Protein kinases as drug targets in parasitic protozoa

The importance of protein kinases in cell signaling and cell cycle control has led to detailed structural and functional studies in various eukaryotes, and hence to the synthesis of specific chemical inhibitors for managing disease. Here, the current progress in applying developments from the wider protein kinase field to parasitic protozoa is reviewed. The availability of genome sequence data for several parasites has led to the identification of many protein kinases. Reverse genetics studies, including gene knockout and 'chemical genetics', can help to define the roles of the protein kinases and validate them as drug targets. In addition, screening chemical libraries with active recombinant protein kinases can identify lead compounds for drug design.     

Protein kinases as targets for anti-parasitic chemotherapy

Parasitic protozoa infecting humans have a staggering impact on public health, especially in the developing world. Furthermore, several protozoan species are major pathogens of domestic animals and have a considerable impact on food production. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in protein kinases inhibitors as potential drugs against a variety of diseases, the possibility that protein kinases may represent targets for novel anti-parasitic agents is being explored. Research into parasite protein kinases has benefited greatly from genome and EST sequencing projects, with the genomes of a few species fully sequenced (notably that of the human malaria parasite Plasmodium falciparum) and several more under way. The overall picture that emerged from research in this area shows that the phylogenetic isolation of parasitic protozoa is reflected by atypical structural and functional properties of many of their protein kinase homologues. Likewise, evidence is emerging, which suggests that the organisation of some otherwise well-conserved signal transduction pathways is divergent in some parasitic species. The differences between protein kinases of a parasite and their homologues in its host cell suggest that specific inhibition of the former can be achieved. The development of anti-parasitic drugs based on protein kinase inhibition is being pursued following two avenues: one consists of screening chemical libraries on recombinant enzymes; several protein kinases from parasitic protozoa are now available for this approach. The second approach relies on the identification of the molecular targets of kinase inhibitors which display anti-parasitic properties. This has led to promising developments in a few instances, in particular regarding PKG as a drug target against Eimeria and Toxoplasma, and purvalanol B, a purine-based CDK inhibitor which appears to affect unexpected targets in several protozoan parasites. The recent resolution of the structure of a Plasmodium protein kinase complexed with small inhibitory molecules opens the way to a rational approach towards the design of anti-parasitic drugs based on kinase inhibition.     

Leishmania amastigotes as targets for drug screening

Human African trypanosomiasis is a threat to millions of people living in sub-Saharan countries and is fatal unless treated. At present, the serological and parasitological tests used in the field for diagnosis of sleeping sickness have low specificity and sensitivity. There is clearly an urgent need for accurate tools for both diagnosis and staging of the disease. The Foundation for Innovative New Diagnostics and the World Health Organization have announced that they will collaborate to develop and evaluate new diagnostic tests for human African trypanosomiasis.     

Rational therapeutic intervention in cancer: kinases as drug targets.

Landmark clinical studies of new drugs developed to target specific forms of cancer were reported in 2001. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her-2/neu positive metastatic breast cancer, when combined with chemotherapy. STI-571, a small molecule inhibitor of the Bcr-Abl, c-kit and platelet derived growth factor receptor tyrosine kinases, produced dramatic clinical responses in patients with Bcr-Abl positive chronic myeloid leukemia and c-kit positive gastrointestinal stromal tumors. These examples have galvanized the cancer research community to extend kinase-inhibitor therapy to other cancers.     

Protein tyrosine kinases as new potential targets against human schistosomiasis

In spite of the numerous efforts made to control their transmission, parasite schistosomes still represent a serious public health concern and a major economic problem in many developing countries. Praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis and the only one that is available for mass chemotherapy. However, its widespread use and its inefficacy on juvenile parasites raise fears that schistosomes will develop drug resistance, and make the development of alternative drugs highly desirable. Protein tyrosine kinases (PTKs) are key molecules that control cell differentiation and proliferation and they already represent important targets for molecular cancer therapy. The recent characterization in Schistosoma mansoni of several cytosolic and receptor PTKs, with properties similar but also divergent from their vertebrate counterparts, opens new perspectives for the development of novel strategies in chemotherapy of schistosomiasis, which could be based on the use of parasite-specific tyrosine phosphorylation inhibitors.     

