Anti-leishmanial Drug Discovery: Rising to the Challenges of a Highly Neglected Disease
Leishmaniasis is a parasitic disease second only to malaria in terms of parasite-related mortality (1). Commonly associated with the developing world, leishmaniasis affects an estimated 10–20 million people, with 1.5–2.5 million new cases (as well as 70,000 deaths) attributed to the disease each year (2, 3). An additional 350 million people are at risk of infection in developing nations. The actual number of cases of leishmaniasis is probably underestimated as leishmaniasis is a reportable disease in only 41 of the the 91 countries where it is known to be present. Increasing migration and travel to leishmaniasis endemic regions as well as global climate and local environmental changes are making leishmaniasis a significant risk for people in the US and other regions previously unaffected by the disease (4, 5). Thus, there has been an expansion in the geographic distribution of leishmaniasis and an increase in the total number of leishmaniasis cases, often in epidemic proportions (6, 7). The impact of leishmaniasis extends beyond the immediate effects on human health to include significant cultural, societal, economic, and psychological repercussions owing to disfiguring lesions, disability, and death (8).
There are three major clinical manifestations of leishmaniasis (Table 1). The most common form is cutaneous (CL), which is caused predominantly by Leishmania (L.) major, L. aethiopica and L. tropica in the Old World and L. braziliensis, L. panamensis, and L. mexicana in the New World (Table 1) (9). CL is typified by self-healing lesions; however, the healing process can involve months or years and varies with the infecting parasite species (9). Muco-cutaneous leishmaniasis (M-CL) is characterized by the destruction of the nasal septum or palate and occurs months or years after the healing of a primary CL lesion (10). The most frequently M-CL-associated infecting species is L. braziliensis. Visceral leishmaniasis (VL) (caused by L. donovani, L. infantum) is the most dangerous form of the disease, targeting the lymph nodes, spleen, liver, and bone marrow (11). In contrast to CL and M-CL, VL can be fatal if left untreated.
Predominant Clinical Manifestations, Characteristics, and Geographic Distribution of Leishmaniasis
Transmission of leishmaniasis occurs through the bite of infected female Phlebotomus (Old World) and Lutzomyia (New World) sandflies (12). In rare instances, leishmaniasis can also be transmitted through blood transfusions, especially to individuals with immature or compromised immune systems (13). The Leishmania parasite exists in two life-cycle states: 1) elongated, flagellated promastigotes in the sand fly vector and 2) rounded, aflagellated intracellular amastigotes found in host cells (12). Upon introduction to human hosts, the Leishmania promastigotes are phagocytosed by macrophages and neutrophils where they subsequently reside in the phagolysosomes as amastigotes (12, 14). As amastigotes, the Leishmania parasites replicate within the host cells causing them to lyse, allowing for the infection of additional cells by free (uninternalized) amastigotes, and perpetuating the parasitization of the host. The life cycle of the Leishmania parasite is completed when a naïve sandfly takes a blood meal from the infected host, allowing the sandfly to infect other human hosts. Currently, there are approximately thirty species of Leishmania with nearly twenty being capable of infecting humans (Table 1) (10, 15). Exposure to a particular Leishmania species is dependent upon geographical location (i.e., Old World or New World) and determines the primary clinical manifestation (10, 16).
