Discovery of trypanocidal thiosemicarbazone inhibitors of rhodesain and TbcatB

Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. The cysteine proteases of T. brucei have been shown to be crucial for parasite replication and represent an attractive point for therapeutic intervention. Herein we describe the synthesis of a series of thiosemicarbazones and their activity against the trypanosomal cathepsins TbcatB and rhodesain, as well as human cathepsins L and B. The activity of these compounds was determined against cultured T. brucei, and specificity was assessed with a panel of four mammalian cell lines.     

Multiple Roles for Cysteine Cathepsins in Cancer

Cysteine cathepsins are a family of proteases that have recently emerged as important players in cancer, and have variously been reported to be involved in apoptosis, angiogenesis, cell proliferation, and invasion. In normal cells, cysteine cathepsins are typically localized in lysosomes and other intracellular compartments, and are involved in protein degradation and processing. However, in certain tumors, cathepsins are translocated from their intracellular compartments to the cell surface, and can even be secreted. In addition, the expression and activity levels of some cysteine cathepsins are upregulated in human and mouse cancers. Understanding which cathepsins are critically involved, what their substrates are, and how they may be mediating these complex roles in cancer are important questions to address. We highlight recent results that begin to answer some of these questions, illustrating in particular the lessons from studying a mouse model of multistage carcinogenesis, which suggests distinctive roles for individual cysteine cathepsins in tumor progression.     

The isolation of nucleic acid from nematodes requires an understanding of the parasite and its cuticular structure

As parasitology increasingly becomes the domain of molecular biologists, it is important to keep in mind that a fundamental understanding of the whole parasite, its structure and behaviour can help to solve complex problems of molecular biology. Hugh Dawkins and Terence Spencer discuss the preparation of DNA from filaform larvae of Trichostrongylus colubriformis and Ostertagia circumcincta and how a detailed knowledge of the morphology and life cycle of each parasite helps in this process.     

The effect of host heterogeneity and parasite intragenomic interactions on parasite population structure

Understanding the processes that shape the genetic structure of parasite populations and the functional consequences of different parasite genotypes is critical for our ability to predict how an infection can spread through a host population and for the design of effective vaccines to combat infection and disease. Here, we examine how the genetic structure of parasite populations responds to host genetic heterogeneity. We consider the well-characterized molecular specificity of major histocompatibility complex binding of antigenic peptides to derive deterministic and stochastic models. We use these models to ask, firstly, what conditions favour the evolution of generalist parasite genotypes versus specialist parasite genotypes? Secondly, can parasite genotypes coexist in a population? We find that intragenomic interactions between parasite loci encoding antigenic peptides are pivotal in determining the outcome of evolution. Where parasite loci interact synergistically (i.e. the recognition of additional antigenic peptides has a disproportionately large effect on parasite fitness), generalist parasite genotypes are favoured. Where parasite loci act multiplicatively (have independent effects on fitness) or antagonistically (have diminishing effects on parasite fitness), specialist parasite genotypes are favoured. A key finding is that polymorphism is not stable and that, with respect to functionally important antigenic peptides, parasite populations are dominated by a single genotype.     

Mining a cathepsin inhibitor library for new antiparasitic drug leads

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ～ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.     

Conformational studies on the specific cleavage site of Type I collagen (alpha-1) fragment (157-192) by cathepsins K and L by proton NMR spectroscopy

Cathepsins K and L are cysteine proteinases which are considered to play an important role in bone resorption. Type I collagen is the most abundant component of the extracellular matrix of bone and regarded as an endogenous substrate for the cysteine proteinases in osteoclastic bone resorption. We have synthesized a fragment of Type I collagen (alpha-1) (157-192) as a substrate for the cathepsins and found that cathepsins K and L cleave the fragment at different specific sites. The major cleavage sites for cathepsin K were Met159-Gly160, Ser162-Gly163 and Arg165-Gly166, while those for cathepsin L were Gly166-Leu167 and Gln180-Gly181. The structure of the fragment was analyzed in aqueous solution by circular dichroism and proton NMR spectroscopy and the difference in the molecular recognition of collagen by cathepsins K and L was discussed from the structural aspect.     

Case-control studies on host factors in severe malaria

Although molecular biology has illustrated the phenotypic heterogeneity of Plasmodium falciparum, there are still no specific markers of virulence. As parasite virulence is an important determinant of severe malaria, the choice of comparison groups in the study of host factors influencing severity is a delicate issue. Ignoring parasite factors in the selection of controls potentially leads to biased comparisons between a majority of cases with virulent parasites and a majority of controls with non-virulent parasites. This article discusses how to avoid this virulence bias in the absence of specific markers of virulence.     

