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Dirofilaria immitis, also known as heartworm, is a major parasitic threat for dogs and cats around the world. Because of its impact on the health and welfare of companion animals, heartworm disease is of huge veterinary and economic importance especially in North America, Europe, Asia and Australia. Within the animal health market many different heartworm preventive products are available, all of which contain active components of the same drug class, the macrocyclic lactones. In addition to compliance issues, such as under-dosing or irregular treatment intervals, the occurrence of drug-resistant heartworms within the populations in the Mississippi River areas adds to the failure of preventive treatments. The objective of this review is to provide an overview of the disease, summarize the current disease control measures and highlight potential new avenues and best practices for treatment and prevention., Graphical abstract Image 1

Coccidiosis is a parasitic disease of a wide variety of animals caused by coccidian protozoa. The coccidia are responsible for major economic losses of the livestock industry. For example, the annual cost due to coccidiosis to the global poultry industry has been estimated to exceed US$ 3 billion annually. Currently available drugs for the control of this disease are either polyether ionophorous antibiotics that are derived from fermentation products, or synthetic compounds, produced by chemical synthesis. Unfortunately, no new drugs in either category have been approved for use for decades. Resistance has been documented for all those of the drugs currently employed and therefore the discovery of novel drugs with unique modes of action is imperative if chemotherapy is to remain the principal means to control this disease. This chapter aims to give an overview of the efficacy and mode of action of the current compounds used to control coccidiosis in livestock and provides a brief outlook of research needs for the future.

Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission., Graphical abstract Image 1, Highlights • Novel drugs against ectoparasites should prohibit vectors to transmit pathogens. • Timing events of most human and animal vector-borne diseases are reviewed. • Most ticks and insects show substantial differences in transmission time. • Drug profile requirements differ between ticks and insects. • Ideal new ectoparasiticides combine fast speed of kill and repellency.

Efficient control of arthropod ectoparasite infestations has a long-standing history in the agriculture and veterinary sectors, aiming to decrease the parasite burden of affected crops and animals. Ligand-gated chloride channels (LGCCs) modulated by γ-aminobutyric acid (GABA) and glutamate have been identified as suitable molecular targets, and several classes of potent parasiticides have been devised. Due to the increase in cross-resistance and decreased development of new chemical entities, an urgent need for new parasiticides or prevention schemes has emerged. In the last decade, an innovative isoxazoline chemotype appears to offer promise for inhibiting LGCCs with a new mode of action and distinct binding site from that of historical agents. Considerable efforts have focused on optimizing the antiparasitic activity of isoxazolines and may provide the potential for future human use.

The extraordinary achievements of genome-based biology in recent years can be explained for the most part by the technological progress in DNA sequencing as well as developments in hardware and software that have made it possible to store and annotate huge amounts of data. The total number of all freely accessible nucleotides in GenBank [genbank], the DNA sequence database at the NCBI, is 218 billion bases within 196 million DNA sequences (Release 215.00, August 2016). The number of all protein sequences in the world’s largest nonredundant protein database UniprotKB [uniprotkb] at the EBI totals 65 million (September 2016).

A new era in genome research started in 1995 with the publication of the first completely sequenced bacterial genome from the human pathogen Haemophilus influenzae. For the first time one could analyze a complete genome, including both genes and their regulatory regions. Three years later, the sequencing of the first multicellular eukaryotic genome, from the nematode Caenorhabditis elegans, was completed. Eukaryotic genomes are larger and far more complex than those from bacteria (► Chap. 7). A comparison of eukaryotic and prokaryotic genomes demonstrated that genes encoding proteins constitute a much smaller proportion of the eukaryotic genome. Thus, in humans and mice just 1.4% of the genome actually encodes genes, and only 5% of both genomes are highly conserved even though both share approximately 80% gene orthology. In addition to protein-encoding genes, conserved regions contain important regulatory elements, non-protein-encoding genes, and regions important for chromosome structure. For the greater proportion of the genome, however, there are few data regarding function (Mouse Genome Sequencing Consortium 2002).

