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Dispatches from the Frontlines of Research—edited by John W. Nelson
Anti-leishmanial Drug Discovery: Rising to the Challenges of a Highly Neglected Disease
Elizabeth R. Sharlow1,2, Max Grögl3, Jacob Johnson3, and John S. Lazo1,2 1 Drug Discovery Institute, 2Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15260; 3 Walter Reed Army Institute of Research, Silver Spring, MD 20910
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 expan- Table 1. Predominant Clinical Manifestations, Characteristics, and Geographic Distribution of Leishmaniasis sion in the geographic distribuPredominant Symptoms Leishmania species Predominant tion of leishmaniasis and an Clinical Geographic increase in the total number Manifestation Distribution of leishmaniasis cases, often in Skin lesions or ulcers that L. aethiopica (Old World) Middle East, Cutaneous epidemic proportions (6, 7). can self heal, with scarNorth and SubL. killicki* (Old World) The impact of leishmaniasis ring and disfigurement. Saharan Africa, L. major (Old World) extends beyond the immediLesions usually appear and South L. tropica* (Old World) on exposed skin such as America ate effects on human health hands, face, or legs. L. amazonensis (New World) to include significant cultural, L. garnhami (New World) societal, economic, and psyL. mexicana (New World) chological repercussions owing L. pifanoi (New World) to disfiguring lesions, disability, L. venezuelensis (New World) and death (8). L. Vianna (V.) braziliensis** (New World) Central and Muco-cutaneous Initiates as skin lesions There are three major L. V. colombiensis (New World) South America that metastasize to L. V. guyanensis (New World) mucous membranes clinical manifestations of leading to the destruction L. V. lainsoni (New World) leishmaniasis (Table 1). The of the oral cavity, larynx, L. V. naiffi (New World) most common form is cutaL. V. panamensis (New World) and pharnyx. Tissue neous (CL), which is caused L. V. peruviana (New World) destruction elevates predominantly by Leishmania L. V. shawi (New World) infection risk. (L.) major, L. aethiopica and L. Visceral Dissemination of L. donovani (Old World) Europe, East tropica in the Old World and parasites to internal L. infantum*** (Old World) Africa, Central organs. Characterized and South L. braziliensis, L. panamensis, by fever, weight loss, L. chagasi*** (New World) America, and L. mexicana in the New hepatosplenomegaly, Central and World (Table 1) (9). CL is anemia and immunosupSoutheast Asia typified by self-healing lesions; pression. Death results if however, the healing process untreated. can involve months or years * L. killiki might be a subspecies of L. tropica; ** Majority of M-CL results from L. braziliensis; and varies with the infect*** L. infantum and L. chagasi are the same species and L. donovani predominates in VL.
ing 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. 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
No known MOA. Recently. pain. 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). of the available antileishmanial treatments. Paromomycin (aminosidine. anemia.e. tion of Leishmania species was Itracanazole Cutaneous Effects ergosterol biosynthesis. Anti-leishmanial Treatments therapeutics and treatment regimens are effective only Drug Primary Use Comments against certain clinical maniPentavalent antimony Cutaneous. indicating that current drug treatments do not completely eliminate parasitic infection and relapse occurs if host immunity fails (21). or exhibit physicochemical liability (i. Newer molecuCutaneous monomycin) nausea. diarrhea. Multiple formulations. 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. 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. 16). Few drugdiscovery efforts have been specifically undertaken to design and develop new chemotherapeutics for leishmaniasis. poor solubility). erythema. Unfortunately. nausea. (polyene antibiotic) Adverse effects include painful administration. nausea. ies. and genomic restriction Visceral. pain. nausea. as there is substantial evidence that some chemoTable 2. April 2010 Volume 10. The life cycle of the Leishmania parasite is completed when a naïve sandfly takes a blood meal from the infected host. Adverse effects include gies are replacing these more hepatotoxicity. there are approximately thirty species of Leishmania with nearly twenty being capable of infecting humans (Table 1) (10. Adverse effects include pain. diarrhea. 