- Email: [email protected]
Drugs against leishmaniasis: a synergy of technology and partnerships
Opinion
TRENDS in Parasitology
Vol.20 No.2 February 2004
Drugs against leishmaniasis: a synergy of technology and partnerships Antony J. Davis1, Henry W. Murray2 and Emanuela Handman1 1
Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3050, Australia 2 Department of Medicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
To date, there are no vaccines against any of the major parasitic diseases, and chemotherapy is the main weapon in our arsenal. There is an urgent need for better drugs against Leishmania. With the completion of the human genome sequence and soon that of Leishmania, for the first time we have the opportunity to identify novel chemotherapeutic treatments. This requires the exploitation of a variety of technologies. The major challenge is to take the process from discovery of drug candidates all the way along the arduous path to the marketplace. A crucial component will be the forging of partnerships between the pharmaceutical industry and publicly funded scientists to ensure that the promise of the current revolution in biology lives up to our hopes and expectations. Leishmaniasis, in each of its three clinical forms (cutaneous, mucosal and visceral), remains a major public health problem throughout much of the tropical and subtropical world. For example, . 100 000 new cases per year of cutaneous leishmaniasis are currently seen in Afghanistan [1]. The prolonged epidemic of visceral leishmaniasis (VL) in the Sudan has killed , 100 000 people and depopulated large areas [2,3]. In northeast India (Bihar State), as many as 100 000–200 000 new cases of visceral leishmaniasis are diagnosed each year [4]. In southern Europe (and probably soon in other regions), the combination of VL and co-infection with HIV has been a major problem (http://www.who.int/inf-fs/en/fact116.html). Attempts to produce an effective vaccine have so far failed, and treatment of the disease in regions other than northeast India still relies primarily on the use of pentavalent antimonials, drugs first introduced in the 1930s [5– 7]. The infamous statement by the US Surgeon General in the 1960s that infectious diseases could be consigned to the history books obviously requires reassessment [8]. Particularly serious issues in the treatment of leishmaniasis are: (i) the escalating prevalence of resistance to antimonials; (ii) the need for affordable alternative drugs; and (iii) well-recognized obstacles to developing new drugs for such neglected diseases in which there is little prospect of financial return [6,9]. Until recently, the search for new antiparasitic drugs has been performed using approaches that are over 50 Corresponding author: Emanuela Handman ([email protected]).
years old, such as inhibition of parasite growth in vitro. The mechanism of action and the interaction of the drugs with humans were often discovered only after the drugs were released for use. This approach could have missed important candidates that did not work in vitro because they might have been unstable or had low penetration into cells. Importantly, it could have missed drugs that would have been specific for the parasite stage present only in the human host, in addition to possible pro-drugs that might be converted to effective forms within the human body. During the late 1980s –1990s, there was a shift in chemical design philosophy because of a massive expansion in databases comprising chemical structures of known drugs, which was accompanied by an increasing database of three-dimensional structures of potential new drug target proteins. These factors raised the hope that rational drug design might replace empirical screening, but the outcomes have been disappointing. Now, with the availability of the complete DNA sequence of the human genome and many of the pathogens of humans, including Leishmania, there is the hope that drug development might evolve from ‘managed serendipity to engineered selection’ [10]. Targeted drug development is lengthy and expensive, and it proceeds through several decision gates [10] from the identification of a potential candidate in in vitro and/or animal experiments to clinical trials and to marketing the product. Here, we examine, from the perspective of nowavailable technology, some of the steps and hurdles at the beginning of the long road to finding new antileishmanial drugs (Figure 1). These technologies include gene knockouts to identify essential targets for disruption by drugs, high-throughput screening of large or selected chemical libraries, and three-dimensional protein structure determination. Targeted drug development also requires the development of new assays linked with functional genomic and proteomics tools to identify essential drug targets and to allow the development of lead compounds. Target selection Identifying suitable potential drug targets is essential for effective drug development (see Ref. [11]). At present, the majority of therapeutic targets are either cell-surface receptors or enzymes [12] because certain protein families are more readily modulated by small molecule interactions than others [13]. It is easier to out-compete an endogenous
www.sciencedirect.com 1471-4922/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2003.11.006
Opinion
74
TRENDS in Parasitology
Genomics
Pre-clinical studies Toxicology Pharmacology
Clinical studies Proof of concept Safety Efficacy
Proteomics
Lead identification and optimization
Product manufacturing and marketing
Target identification
Target validation
Long-term monitoring (post marketing)
Vol.20 No.2 February 2004
relevant life cycle stages of many organisms to obtain enough material for proteomic analysis. In leishmaniasis, although the obligatory intracellular amastigote is the target for any chemotherapeutic agent, very little proteomics data are available for this form [15]; most data focuses on the promastigote, which is easy to culture [16,17]. There is a need to develop fractionation procedures to purify or enrich for parasite material obtained from infected hosts; this could reveal pathways that are activated only when the parasite is within the host. Understanding the mechanisms of drug resistance in Leishmania is important and should lead to improved drug design. An extensive proteomics approach investigating potential mechanisms of drug resistance in L. major [18] has identified pteridine reductase (PTR1) as a major candidate.