The first human genome was published in 2001 by the Human Genome Project. At that time it was estimated that the number of human genes was in a range between 30,000 and 35,000. It is known today that the human genome is quite young from a phylogenetic point of view and shows an enormous difference between the number of genes and the size of the genome. It contains 19,000–20,000 genes (Ezkurdia et al. 2014), with an overall size of 3.3 Gigabases (see also ► Chaps. 4 and 7). Each human cell, except for sperm and eggs, has a complete set of genes. Obviously, however, a blood cell differs in its morphology and physiology from a liver cell. How, therefore, can these differences be explained if all cells have the same genetic material? The answer is simple. Not every gene is transcribed and expressed in every cell. It follows that only those proteins that are required are present in a cell at a given time during the cell’s lifetime. The proteome of a cell or tissue is therefore dependent on the cell type and its current state.

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of, Author Summary Trypanosomatidae are responsible for approximately half a million human fatalities per annum and the situation is compounded by substantial economic losses due to affecting live stock as well. Trypanothione reductase (TR) is an essential key enzyme of the unique trypanothione-based thiol metabolism of the trypanosomatidae and TR is a promising target for the development of selective inhibitors. However, TR is a very hard to attack target in standard drug discovery approaches. Therefore, we developed a combined and iterative in vitro and in silico screening approach, which led to a high number of novel TR inhibitors. 82 of those showed activities down to the nM range against T. cruzi TR. Moreover, the four most active compounds were selected for determining the inhibitor constant. In first on parasite efficacy studies, three of those compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 μM.

Haemophilus parasuis is the causative agent of polyserositis in pigs, a mostly fatal disease on the rise especially in early-weaned pigs and in pig herds with a high-health status. The mechanisms by which H. parasuis propagates through the body and colonizes the serous membranes are unknown. We have used an H. parasuis microarray to identify virulence genes involved in host adaptation. H. parasuis gene expression was analysed under in vitro growth conditions mimicking the environmental conditions encountered during an infection. These included iron-limitation, acidic and temperature stress and growth under microaerobic conditions. A kinetic impression of the gene regulation was obtained by analysing the transcription 10, 30 and 60 min after induction of the altered growth conditions. A total of 75 regulated H. parasuis genes were identified, most of which coded for transporters of iron and sugar metabolites, metabolic enzymes, DNA metabolism and hypothetical proteins with unknown functions. Furthermore, H. parasuis genes were identified that have homology to known virulence factors in other pathogenic bacteria. Homologues of some of the identified H. parasuis genes are known to be expressed during natural and experimental infections in pathogens of the Pasteurellaceae family.

With rising numbers of fully sequenced genomes the importance of comparative genomics is constantly increasing. Although several software systems for genome comparison analyses do exist, their functionality and flexibility is still limited, compared to the manifold possible applications. Therefore, we developed Genlight, a Client/Server based program suite for large scale sequence analysis and comparative genomics. Genlight uses the object relational database system PostgreSQL together with a state of the art data representation and a distributed execution approach for large scale analysis tasks. The system includes a wide variety of comparison and sequence manipulation methods and supports the management of nucleotide sequences as well as protein sequences. The comparison methods are complemented by a large variety of visualization methods for the assessment of the generated results. In order to demonstrate the suitability of the system for the treatment of biological questions, Genlight was used to identify potential drug and vaccine targets of the pathogen Helicobacter pylori. Availability: The Genlight system is publicly available for non-commercial use on http://piranha.techfak.uni-bielefeld.de. To retrieve a personal user account, please use the contact address mentioned on this page.

The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1′-S3′ subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3′ by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3′ subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2′ position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.

The binding modes of E-64c to papain combined with molecular modeling and ligand design using the crystal structure of cruzain have been used to develop new, potent d -Homophenylalanyl epoxysuccinate inhibitors of cruzain, the major cysteine protease of Trypanosoma cruzi . The most potent inhibitor 47 contains an O -benzyl hydroxamate unit and is highly specific for cruzain relative to other cysteine proteases tested.