25). analysis (27). festations of leishmaniasis and (sodium stibogluconate. diarrhea. many are decades old. renal toxicity. vomiting. Impacts protein synthesis. there are limited chemotherapeutics available for treatment and no vaccines or prophylactic drugs for any form of the disease (Table 2). making leishmaniasis one of the most neglected diseases (8). nausea. Most regimens rely heavily on the pentavalent antimonials and amphotericin B formulations. MOA. mechanism of action. allowing the sandfly to infect other human hosts. however. The failure to focus some resources on the most common form of leishmaniasis is unfortunate. against particular Leishmania species or strains. nausea. No known MOA. Amphotericin B formulations Visceral. liver toxicity. Muco-cutaneous. and Leishmania species should Visceral be pursued. endocrine dysfunction. This prompts the question: What specific attributes of the Leishmania parasites and screening assay formats can be exploited to develop the next generation of antileishmanial therapeutics? A secondary question arises as to which species should be targeted for screening. are painful to administer. mild edema. Newer compounds are the oral agent miltefosine and both parenteral and topical paromomycin (Table 2). complementary and evolving strategy uses methods that assay changes to the phenotype of the whole parasite as the platform for anti-leishmanial drug discovery. antileishmanial drug discovery programs have not had the screening throughput and the commitment of resources seen with other diseases. Although there is a need to identify new drug treatments for all manifestations of leishmaniasis. monoclonal antibodpain. Will no longer be manufactured by GlaxoSmith Kline. Fluconazole Cutaneous Effects ergosterol biosynthesis. Therefore. traditional methods and tend Miltefosine Visceral Impacts cell membrane integrity. 23).. 15). Substantial evidence suggests that Leishmania parasites can persist at the primary sites of infection and in lymph nodes even after therapeutic intervention.e. diarrhea. Moreover.the parasitization of the host. multiple anti-leishmanial drugCutaneous hypokalemia. diarrhea. Rare adverse effects but include specific clinical manifestations Muco-cutaneous. ago. No known MOA. reliable (27). to be more standardized and (hexadecylphosphocholine) Adverse effects include nausea. as do the antimonials and amphotericin B (17–20). vomiting. Therefore. As a result. cause some form of systemic toxicity. most drug discovery research has been biased toward the species that cause VL because of the higher risk of mortality associated with VL. Despite the prevalence of leishmaniasis and its substantial impact on human life. Impacts cell membrane integrity. Rare adverse effects but The original classificainclude pain. An attractive. the identification of potential new leishmanicidal chemotypes has been constrained by the limited diversity of the compound library. Exposure to a particular Leishmania species is dependent upon geographical location (i. lar biology–based methodoloKetoconazole Cutaneous Effects ergosterol biosynthesis. In addition. there is a pressing need for new efficacious agents. Adverse effects include phoresis. Muco-cutaneous. genomic sequencing information for several Leishmania species has enabled molecular target– driven approaches to anti-leishmanial drug discovery (24. Currently. Issue 2 73 . Old World or New World) and determines the primary clinical manifestation (10. meglumine antimoniate) Visceral UK after 2012. Thus. using isoenzyme electroAllopurinol Cutaneous Inhibits xanthine oxidase.. Adverse effects include cardiotoxicity. rash. discovery strategies that target Pentamidine (diamidine) Cutaneous. Adverse effects include established over twenty years pain.
be relatively inexpensive. suggesting that multiple drug discovery and development strategies should be embraced. 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. References 1. and Alvar J (2008). 14. Maguire JH. 12. 6. Schwartz E. 2. 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. Moreover.4 Acknowledgments We thank John Nelson for editorial assistance. Mem Inst Oswaldo Cruz 102:541–547. 13. Ameen M (2007) Cutaneous leishmaniasis: Therapeutic strategies and future directions. Lange UG. and McConville MJ (2008) Role of hexosamine biosynthesis in Leishmania growth and virulence. Dermatol Ther 22:491–502. Reed S. 17. 10. and Blackwell JM. Ezzati M. and Lopez AD (2007) Measuring the burden of neglected tropical diseases: The global burden of disease framework. and Foote SJ (2000) Leishmaniasis. strains. they are easy to culture and can be readily adapted to HTS. promastigotes are considered to be less pathologically meaningful. will be a major pharmacological challenge. 