatively to break down haemoglobin and that inhibition of both classes of enzymes may offer a potent means of blocking parasite development.

Older studies demonstrated potent antimalarial activity of a number of classes of cysteine protease inhibitors. This current project built on interest at SmithKline Beecham (now GlaxoSmithKline – GSK) in the inhibition of human cysteine proteases in the treatment of osteoporosis. Compounds from corporate inhibitor libraries demonstrated potent antimalarial activity, providing initial leads for a drug discovery programme. The project was initiated in October 2001, and shortly thereafter, in March 2002, the GSK Diseases of the Developing World group at Tres Cantos, Spain, took on a principal role in drug discovery. Since then, the project has explored multiple classes of cysteine protease inhibitors as potential antimalarial drugs.

Initial classes of cysteine protease inhibitors demonstrated potent activity against falcipain cysteine proteases and against cultured malaria parasites. Some compounds also demonstrated activity against animal models of malaria, including a standard Plasmodium berghei mouse model and a new model, optimized at GSK, utilizing immunocompromised mice infected with P. falciparum. However, these initial classes were limited by expensive synthetic routes and, in some cases, compound instability, leading to searches for new lead classes. In May 2004, the study of a new promising class began, and this class remains of principal interest at this time.

The new lead class offers simple routes of synthesis, potent inhibition of falcipains, and in limited studies available to date, good drug properties, including pharmacokinetics, oral bioavailability and safety. An initial lead compound was recently studied in vivo. This compound demonstrated antimalarial effects in the P. berghei mouse model, and in the P. falciparum immunocompromised mouse model it cured animals after a four-day treatment course. Importantly, parasites collected from these animals during treatment demonstrated an obvious block in haemoglobin hydrolysis, with swollen, dark-staining food vacuoles, proving that the lead compound was acting against the intended falcipain target in exerting its antimalarial activity. Project efforts are now concentrated on optimizing members of this class as antimalarial falcipain inhibitors with optimal properties for new antimalarial drugs.

The current project goal is to progress through lead optimization and to generate compounds with in vitro and in vivo antimalarial activity along with appropriate pharmacokinetic properties and reasonable potential cost of goods. Building on advances with the lead class since 2004, it is hoped that a candidate for preclinical development will be available in 2006.