TRENDS in Parasitology
Figure 1. Key steps in drug development. The steps on the road to drug development, from the discovery of drug targets, which are achievable in a publicly funded laboratory, to the expensive process of clinical evaluation, possible only through a partnership with industry.
ligand (e.g. the substrate of an enzyme) with a small chemical molecule than it is to interfere with protein – protein interactions over large surface areas such as those found on large protein receptors [13]. For example, the active site of the influenza virus enzyme neuraminidase is bound by the transition state analogue and antiviral drug zanamivir [14]. A potential drug target must be validated to elucidate its role in the disease process before it becomes the focus of a screening program. Target validation using mutant parasites with specific gene deletions in an animal model of disease is the most useful approach for predicting drug action because it mimics the effect of a highly specific inhibitor of the target protein in vivo [11]. In the case of leishmaniasis, most species that cause disease in humans also infect laboratory animals, thus providing experimental models of infection for drug target validation. The imminent completion of the Leishmania major and in the near future Leishmania infantum genome sequence should provide many new potential target proteins to be used in conjunction with comparative and functional genomics studies. Such comparative genomics studies will also allow the identification of molecules or biochemical pathways that have already been targeted successfully in other pathogens. In addition to blocking essential pathways, opportunities could become apparent in which a non-essential enzyme, which is not present in the host, could be used to convert a pro-drug into an active compound within the parasite, but not in the host. Pathways absent from humans could include mechanisms that control parasite virulence. Parasite virulence is an attractive target because parasite attenuation could reduce disease severity and allow the immune system to mount a host-protective response and synergize with chemotherapy. The availability of an annotated genome sequence will also aid in large-scale proteomics studies of Leishmania. The application of proteomics techniques to generate expression profiles of protozoan parasites is starting to catch up with more advanced studies in viruses and bacteria. This lag is due to the difficulty in culturing the www.sciencedirect.com
High-throughput screening After the selection of a suitable target molecule, the next step is to identify compounds that can modulate the activity of the target in a relevant assay. There are many methods that can be employed to generate these initial hits (for review, see Ref. [13]). These approaches rely either on detailed structural data of the target or ligand, or could be more pragmatic such as high-throughput screening (HTS) of vast chemical libraries. HTS has become one of the most commonly used techniques for drug discovery in pharmaceutical research (for reviews, see Refs [13,19]). HTS is often the first method of choice for the interrogation of a new target molecule and has the distinct advantage that no structural information concerning the drug target is required. It is now possible to screen 100 000 compounds per day, usually in a 384-well plate format. Recently, the focus has shifted from trying to screen as many compounds as possible to the selection of morefocused libraries. Computational filtering methods are now employed to optimize the physical properties of compounds for ‘drug-likeness’ and to avoid molecules with unfavorable characteristics [13,19]. Following an analysis of . 2000 compounds from the World Drug Index (http://www.derwent.com/products/ir/wdi), Lipinski et al. were able to define a set of rules based on properties of a molecule, such as the number of hydrogen-bond donors and acceptors, the molecular mass and logP (lipophilicity), to aid in the prediction of drug-like compounds [20]. However, fungicides, protozoacides and antiseptics all fell outside this set of rules because they contained structural features that allowed them to act as substrates for naturally occurring transporters, and this will have to be taken into consideration when designing screens against Leishmania targets. Another similar filtering approach known as rapid elimination of swill (REOS) has been designed to remove from a library those compounds that have potentially toxic or reactive properties [21]. Screening with compound libraries based on quality, rather than quantity, should result in an increase in the quality of data obtained from HTS. Structure-based lead discovery and virtual screening The three-dimensional structure of a protein can provide a chemist with the necessary data to synthesize compounds that exhibit better potency and selectivity for a given
Opinion
TRENDS in Parasitology
target. This is achieved by optimizing the interaction between the compound and the protein by computer-based molecular modelling (compound docking [22]), followed by laboratory experimentation to test the prediction. Modelling based on the crystal structure of neuraminidase resulted in the development of the anti-influenza drug zanamivir (Relenzaw, GlaxoSmithKline; http://www.gsk. com) (reviewed in Ref. [23]). Two HIV drugs, amprenavir and nelfinavir, were developed using the crystal structure of HIV protease [24]. In the case of the HIV protease, the enzyme was first identified via comparative genomics and backed up by comparative modelling of the protein on the structures of aspartic proteases. The foregoing principles form the basis of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium (http://depts.washington.edu/sgpp/), which aims to apply high-throughput methods to express large numbers of proteins, and to determine three-dimensional