15. 4. different diseases. 3. whole-parasite HTS assays. Davies CR. Brun R. Comparative gene-expression data indicate that there are relatively few life cycle–dependent expressed genes. and Hatz C (2004) Treatment of cutaneous leishmaniasis among travelers. and the antiquated and toxic pharmacological agents available for its treatment are deplorable. 29). Berman JD. previously classified Leishmania species or strains. These evolving target-based drug-discovery strategies should be complemented with unbiased. identified in whole-parasite screens. 31).10. suggesting that the promastigote and amastigote life-cycle forms are very similar (30. inexpensive. Lesho EP. JAMA 292:1294. (2005) IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection. Cell-based amastigotes are the pathologically relevant form of the Leishmania parasite for humans. Fairlamb A. BMJ 321:801–804. Clin Dermatol 17:279–289. promastigote. few validated large-scale anti-leishmanial HTS assays have been reported. 5. Expert Opin Pharmacother 8:2689–2699. McKerrow J. There is ongoing controversy as to which life-cycle form (i. as they are the form found in the sandfly vector. There is even evidence that promastigotes provide a good model for gauging a compound’s leishmanicidal activity with the exception of the immunomodulators. but.e. Bern C. PLoS Negl Trop Dis 2:e313. Desjeux P. Rev Sci Tech 27:399–412. J Immunol 175:2517–2524. have a simple assay format. Alexander B. 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. and Brooker S (2007) Cutaneous leishmaniasis. it is important to determine conclusively the species of parasite being used for purposes of drug discovery especially when using older. Roberts LJ. Blum J. Naderer T. the effectiveness of the currently available drugs is highly variable and depends upon the infecting Leishmania species. Lancet Infect Dis 7:581–596. Gürtler RE. Roberts MTM. however. Stuart K. 18. For maximum utility. and be coupled with secondary assays to expedite confirmation of the activity and specificity of novel chemotypes (26). they are currently not amenable to easy conversion into an HTS assay format. by comparison. Pirmez C. Mathers CD. Cardo LJ (2006) Leishmania: Risk to the blood supply. and Tarleton R (2008) Kinetoplastids: Related protozoan pathogens. Thus far. J Antimicrob Chemother 53:158–166. doi:10. 11. PLoS Negl Trop Dis 1:e114. it will be imperative that whole parasite-based assays be founded on the appropriately speciated Leishmania population. Lancet 366:1561–1577. Ready PD (2008) Leishmaniasis emergence and climate change. Handman E. Thus. 9. Uncovering the mechanism of action of promising anti-leishmanial compounds. Killick-Kendrick R (1999) The biology and control of phlebotomine sand flies. Wee E. (2002) CMAJ 167:536. amastigote) of the Leishmania parasite is the most suitable to use for HTS activities.. Complexities of assessing the disease burden attributable to leishmaniasis. making advancements challenging. J Clin Invest 118:1301–1310. Reithinger R. Neafie R. Louzir H. Alcami A.2. based on the promastigote and amastigote life-cycle forms of the Leishmania parasite. Dujardin JC. 8. and Saravia NG (2005) Advances in leishmaniasis. Croft S. High-throughput screening (HTS) assays provide one approach to rapidly expand the diversity of available anti-leishmanial chemotypes for development. integrated with data management and capture systems. 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. Beck B. Shaw J (2007) The leishmaniasis––survival and expansion in a changing world. Stephenson J (2004) Leishmaniasis epidemic. Thus. 7. Leishmaniasis is a profound global health problem. the HTS assays must be well-validated. Leishmaniasis epidemic hits Afghanistan. Transfusion 46:1641–1645. David CV and Craft N (2009) Cutaneous and mucocutaneous leishmaniasis. The molecular targets of the commonly available drugs are unclear. Mol Micro 69:858–869. Murray HW. Stober CB. and Aronson N (2005) Nonhealing skin lesions in a sailor and a journalist returing from Iraq.groups are revisiting the speciation of the Leishmania parasite lending to the dynamic nature of the field. 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. as our understanding of the classification and involvement of the various Leishmania species in the clinical manifestations of leishmaniasis grows. Moreover. such as the pentavalent antimonials (28. and geographic area. Wortmann G.1124/mi. 16. Conversely. Cleve Clin J Med 72:93–106. clinical manifestation. 74 .