Despite the prevalence of leishmaniasis and its substantial impact on human life, there are limited chemotherapeutics available for treatment and no vaccines or prophylactic drugs for any form of the disease (Table 2). Moreover, of the available anti-leishmanial treatments, many are decades old, cause some form of systemic toxicity, are painful to administer, or exhibit physicochemical liability (i.e., poor solubility), as do the antimonials and amphotericin B (17–20). Most regimens rely heavily on the pentavalent antimonials and amphotericin B formulations. Newer compounds are the oral agent miltefosine and both parenteral and topical paromomycin (Table 2). Substantial evidence suggests that Leishmania parasites can persist at the primary sites of infection and in lymph nodes even after therapeutic intervention, indicating that current drug treatments do not completely eliminate parasitic infection and relapse occurs if host immunity fails (21). Thus, there is a pressing need for new efficacious agents. Few drug-discovery efforts have been specifically undertaken to design and develop new chemotherapeutics for leishmaniasis. Those efforts that have done so have concentrated on compounds with known pharmacological actions and on chemical libraries with a small number of compounds (22, 23). Therefore, the identification of potential new leishmanicidal chemotypes has been constrained by the limited diversity of the compound library. In addition, anti-leishmanial drug discovery programs have not had the screening throughput and the commitment of resources seen with other diseases, making leishmaniasis one of the most neglected diseases (8). Recently, however, genomic sequencing information for several Leishmania species has enabled molecular target–driven approaches to anti-leishmanial drug discovery (24, 25). Unfortunately, these approaches have not yet reached their full potential because of the complexities of leishmaniasis and because of an incomplete understanding of the roles played by the hypothesized molecular targets in parasite growth and survival. An attractive, complementary and evolving strategy uses methods that assay changes to the phenotype of the whole parasite as the platform for anti-leishmanial drug discovery. This prompts the question: What specific attributes of the Leishmania parasites and screening assay formats can be exploited to develop the next generation of anti-leishmanial therapeutics? A secondary question arises as to which species should be targeted for screening.
Anti-leishmanial Treatments
Although there is a need to identify new drug treatments for all manifestations of leishmaniasis, most drug discovery research has been biased toward the species that cause VL because of the higher risk of mortality associated with VL. These VL-centric efforts are commendable but they effectively relegate CL and M-CL to a position of being the “most neglected of neglected diseases” (26). The failure to focus some resources on the most common form of leishmaniasis is unfortunate, as there is substantial evidence that some chemo-therapeutics and treatment regimens are effective only against certain clinical manifestations of leishmaniasis and against particular Leishmania species or strains. Therefore, multiple anti-leishmanial drug-discovery strategies that target specific clinical manifestations and Leishmania species should be pursued.
The original classification of Leishmania species was established over twenty years ago, using isoenzyme electrophoresis, monoclonal antibodies, and genomic restriction analysis (27). Newer molecular biology–based methodologies are replacing these more traditional methods and tend to be more standardized and reliable (27). As a result, groups are revisiting the speciation of the Leishmania parasite lending to the dynamic nature of the field. Thus, it is important to determine conclusively the species of parasite being used for purposes of drug discovery especially when using older, previously classified Leishmania species or strains.
High-throughput screening (HTS) assays provide one approach to rapidly expand the diversity of available anti-leishmanial chemotypes for development. For maximum utility, the HTS assays must be well-validated, integrated with data management and capture systems, have a simple assay format, be relatively inexpensive, and be coupled with secondary assays to expedite confirmation of the activity and specificity of novel chemotypes (26). Thus far, few validated large-scale anti-leishmanial HTS assays have been reported.
There is ongoing controversy as to which life-cycle form (i.e., promastigote, amastigote) of the Leishmania parasite is the most suitable to use for HTS activities. Cell-based amastigotes are the pathologically relevant form of the Leishmania parasite for humans; however, they are currently not amenable to easy conversion into an HTS assay format. Conversely, promastigotes are considered to be less pathologically meaningful, as they are the form found in the sandfly vector, but, by comparison, they are easy to culture and can be readily adapted to HTS. There is even evidence that promastigotes provide a good model for gauging a compound’s leishmanicidal activity with the exception of the immunomodulators, such as the pentavalent antimonials (28, 29). Comparative gene-expression data indicate that there are relatively few life cycle–dependent expressed genes, suggesting that the promastigote and amastigote life-cycle forms are very similar (30, 31). Thus, we posit that HTS assays using promastigotes will be an indispensable resource for the initial screening of chemotypes for anti-leishmanial activity and that they should complement HTS assays using mammalian cell-based amastigotes.