crystal structures of proteins from Leishmania and other parasites. These structures are available to all researchers and will probably provide targets for structure-based lead discovery in the future. Having high-resolution structural data of target proteins also enables the use of virtual screening (VS) of large databases of compounds in silico (for review, see Ref. [25]), which could allow the selection of a manageable number of candidate molecules that can be tested for biological activity. VS can select a small subset of compounds from a large library for use in more complex, low-throughput assays such as cell-based assays. The main components of VS are compound docking based on a protein’s structure, and chemical-similarity searching on small molecules if an active compound is known. The computational docking approach was instrumental in the identification of 40 parasite-specific inhibitors of the L. mexicana glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [26]. Although several drugs have been discovered through structure-based design, there are limitations in the use of X-ray crystallographic data in drug discovery. These limitations include uncertainties in atomic models of proteins generated by X-ray diffraction data and the failure of structure-based design to adequately address drug optimization properties such as absorption, metabolic stability, plasma – protein binding, elimination and toxicological profile [27]. Despite these limitations, the combination of VS and HTS is predicted to increase the success rate of screening experiments. Development of a drug candidate A major decision gate on the road to drug development is whether there are any properties of the drug candidate that would make its development difficult. Issues of bioavailability, metabolic processing, toxicity and interactions with other drugs have to be considered. An interesting by-product of the advances in genetics and proteomics has been the identification and modification of potentially therapeutic macromolecules, such as hormones and growth factors, from biological sources, rather than small-molecule synthetic drugs [10]. However, the existing assays for toxicity, metabolic processing and absorption, usually performed in animal models, might not www.sciencedirect.com
Vol.20 No.2 February 2004
75
be appropriate because these molecules are cell receptors or ligands, or antibodies and peptides specifically engineered to affect human targets. Developing new pre-clinical tests for the drugs of the future is a new challenge not only for pharmaceutical companies, but also for basic research scientists embarking on this road. Synergy between drugs and host immunity Antimicrobial drugs generally work in conjunction with the host immune system; antibiotic therapy is far less effective in the setting of immunodeficiency. This is also relevant to the development of new antileishmanial agents because the T cell-dependent immune response in the infected host is directed at activating tissue macrophages, the parasite’s host cells. Work in the past decade has shown that some drugs, such as pentavalent antimonials, require an intact cell-mediated immune response for expression of their antileishmanial effect in vivo, whereas amphotericin B acts directly on the host cell [6]. Irrespective of whether host T-cell responsiveness is required, the in vivo efficacy of antileishmanial drugs can be enhanced by simultaneously triggering proinflammatory T helper cell Type 1 (Th1) immune responses [6,9,28] with the secretion of cytokines such as interleukin 12 (IL-12) and interferon g (IFN-g). Combining drugs with immunostimulation (immunochemotherapy) to enhance antileishmanial efficacy and improve outcome adds a new level of complexity to drug development [6]. Conversely, inhibition of suppressive Th2-type cytokines such as IL-4 and IL-10 might prove useful for individuals with severe disease because of their propensity to turn on these deleterious cytokines, rather than the host-protective ones [6,9,29,30]. This could be accomplished by injecting neutralizing anti-IL-4 or anti-IL-10 receptor antibodies along with drug. Interdigitation of chemotherapy with immunomodulators represents another avenue to consider for new antileishmanial drug development [31,32]. Funding of drug discovery and development When discussing drug development, we cannot ignore the economic realities. Should the limited resources be used to improve the existing drugs or to search for new ones? Even if available, will the people most in need be able to afford them? There is no doubt that drug discovery and development is an expensive and risky business under the best circumstances. Unfortunately, drug companies seem to be pulling out of antibiotics development [33,34], and the cost and resources associated with drug development are generally outside the scope of academic laboratories. While HTS is a key part of modern pharmaceutical development, most publicly funded laboratories cannot afford access to such technology. It is imperative that publicly funded laboratories forge partnerships with industry. The WHO/TDR (http://www.who.int/tdr/) and National Institutes of Health (http://www.niaid.nih.gov/ dmid/drug) are providing funding and guidance with the aim of establishing links with industry partners, and Me´dicins Sans Frontie`res has established the Drugs for Neglected Diseases Initiative (http://www.accessmed-msf. org/dndi.asp). It is hoped that such relationships will
Opinion
76
TRENDS in Parasitology
greatly expedite the discovery and implementation of novel therapies for leishmaniasis. Acknowledgements We are indebted to Jim Goding for his critical review of the article. The work of H.W.M. is supported by NIH grant AI 16393. E.H. and A.J.D. are supported by the Australian National Health and Medical Research Council and UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.