Yamamoto Y. Falcão CA. Sharlow ER. Aert R. Marco JD. Hashiguchi Y. PhD. She completed her PhD in Genetics at the Pennsylvania State University. Callahan HL. Grögl currently holds Adjunct Professorships at the University of Hawaii and the Uniformed Services University of the Health Sciences (Bethesda. et al. Donhauser N. Smith DF. Murphy L. Peacock CS. serving as a member of the malaria and leishmaniasis drug discovery teams. Berriman M. focusing on natural product drug discovery. She joined Johnson & Johnson CPWW as a post-doctoral fellow and was a Senior Scientist at ProlX Pharmaceuticals. Dr. et al. Dr. Oshiro M. WV) and his PhD at the University of Cincinnati College of Medicine (Biomedical Research) in 2003. Bioorg Med Chem 14:3467–3480. Shun T. 21. Issue 2 75 . Spinelli R. (2005) The genome of the kinetoplastid parasite. small molecule screen. 27. Adlem E. when he became Chairman of Pharmacology at the University of Pittsburgh. Piro OE. Nat Gen 39:839–847. Muzitano MF. Max Grögl. Berman JD (1988) Chemotherapy for leishmaniasis: Biochemical mechanisms. Int J Parasitol 37:1173–1186. Tivey A. fax 412-648-9009. and Grögl M (1997) An axenic amastigote system for drug screening. PhD. J Exp Med 191:2121–2130. and future strategies. Portal AC. Rigol C. Rev Infect Dis 10:560–586. MD). Johnson completed a post-doctoral fellowship at the Harbor Branch Oceanographic Institution (Fort Pierce. 23. is the Chief of Parasite Assay Development with the Division of Experimental Therapeutics at Walter Reed Institute of Research (WRAIR) (Silver Spring. After graduating with a bachelor degree in Chemistry from the Johns Hopkins University. 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. 25.edu. Peacock CS. Am J Trop Med Hyg 46:296–306. 20. 26. In 2004. Sharlow. Peacock CS. Leimgruber S. Gerpe A. et al. Wipf P. lowstringency. 22. Devereaux R. clinical efficacy. Cpt. Exp Parasitol 121:352–361. Quail MA. de Souza R. PhD. Antimicrob Agents Chem 41:818– 822. biological evaluation and mechanism of action studies. PLoS Negl Trop Dis 3:e540. Olea-Azar C. Res Microbiol 155: 224–230. Colombia). and Vasconcellos ML (2007) High selective leishmanicidal activity of 3-hydroxy-2-methylene-3-(4-bromophenyl)propanenitrile and analogous compounds. Science 309:436–442. Peter N. 31. Rossi-Bergmann B. she joined the Drug Discovery Institute and her research focuses on the development and implementation of high throughput and high content screening assays. Asato Y. is the Allegheny Foundation Professor of Pharmacology and the Director of the University of Pittsburgh Drug Discovery Institute. Döring R. His laboratory is currently interested in the biological role of protein phosphatases and in the mechanism of action of novel agents. Reed R. He joined WRAIR in 2005. He joined WRAIR in 1984 and has been studying leishmaniasis and focusing on leishmaniasis drug discovery for over 25 years. Ivens. Herwaldt BL and Berman JD (1992) Recommendations for treating leishmaniasis with sodium stibogluconate (Pentostam) and review of pertinent clinical studies. Diefenbach A. AC. computationally enhanced. Elizabeth R. Bogdan C. Maya JD. Lynn MA and McMaster WR (2008) Leishmania: Conserved evolution– –diverse diseases. Harris D. Fumarola L. MD). Trends Parasitol 24:103–105. Mustata G. Gomez EA. Sisk E. Eur J Med Chem 42:99–102.19. Boiani L. Aslett M et al (2007) Comparative genomic analysis of three Leishmania species that cause diverse human disease. MSc (Microbiology) at the Institute of Tropical Medicine (Sao Paulo. O’Neil M. Rajandream MA. Brazil) and his PhD (Immunology) at Wake Forest University. Col. and Rittig MG (2000) Fibroblasts as host cells in latent leishmaniosis. is the Acting Director of the Division of Experimental Therapeutics at Walter Reed Institute of Research (WRAIR) (Silver Spring. Myint CK. Aguirre G. Worthey EA. Close D. Morello A. Filho EB. 30. Lazo. 29. and Cruz AK (2007) Comparative genomics: From genotype to disease phenotype in the leishmaniases. Kato H. Röllinghoff M. Adlem E. Gonzalez M. Pereira VLP. he completed his PhD in Pharmacology with Raymond Ruddon at the University of Michigan. John S. Mimori T. 24. He completed his BSc (Biology) at the Andes University (Bogota. Aggarwal G. Johnson J. Seeger K. Cerecetto H. PhD. Leishmania major. and Brandonisio O (2004) In vitro assays for evaluation of drug activity against Leishmania spp. Jacob Johnson. Ruiz JC. Grögl M. FL). E-mail lazo@pitt. April 2010 Volume 10. He completed his BSc (Molecular Biology and Biotechnology) at Salem-Teikyo University (Salem. Murphy L. 28. MD). (2009) Identification of potent chemotypes targeting Leishmania major using a high-throughput. and Uezato H (2009) Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing. 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. (2006) Indazole N-oxide derivatives as antiprotozoal agents: Synthesis.
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