In the 20th century and especially during the last 50 years, antiinfectives have been increasingly used to control and prevent infectious diseases. Unfortunately the resistance of microorganisms to these Pharmaceuticals has increased as well. At the same time the discovery process for novel antiinfectives, the so-called “conventional” screening approach, involves testing natural products or derivatives of known compounds in in vitro cultures. By now it is obvious that this screening approach did not meet the expectations to generate a sufficient number of novel drug candidates. Consequently, studies for selective antiinfectives with new modes of action, which are able to break resistance, are highly desirable for human and animal health. The enormous advance in sequencing technologies — leading to a constantly growing number of known microbial genomes — together with the rapid development of computer power and bioinformatic software tools, now makes it possible to identify genes and gene products that are essential to the pathogenic organisms and are therefore considered to be novel targets for the development of new antiinfectives. When these potential targets have been validated by sophisticated laboratory methods, large diverse compound libraries can be tested in in vitro assays using high-throughput screening. This approach will most likely generate an increasing number of novel lead structures that will be specifically optimized by modern combinatorial chemistry and subsequently lead to new antiinfective candidates strengthening the armoury of weapons available to fight infectious diseases in humans and animals.

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

We used an axenic cultivation system to grow African trypanosomes in vitro. Long-term cultivation for more than 60 days has been achieved by replacing the culture medium at regular intervals between 6 and 48 h. In contrast to a control culture without medium replacement, increasing amounts of maximum cell concentrations have been obtained, ranging from 5 x 10(6) to 2 x 10(7) trypanosomes ml-1, whereas the generation doubling time remained constant (about 6 h). Higher cell concentrations have only been obtained by total medium replacement; neither addition of fresh medium nor serum led to a higher cell yield, suggesting that a trypanosome-derived factor or metabolite accumulated in the medium rather than medium was depleted of an essential nutrient. Most interestingly, however, successive waves have been obtained which eventually led to a damped oscillation curve with a constant high population density after about 40 days of cultivation. Cultures were started with a homogeneous population of the long-slender form. As judged by light microscopy, cells showed a stumpy morphology during the declining phase and became slender again in the following growth phase. At later time points, when cells remained in a stationary phase at high population density, many different morphological stages have been observed, similar to those described by early authors as intermediate forms [Ormerod, W. E. (1979) In: Biology of the Kinetoplastida, Vol. 2, pp. 340-393], although many dividing forms are still present at that time. In contrast, identically treated procyclic cultures were unable to produce cyclic growth waves. Based on these results, a novel concept considering a possible differentiation mechanism is discussed.

The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two \(Mg^{2+}\) ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows \(N^1\)-glutathionylation of \(N^8\)-glutathionylspermidine, classifying \(N^8\)-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for \(N^8\)-glutathionylspermidine was characterised as druggable.

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria . We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella ( EtCatB ). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris . Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria . The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

Apicomplexan parasites encompass several human- and animal-pathogenic protozoans such as Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella causes coccidiosis, a disease that afflicts chickens, leading to tremendous economic losses to the global poultry industry. The considerable increase in drug resistance makes it necessary to develop new therapeutic strategies against this parasite. Cyclin-dependent kinases (CDKs) are key molecules in cell-cycle regulation and are therefore prominent target proteins in parasitic diseases. Bioinformatics analysis revealed four potential CDK-like proteins, of which one—E. tenella CDK-related kinase 2 (EtCRK2)—has already been characterized by gene cloning and expression.1 By using the CDK-specific inhibitor flavopiridol in EtCRK2 enzyme assays and schizont maturation assays (SMA), we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as a template to build protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP binding site between EtCRK2 and HsCDK2, as well as chicken CDK3, were addressed for the optimization of selective ATP-competitive inhibitors. Virtual screening and “wet-bench” high-throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized, leading to a set of four promising lead compounds for development as EtCRK2 inhibitors.