References 1 Reithinger, R. et al. (2003) Anthroponotic cutaneous leishmaniasis, Kabul, Afghanistan. Emerg. Infect. Dis. 9, 727– 729 2 Seaman, J. et al. (1996) The epidemic of visceral leishmaniasis in western upper Nile, southern Sudan: course and impact from 1984 to 1994. Int. J. Epidemiol. 25, 862 – 871 3 Zijlstra, E.E. and el-Hassan, A.M. (2001) Leishmaniasis in Sudan. Visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 95 (Suppl. 1), S27– S58 4 Herwaldt, B.L. (1999) Leishmaniasis. Lancet 354, 1191 – 1199 5 Yardley, V. and Croft, S.L. (2000) A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis. Int. J. Antimicrob. Agents 13, 243 – 248 6 Murray, H.W. (2001) Clinical and experimental advances in treatment of visceral leishmaniasis. Antimicrob. Agents Chemother. 45, 2185 – 2197 7 Croft, S. et al. (2003) Leishmaniasis – current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol. 19, 502 – 508 8 Garrett, L. (1992) The next epidemic. In AIDS in the World (Mann, J.M. et al., eds), pp. 825 – 839, Harvard University Press 9 Murray, H.W. et al. (2003) Immunoenhancement combined with amphotericin B as treatment in experimental visceral leishmaniasis. Antimicrob. Agents Chemother. 47, 2513 – 2517 10 Pritchard, J.F. et al. (2003) Making better drugs: decision gates in nonclinical drug development. Nat. Rev. Drug Discov. 2, 542– 553 11 Smith, C. (2003) Drug target validation: hitting the target. Nature, 422341, 343, 345 12 Drews, J. (2000) Drug discovery: a historical perspective. Science 287, 1960 – 1964 13 Bleicher, K.H. et al. (2003) Hit and lead generation: beyond highthroughput screening. Nat. Rev. Drug Discov. 2, 369 – 378 14 von Itzstein, M. et al. (1993) Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363, 418 – 423 15 Bente, M. et al. (2003) Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani. Proteomics 3, 1811 – 1829
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16 El Fakhry, Y. et al. (2002) A proteomic approach to identify developmentally regulated proteins in Leishmania infantum. Proteomics 2, 1007– 1017 17 Acestor, N. et al. (2002) Establishing two-dimensional gels for the analysis of Leishmania proteomes. Proteomics 2, 877 – 879 18 Drummelsmith, J. et al. (2003) Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms. Mol. Cell. Proteomics 2, 146– 155 19 Walters, W.P. and Namchuk, M. (2003) Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov. 2, 259 – 266 20 Lipinski, C.A. et al. (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3 – 26 21 Walters, W.P. et al. (1999) Recognizing molecules with drug-like properties. Curr. Opin. Chem. Biol. 3, 384 – 387 22 Halperin, I. et al. (2002) Principles of docking: An overview of search algorithms and a guide to scoring functions. Proteins 47, 409 – 443 23 Blundell, T.L. et al. (2002) High-throughput crystallography for lead discovery in drug design. Nat. Rev. Drug Discov. 1, 45 – 54 24 Greer, J. et al. (1994) Application of the three-dimensional structures of protein target molecules in structure-based drug design. J. Med. Chem. 37, 1035 – 1054 25 Bajorath, J. (2002) Integration of virtual and high-throughput screening. Nat. Rev. Drug Discov. 1, 882 – 894 26 Bressi, J.C. et al. (2001) Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design. J. Med. Chem. 44, 2080 – 2093 27 Davis, A.M. et al. (2003) Application and limitations of x-ray crystallographic data in structure-based ligand and drug design. Angew. Chem. Int. Ed. Engl. 42, 2718– 2736 28 Masihi, K.N. (2003) Concepts of immunostimulation to increase antiparasitic drug action. Parasitol. Res. 90 (Suppl. 