Leishmaniasis is a profound global health problem, and the antiquated and toxic pharmacological agents available for its treatment are deplorable. Moreover, the effectiveness of the currently available drugs is highly variable and depends upon the infecting Leishmania species, strains, clinical manifestation, and geographic area, suggesting that multiple drug discovery and development strategies should be embraced. The molecular targets of the commonly available drugs are unclear, making advancements challenging. The various Leishmania genome projects offer promise in terms of the future development of drugs that target specific genes or pathways essential for survival of the parasite. These evolving target-based drug-discovery strategies should be complemented with unbiased, inexpensive, whole-parasite HTS assays, based on the promastigote and amastigote life-cycle forms of the Leishmania parasite. The success of these whole-parasite drug-discovery strategies depends on the development of validated HTS assay formats and the accessibility to a series of standardized secondary confirmation assays that will progress compounds through initial screening processes through lead optimization and pre-clinical efficacy studies. Uncovering the mechanism of action of promising anti-leishmanial compounds, identified in whole-parasite screens, will be a major pharmacological challenge. Moreover, as our understanding of the classification and involvement of the various Leishmania species in the clinical manifestations of leishmaniasis grows, it will be imperative that whole parasite-based assays be founded on the appropriately speciated Leishmania population.
Acknowledgments
We thank John Nelson for editorial assistance. This work was funded in part by United States Army Medical Research Acquisition Activity (USAMRAA) grants W81XWH-07-1-0396 and W81WH-10-2-0001. The opinions or assertions contained herein are our private views and are not to be construed as official or as reflecting views of the Department of the Army or the Department of Defense.
- Copyright © 2010
References
John S. Lazo, PhD, is the Allegheny Foundation Professor of Pharmacology and the Director of the University of Pittsburgh Drug Discovery Institute. After graduating with a bachelor degree in Chemistry from the Johns Hopkins University, he completed his PhD in Pharmacology with Raymond Ruddon at the University of Michigan. He joined Alan Sartorelli’s laboratory as a postdoctoral fellow and remained on the faculty in the Department of Pharmacology at Yale University until 1987, when he became Chairman of Pharmacology at the University of Pittsburgh. His laboratory is currently interested in the biological role of protein phosphatases and in the mechanism of action of novel agents. E-mail lazo{at}pitt.edu; fax 412-648-9009.
Cpt. Jacob Johnson, PhD, is the Chief of Parasite Assay Development with the Division of Experimental Therapeutics at Walter Reed Institute of Research (WRAIR) (Silver Spring, MD). He completed his BSc (Molecular Biology and Biotechnology) at Salem-Teikyo University (Salem, WV) and his PhD at the University of Cincinnati College of Medicine (Biomedical Research) in 2003. Dr. Johnson completed a post-doctoral fellowship at the Harbor Branch Oceanographic Institution (Fort Pierce, FL), focusing on natural product drug discovery. He joined WRAIR in 2005, serving as a member of the malaria and leishmaniasis drug discovery teams.
Col. Max Grögl, PhD, is the Acting Director of the Division of Experimental Therapeutics at Walter Reed Institute of Research (WRAIR) (Silver Spring, MD). He completed his BSc (Biology) at the Andes University (Bogota, Colombia), MSc (Microbiology) at the Institute of Tropical Medicine (Sao Paulo, Brazil) and his PhD (Immunology) at Wake Forest University. Dr. Grögl currently holds Adjunct Professorships at the University of Hawaii and the Uniformed Services University of the Health Sciences (Bethesda, MD). He joined WRAIR in 1984 and has been studying leishmaniasis and focusing on leishmaniasis drug discovery for over 25 years.
Elizabeth R. Sharlow, PhD, is a Research Assistant Professor in the Department of Pharmacology and Chemical Biology and a faculty member with the University of Pittsburgh Drug Discovery Institute. She completed her PhD in Genetics at the Pennsylvania State University. She joined Johnson & Johnson CPWW as a post-doctoral fellow and was a Senior Scientist at ProlX Pharmaceuticals. In 2004, she joined the Drug Discovery Institute and her research focuses on the development and implementation of high throughput and high content screening assays.