The resurgence of Tuberculosis, caused primarily by Mycobacterium tuberculosis, and the appearance of drug resistant tuberculosis strains encourage the need for new drugs with alternative mode of actions [1]. An interesting drug-target is the Thioredoxin-Reductase (TrxR)/Thioredoxin (Trx) System of M. tuberculosis, which is responsible for providing reducing equivalents for many cellular processes including bacterial antioxidant defence [2]. We performed a virtual screening approach for identifying novel drugs for the treatment of Tuberculosis that used the hydrogen bonding constraint functionality of GOLD for pre-processing instead of using pharmacophore filtering. Two important hydrogen bonding interactions could be identified at the protein-protein interface of the TrxR-Trx complex. A high-throughput docking was applied to filter the Intervet in-house compound library (~6.5 million compounds) for possible hits that interact with these two hydrogen bonding acceptors. This reduced the number of interesting compounds to 151768 that were redocked without any constraints and more accurate docking settings. Finally, the ranking of the reduced compound set was obtained using a normalisation based on molecular weight [3] and a consensus scoring in a restrictive way using Chemscore and ASPscore. So far, six out of the first 25 of the ranked compound list showed an activity with an IC50 value upto μM range. Especially three compounds with different scaffolds and a low molecular weight are promising candidates for further developments.

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate → 1 lactate + 1 acetate + 1 CO 2 ). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture orin vivo. We now report that specific cysteine protease inhibitors killLeishmaniaparasitesin vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition ofLeishmaniacysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stablein vivoto ameliorate the pathology associated with a mouse model ofLeishmaniainfection.

The crystal structures of papain, cruzain, and human liver cathepsin B were used to build homology-based enzyme models of a cathepsin L-like cysteine protease (cpL) and a cathepsin B-like cysteine protease (cpB) from the protozoan parasite Leishmania major. Although structurally a member of the cathepsin B subfamily, the L. major cpB is not able to cleave synthetic substrates having an arginine in position P2. This biochemical property correlates with the prediction of a glycine instead of a glutamic acid at position 205 (papain numbering). The modeled active sites of the L. major cpB and cpL were used to screen the Available Chemicals Directory (a database of about 150,000 commercially available compounds) for potential cysteine protease inhibitors, using DOCK3.5. Based on both steric and force field considerations, 69 compounds were selected. Of these, 18 showed IC50's between 50 and 100 microM and 3 had IC50's below 50 microM. A secondary library of compounds, originally derived from a structural screen against the homologous protease of Plasmodium falciparum (falcipain), and subsequently expanded by combinatorial chemistry, was also screened. Three inhibitors were identified which were not only effective against the L. major protease but also inhibited parasite growth at 5-50 microM.

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

The protozoan parasites of the genus Trypanosoma sp. and Leishmania sp. are responsible for neglected diseases like Chagas’ disease, African sleeping sickness or Leishmaniasis. The trypanothione synthetase (TryS) is an attractive new drug target for the development of trypanocidal and antileishmanial drugs [1]. In our virtual screening campaign for targeting the trypanothione synthetase (TryS) we used representative protein conformations derived from a computational analysis using molecular dynamics (MD) simulations of this key component of trypanothione biosynthesis. The publicly available crystal structure lacks a variable loop region that is known to be important for trypanothione biosynthesis. MD simulations turned out to be a good tool to model this loop region and obtain a more complete set of protein conformations for subsequent use in virtual screening [2]. For creating a structure-based consensus pharmacophore model, Superstar [3] was deployed to generate favourable non-bonded interaction maps of different functional groups (probes) for all representative protein conformations. The pharmacophore model was then created for the rigid part of the binding pocket based on high- propensity peaks of these maps. The variable loop region was left out since it can not be depicted by this approach. To include also multiple conformations of the variable loop region, the new ensemble docking feature of Gold was used [4]. After a pharmacophore search within the ZINC database the retrieved molecules were simultaneously docked to the different protein conformations to identify the best combination of ligand pose and protein conformer. Finally, several high-scoring molecules were selected for further testing. We will discuss in detail this combined pharmacophore/ensemble docking approach based on MD simulations and will present the results of the compound selection for testing.