2), S97– S104 29 Murray, H.W. et al. (2002) Interleukin-10 (IL-10) in experimental visceral leishmaniasis and IL-10 receptor blockade as immunotherapy. Infect. Immun. 70, 6284 – 6293 30 Murray, H.W. et al. (2003) Modulation of T cell costimulation as immunotherapy or immunochemotherapy in experimental visceral leishmaniasis. Infect. Immun. 71, 6453– 6462 31 Buates, S. and Matlashewski, G. (1999) Treatment of experimental leishmaniasis with the immunomodulators imiquimod and S-28463: efficacy and mode of action. J. Infect. Dis. 179, 1485– 1494 32 Arevalo, I. et al. (2001) Successful treatment of drug-resistant cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator. Clin. Infect. Dis. 33, 1847– 1851 33 Walsh, C. (2003) Where will new antibiotics come from? Nat. Rev. Microbiol. 1, 65 – 70 34 Clarke, T. (2003) Drug companies snub antibiotics as pipeline threatens to run dry. Nature 425, 225
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TRENDS in Parasitology
Vol.20 No.2 February 2004
Drugs against leishmaniasis: a synergy of technology and partnerships Antony J. Davis1, Henry W. Murray2 and Emanuela Handman1 1
Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3050, Australia 2 Department of Medicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
To date, there are no vaccines against any of the major parasitic diseases, and chemotherapy is the main weapon in our arsenal. There is an urgent need for better drugs against Leishmania. With the completion of the human genome sequence and soon that of Leishmania, for the first time we have the opportunity to identify novel chemotherapeutic treatments. This requires the exploitation of a variety of technologies. The major challenge is to take the process from discovery of drug candidates all the way along the arduous path to the marketplace. A crucial component will be the forging of partnerships between the pharmaceutical industry and publicly funded scientists to ensure that the promise of the current revolution in biology lives up to our hopes and expectations. Leishmaniasis, in each of its three clinical forms (cutaneous, mucosal and visceral), remains a major public health problem throughout much of the tropical and subtropical world. For example, . 100 000 new cases per year of cutaneous leishmaniasis are currently seen in Afghanistan [1]. The prolonged epidemic of visceral leishmaniasis (VL) in the Sudan has killed , 100 000 people and depopulated large areas [2,3]. In northeast India (Bihar State), as many as 100 000–200 000 new cases of visceral leishmaniasis are diagnosed each year [4]. In southern Europe (and probably soon in other regions), the combination of VL and co-infection with HIV has been a major problem (http://www.who.int/inf-fs/en/fact116.html). Attempts to produce an effective vaccine have so far failed, and treatment of the disease in regions other than northeast India still relies primarily on the use of pentavalent antimonials, drugs first introduced in the 1930s [5– 7]. The infamous statement by the US Surgeon General in the 1960s that infectious diseases could be consigned to the history books obviously requires reassessment [8]. Particularly serious issues in the treatment of leishmaniasis are: (i) the escalating prevalence of resistance to antimonials; (ii) the need for affordable alternative drugs; and (iii) well-recognized obstacles to developing new drugs for such neglected diseases in which there is little prospect of financial return [6,9]. Until recently, the search for new antiparasitic drugs has been performed using approaches that are over 50 Corresponding author: Emanuela Handman ([email protected]).