Molecular studies of Biomphalaria glabrata, an intermediate host of Schistosoma mansoni

The freshwater gastropod Biomphalaria glabrata is one of the most important invertebrate hosts of the helminth parasite Schistosoma mansoni. Investigators are using different strategies to determine the molecular basis of this snail-parasite relationship. Of particular interest are the identification of parasite resistance genes in the snail, and the application of molecular probes to better understand the epidemiology of schistosomiasis. This review will focus on recent advances that have been made on genome analysis of B. glabrata. Much of this work has centred on the use of random amplification of polymorphic DNA-PCR-based technology, with restriction fragment length polymorphism analysis and the generation of expressed sequence tags from the snail. A brief discussion of how parasite products may complicate this analysis is also given, along with an indication of the scope of the problems that lie ahead.     

Inhibition of mammalian cathepsins byPlesiomonas shigelloides

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.     

Helminth pathogen cathepsin proteases: it's a family affair

Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.     

An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest.     

Acid proteinase activity in fish II. Purification and characterization of cathepsins B and D from Mujil auratus muscle

Two cathepsins were detected in Mujil auratus muscle extracts. They were classified as a thiol- and aspartyl-proteinase (cathepsins B and D, respectively) on the basis of their catalytic behaviour in presence of specific inhibitors. Following extraction in 1% KCl, the proteinases were purified by autolysis, acetone fractionation, affinity chromatography, and gel permeation chromatography. The haemoglobin-agarose column chromatography allowed us to separate the two activities. Sephadex G-75 column chromatography resulted in apparent molecular weights of 25,000 (cathepsin B) and 35,000 (cathepsin D). The molecular size, together with pH-activity profiles and kinetic parameters are similar to those reported for mammalian cathepsins B and D. This was not the case with the temperature-activity profiles, the optimum temperature as well as the heat stability being higher for fish cathepsins than for those obtained from other sources. Cathepsin B was characterized by its ability to inactivate aldolase. Fluorescence quenching experiments showed that tryptophyl residues of cathepsin B were less occluded and located in a more electronegative microenvironment that those pertaining to cathepsin D.     

组织蛋白酶及其抑制剂研究进展

组织蛋白酶是半胱氨酸蛋白酶家族的主要成员,在生物界已发现20余种,人体中主要存在11种,它们与人类肿瘤、骨质疏松、关节炎等多种重大疾病密切相关,是近年来备受关注的一类靶标蛋白酶。自从20世纪90年代以来,多种组织蛋白酶的晶体结构陆续明确,有关其研究进展较快。本文以人类组织蛋白酶为重点,主要介绍近15年来组织蛋白酶结构、功能和抑制剂研究方面的一些重要进展。     

Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma

Recent years have seen tremendous progress in our understanding of malaria parasite molecular biology. To a large extent, this progress follows significant developments in genetic, molecular and chemical tools available to study the malaria parasites and related Apicomplexa, in particular Toxoplasma gondii. One area of major advancement has been in understanding parasite host-cell invasion, a process that utilizes several essential molecular mechanisms that are conserved across the different lifecycle stages. Here, we summarize some of the most recent experimental data that shed light on the events underlying preparation and execution of malaria parasite invasion and how these insights might relate to the development of new antimalarial drugs.     

Resistance to antiparasitic drugs: the role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.     

The evolutionary ecology of Plasmodium

Plasmodium, the aetiological agent of malaria, imposes a substantial public health burden on human society and one that is likely to deteriorate. Hitherto, the recent Darwinian medicine movement has promoted the important role evolutionary biology can play in issues of public health. Recasting the malaria parasite two‐host life cycle within an evolutionary framework has generated considerable insight into how the parasite has adapted to life within both vertebrate and insect hosts. Coupled with the rapid advances in the molecular basis to host–parasite interactions, exploration of the evolutionary ecology of Plasmodium will enable identification of key steps in the life cycle and highlight fruitful avenues of research for developing malaria control strategies. In addition, elucidating the extent to which Plasmodium can respond to short‐ and long‐term changes in selection pressures, i.e. its adaptive capacity, is even more crucial in predicting how the burden of malaria will alter with our rapidly evolving ecology.     

Apoptosis caused by cathepsins does not require Bid signaling in an in vivo model of progressive myoclonus epilepsy (EPM1)

Apoptosis can be mediated by mechanisms other than the traditional caspase-mediated cleavage cascade. There is growing recognition that alternative proteolytic enzymes such as the lysosomal cathepsin proteases can initiate or propagate proapoptotic signals, but it is currently unclear how cathepsins achieve these actions. Recent in vitro evidence suggests that cathepsins cleave the proapoptotic Bcl-2 family member Bid, thereby activating it and allowing it to induce the mitochondrial release of cytochrome c and subsequent apoptosis. We have tested this hypothesis in vivo by breeding mice that lack cathepsin inhibition (cystatin B-deficient mice) to Bid-deficient mice, to determine whether the apoptosis caused by cathepsins is dependent on Bid signaling. We found that cathepsins are still able to promote apoptosis even in the absence of Bid, indicating that these proteases mediate apoptosis via a different pathway, or that some other molecule can functionally substitute for Bid in this system.