years old, such as inhibition of parasite growth in vitro. The mechanism of action and the interaction of the drugs with humans were often discovered only after the drugs were released for use. This approach could have missed important candidates that did not work in vitro because they might have been unstable or had low penetration into cells. Importantly, it could have missed drugs that would have been specific for the parasite stage present only in the human host, in addition to possible pro-drugs that might be converted to effective forms within the human body. During the late 1980s –1990s, there was a shift in chemical design philosophy because of a massive expansion in databases comprising chemical structures of known drugs, which was accompanied by an increasing database of three-dimensional structures of potential new drug target proteins. These factors raised the hope that rational drug design might replace empirical screening, but the outcomes have been disappointing. Now, with the availability of the complete DNA sequence of the human genome and many of the pathogens of humans, including Leishmania, there is the hope that drug development might evolve from ‘managed serendipity to engineered selection’ [10]. Targeted drug development is lengthy and expensive, and it proceeds through several decision gates [10] from the identification of a potential candidate in in vitro and/or animal experiments to clinical trials and to marketing the product. Here, we examine, from the perspective of nowavailable technology, some of the steps and hurdles at the beginning of the long road to finding new antileishmanial drugs (Figure 1). These technologies include gene knockouts to identify essential targets for disruption by drugs, high-throughput screening of large or selected chemical libraries, and three-dimensional protein structure determination. Targeted drug development also requires the development of new assays linked with functional genomic and proteomics tools to identify essential drug targets and to allow the development of lead compounds. Target selection Identifying suitable potential drug targets is essential for effective drug development (see Ref. [11]). At present, the majority of therapeutic targets are either cell-surface receptors or enzymes [12] because certain protein families are more readily modulated by small molecule interactions than others [13]. It is easier to out-compete an endogenous
www.sciencedirect.com 1471-4922/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2003.11.006
Opinion
74
TRENDS in Parasitology
Genomics
Pre-clinical studies Toxicology Pharmacology
Clinical studies Proof of concept Safety Efficacy
Proteomics
Lead identification and optimization
Product manufacturing and marketing
Target identification
Target validation
Long-term monitoring (post marketing)
Vol.20 No.2 February 2004
relevant life cycle stages of many organisms to obtain enough material for proteomic analysis. In leishmaniasis, although the obligatory intracellular amastigote is the target for any chemotherapeutic agent, very little proteomics data are available for this form [15]; most data focuses on the promastigote, which is easy to culture [16,17]. There is a need to develop fractionation procedures to purify or enrich for parasite material obtained from infected hosts; this could reveal pathways that are activated only when the parasite is within the host. Understanding the mechanisms of drug resistance in Leishmania is important and should lead to improved drug design. An extensive proteomics approach investigating potential mechanisms of drug resistance in L. major [18] has identified pteridine reductase (PTR1) as a major candidate.
TRENDS in Parasitology
Figure 1. Key steps in drug development. The steps on the road to drug development, from the discovery of drug targets, which are achievable in a publicly funded laboratory, to the expensive process of clinical evaluation, possible only through a partnership with industry.
ligand (e.g. the substrate of an enzyme) with a small chemical molecule than it is to interfere with protein – protein interactions over large surface areas such as those found on large protein receptors [13]. For example, the active site of the influenza virus enzyme neuraminidase is bound by the transition state analogue and antiviral drug zanamivir [14]. A potential drug target must be validated to elucidate its role in the disease process before it becomes the focus of a screening program. Target validation using mutant parasites with specific gene deletions in an animal model of disease is the most useful approach for predicting drug action because it mimics the effect of a highly specific inhibitor of the target protein in vivo [11]. In the case of leishmaniasis, most species that cause disease in humans also infect laboratory animals, thus providing experimental models of infection for drug target validation. The imminent completion of the Leishmania major and in the near future Leishmania infantum genome sequence should provide many new potential target proteins to be used in conjunction with comparative and functional genomics studies. Such comparative genomics studies will also allow the identification of molecules or biochemical pathways that have already been targeted successfully in other pathogens. In addition to blocking essential pathways, opportunities could become apparent in which a non-essential enzyme, which is not present in the host, could be used to convert a pro-drug into an active compound within the parasite, but not in the host. Pathways absent from humans could include mechanisms that control parasite virulence. Parasite virulence is an attractive target because parasite attenuation could reduce disease severity and allow the immune system to mount a host-protective response and synergize with chemotherapy. The availability of an annotated genome sequence will also aid in large-scale proteomics studies of Leishmania. The application of proteomics techniques to generate expression profiles of protozoan parasites is starting to catch up with more advanced studies in viruses and bacteria. This lag is due to the difficulty in culturing the www.sciencedirect.com
High-throughput screening After the selection of a suitable target molecule, the next step is to identify compounds that can modulate the activity of the target in a relevant assay. There are many methods that can be employed to generate these initial hits (for review, see Ref. [13]). These approaches rely either on detailed structural data of the target or ligand, or could be more pragmatic such as high-throughput screening (HTS) of vast chemical libraries. HTS has become one of the most commonly used techniques for drug discovery in pharmaceutical research (for reviews, see Refs [13,19]). HTS is often the first method of choice for the interrogation of a new target molecule and has the distinct advantage that no structural information concerning the drug target is required. It is now possible to screen 100 000 compounds per day, usually in a 384-well plate format. Recently, the focus has shifted from trying to screen as many compounds as possible to the selection of morefocused libraries. Computational filtering methods are now employed to optimize the physical properties of compounds for ‘drug-likeness’ and to avoid molecules with unfavorable characteristics [13,19]. Following an analysis of . 2000 compounds from the World Drug Index (http://www.derwent.com/products/ir/wdi), Lipinski et al. were able to define a set of rules based on properties of a molecule, such as the number of hydrogen-bond donors and acceptors, the molecular mass and logP (lipophilicity), to aid in the prediction of drug-like compounds [20]. However, fungicides, protozoacides and antiseptics all fell outside this set of rules because they contained structural features that allowed them to act as substrates for naturally occurring transporters, and this will have to be taken into consideration when designing screens against Leishmania targets. Another similar filtering approach known as rapid elimination of swill (REOS) has been designed to remove from a library those compounds that have potentially toxic or reactive properties [21]. Screening with compound libraries based on quality, rather than quantity, should result in an increase in the quality of data obtained from HTS. Structure-based lead discovery and virtual screening The three-dimensional structure of a protein can provide a chemist with the necessary data to synthesize compounds that exhibit better potency and selectivity for a given
Opinion
TRENDS in Parasitology
target. This is achieved by optimizing the interaction between the compound and the protein by computer-based molecular modelling (compound docking [22]), followed by laboratory experimentation to test the prediction. Modelling based on the crystal structure of neuraminidase resulted in the development of the anti-influenza drug zanamivir (Relenzaw, GlaxoSmithKline; http://www.gsk. com) (reviewed in Ref. [23]). Two HIV drugs, amprenavir and nelfinavir, were developed using the crystal structure of HIV protease [24]. In the case of the HIV protease, the enzyme was first identified via comparative genomics and backed up by comparative modelling of the protein on the structures of aspartic proteases. The foregoing principles form the basis of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium (http://depts.washington.edu/sgpp/), which aims to apply high-throughput methods to express large numbers of proteins, and to determine three-dimensional crystal structures of proteins from Leishmania and other parasites. These structures are available to all researchers and will probably provide targets for structure-based lead discovery in the future. Having high-resolution structural data of target proteins also enables the use of virtual screening (VS) of large databases of compounds in silico (for review, see Ref. [25]), which could allow the selection of a manageable number of candidate molecules that can be tested for biological activity. VS can select a small subset of compounds from a large library for use in more complex, low-throughput assays such as cell-based assays. The main components of VS are compound docking based on a protein’s structure, and chemical-similarity searching on small molecules if an active compound is known. The computational docking approach was instrumental in the identification of 40 parasite-specific inhibitors of the L. mexicana glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [26]. Although several drugs have been discovered through structure-based design, there are limitations in the use of X-ray crystallographic data in drug discovery. These limitations include uncertainties in atomic models of proteins generated by X-ray diffraction data and the failure of structure-based design to adequately address drug optimization properties such as absorption, metabolic stability, plasma – protein binding, elimination and toxicological profile [27]. Despite these limitations, the combination of VS and HTS is predicted to increase the success rate of screening experiments. Development of a drug candidate A major decision gate on the road to drug development is whether there are any properties of the drug candidate that would make its development difficult. Issues of bioavailability, metabolic processing, toxicity and interactions with other drugs have to be considered. An interesting by-product of the advances in genetics and proteomics has been the identification and modification of potentially therapeutic macromolecules, such as hormones and growth factors, from biological sources, rather than small-molecule synthetic drugs [10]. However, the existing assays for toxicity, metabolic processing and absorption, usually performed in animal models, might not www.sciencedirect.com
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be appropriate because these molecules are cell receptors or ligands, or antibodies and peptides specifically engineered to affect human targets. Developing new pre-clinical tests for the drugs of the future is a new challenge not only for pharmaceutical companies, but also for basic research scientists embarking on this road. Synergy between drugs and host immunity Antimicrobial drugs generally work in conjunction with the host immune system; antibiotic therapy is far less effective in the setting of immunodeficiency. This is also relevant to the development of new antileishmanial agents because the T cell-dependent immune response in the infected host is directed at activating tissue macrophages, the parasite’s host cells. Work in the past decade has shown that some drugs, such as pentavalent antimonials, require an intact cell-mediated immune response for expression of their antileishmanial effect in vivo, whereas amphotericin B acts directly on the host cell [6]. Irrespective of whether host T-cell responsiveness is required, the in vivo efficacy of antileishmanial drugs can be enhanced by simultaneously triggering proinflammatory T helper cell Type 1 (Th1) immune responses [6,9,28] with the secretion of cytokines such as interleukin 12 (IL-12) and interferon g (IFN-g). Combining drugs with immunostimulation (immunochemotherapy) to enhance antileishmanial efficacy and improve outcome adds a new level of complexity to drug development [6]. Conversely, inhibition of suppressive Th2-type cytokines such as IL-4 and IL-10 might prove useful for individuals with severe disease because of their propensity to turn on these deleterious cytokines, rather than the host-protective ones [6,9,29,30]. This could be accomplished by injecting neutralizing anti-IL-4 or anti-IL-10 receptor antibodies along with drug. Interdigitation of chemotherapy with immunomodulators represents another avenue to consider for new antileishmanial drug development [31,32]. Funding of drug discovery and development When discussing drug development, we cannot ignore the economic realities. Should the limited resources be used to improve the existing drugs or to search for new ones? Even if available, will the people most in need be able to afford them? There is no doubt that drug discovery and development is an expensive and risky business under the best circumstances. Unfortunately, drug companies seem to be pulling out of antibiotics development [33,34], and the cost and resources associated with drug development are generally outside the scope of academic laboratories. While HTS is a key part of modern pharmaceutical development, most publicly funded laboratories cannot afford access to such technology. It is imperative that publicly funded laboratories forge partnerships with industry. The WHO/TDR (http://www.who.int/tdr/) and National Institutes of Health (http://www.niaid.nih.gov/ dmid/drug) are providing funding and guidance with the aim of establishing links with industry partners, and Me´dicins Sans Frontie`res has established the Drugs for Neglected Diseases Initiative (http://www.accessmed-msf. org/dndi.asp). It is hoped that such relationships will
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greatly expedite the discovery and implementation of novel therapies for leishmaniasis. Acknowledgements We are indebted to Jim Goding for his critical review of the article. The work of H.W.M. is supported by NIH grant AI 16393. E.H. and A.J.D. are supported by the Australian National Health and Medical Research Council and UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.
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16 El Fakhry, Y. et al. (2002) A proteomic approach to identify developmentally regulated proteins in Leishmania infantum. Proteomics 2, 1007– 1017 17 Acestor, N. et al. (2002) Establishing two-dimensional gels for the analysis of Leishmania proteomes. Proteomics 2, 877 – 879 18 Drummelsmith, J. et al. (2003) Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms. Mol. Cell. Proteomics 2, 146– 155 19 Walters, W.P. and Namchuk, M. (2003) Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov. 2, 259 – 266 20 Lipinski, C.A. et al. (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3 – 26 21 Walters, W.P. et al. (1999) Recognizing molecules with drug-like properties. Curr. Opin. Chem. Biol. 3, 384 – 387 22 Halperin, I. et al. (2002) Principles of docking: An overview of search algorithms and a guide to scoring functions. Proteins 47, 409 – 443 23 Blundell, T.L. et al. (2002) High-throughput crystallography for lead discovery in drug design. Nat. Rev. Drug Discov. 1, 45 – 54 24 Greer, J. et al. (1994) Application of the three-dimensional structures of protein target molecules in structure-based drug design. J. Med. Chem. 37, 1035 – 1054 25 Bajorath, J. (2002) Integration of virtual and high-throughput screening. Nat. Rev. Drug Discov. 1, 882 – 894 26 Bressi, J.C. et al. (2001) Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design. J. Med. Chem. 44, 2080 – 2093 27 Davis, A.M. et al. (2003) Application and limitations of x-ray crystallographic data in structure-based ligand and drug design. Angew. Chem. Int. Ed. Engl. 42, 2718– 2736 28 Masihi, K.N. (2003) Concepts of immunostimulation to increase antiparasitic drug action. Parasitol. Res. 90 (Suppl. 2), S97– S104 29 Murray, H.W. et al. (2002) Interleukin-10 (IL-10) in experimental visceral leishmaniasis and IL-10 receptor blockade as immunotherapy. Infect. Immun. 70, 6284 – 6293 30 Murray, H.W. et al. (2003) Modulation of T cell costimulation as immunotherapy or immunochemotherapy in experimental visceral leishmaniasis. Infect. Immun. 71, 6453– 6462 31 Buates, S. and Matlashewski, G. (1999) Treatment of experimental leishmaniasis with the immunomodulators imiquimod and S-28463: efficacy and mode of action. J. Infect. Dis. 179, 1485– 1494 32 Arevalo, I. et al. (2001) Successful treatment of drug-resistant cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator. Clin. Infect. Dis. 33, 1847– 1851 33 Walsh, C. (2003) Where will new antibiotics come from? Nat. Rev. Microbiol. 1, 65 – 70 34 Clarke, T. (2003) Drug companies snub antibiotics as pipeline threatens to run dry. Nature 425, 225
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