Recent advances in identifying and validating drug targets in trypanosomes and leishmanias

The unique aspects of the biochemistry of trypanosomatids make rational drug design an attractive approach, but targets must be selected carefully. Genetic manipulation provides a valuable means of mimicking loss of function attributable to therapeutic intervention, but caution must be exercised when interpreting such data with respect to target validation.     

Inhibitors of dihydrofolate reductase in Leishmania and trypanosomes

The protozoan diseases leishmaniasis, Chagas' disease and African trypanosomiasis are major health problems in many countries, particularly developing countries, and there are few drugs available to treat these diseases. Dihydrofolate reductase (DHFR) inhibitors have been used successfully in the treatment of a number of other diseases such as cancer, malaria and bacterial infections; however they have not been used for the treatment of these diseases. This article summarises studies on leishmanial and trypanosomal DHFR inhibitor development and evaluation. Possible mechanisms of resistance to DHFR inhibitors are also discussed.     

Proteases as drug targets

The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.     

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP-independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double-stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP-independent topoisomerase II provides a potential target for chemotherapy.     

Histidine kinases as targets for new antimicrobial agents

The emergence and spread of hospital acquired multi drug resistant bacteria present a need for new antibiotics with innovative mode of action. Advances in molecular microbiology and genomics have led to the identification of numerous bacterial genes coding for proteins that could potentially serve as targets for antibacterial compounds. Histidine kinase promoted two-component systems are extremely common in bacteria and play an important role in essential signal transduction for adapting to bacterial stress. Since signal transduction in mammals occurs by a different mechanism, inhibition of histidine kinases could be a potential target for antimicrobial agents. This review will summarize our current knowledge of the structure and function of histidine kinase and the development of antibiotics with a new mode of action: targeting histidine kinase promoted signal transduction and its subsequent regulation of gene expression system.     

Adenosine receptors as drug targets

There are four adenosine receptors, A₁, A_2A, A_2B and A₃, together forming a defined subgroup of G protein coupled receptors. They are well conserved and widely expressed. The endogenous agonist, adenosine, has a minimal concentration in body fluids (20-200 nM) that is sufficient to slightly activate the receptors where they are very highly expressed—as in the basal ganglia, on fat cells and in the kidney. Here adenosine can play a physiological role and here antagonists such as caffeine can have effects in healthy individuals. Adenosine levels rise in stress and distress (up to 30 μM in ischemia) and tend to minimize the risk for adverse outcomes by increasing energy supply and decreasing cellular work, by stimulating angiogenesis, mediating preconditioning and having multiple effects on immune competent cells. These pathophysiological roles of adenosine also offer some potential drug targets, but the fact that adenosine receptors are involved in so many processes does not simplify drug development.     

Microbial proteasomes as drug targets

Proteasomes are compartmentalized, ATP-dependent, N-terminal nucleophile hydrolases that play essentials roles in intracellular protein turnover. They are present in all 3 kingdoms. Pharmacological inhibition of proteasomes is detrimental to cell viability. Proteasome inhibitor rugs revolutionize the treatment of multiple myeloma. Proteasomes in pathogenic microbes such as Mycobacterium tuberculosis (Mtb), Plasmodium falciparum (Pf), and other parasites and worms have been validated as therapeutic targets. Starting with Mtb proteasome, efforts in developing inhibitors selective for microbial proteasomes have made great progress lately. In this review, we describe the strategies and pharmacophores that have been used in developing proteasome inhibitors with potency and selectivity that spare human proteasomes and highlight the development of clinical proteasome inhibitor candidates for treatment of leishmaniasis and Chagas disease. Finally, we discuss the future challenges and therapeutical potentials of the microbial proteasome inhibitors.     

Riboswitches as antibacterial drug targets

New validated cellular targets are needed to reinvigorate antibacterial drug discovery. This need could potentially be filled by riboswitches-messenger RNA (mRNA) structures that regulate gene expression in bacteria. Riboswitches are unique among RNAs that serve as drug targets in that they have evolved to form structured and highly selective receptors for small drug-like metabolites. In most cases, metabolite binding to the receptor represses the expression of the gene(s) encoded by the mRNA. If a new metabolite analog were designed that binds to the receptor, the gene(s) regulated by that riboswitch could be repressed, with a potentially lethal effect to the bacteria. Recent work suggests that certain antibacterial compounds discovered decades ago function at least in part by targeting riboswitches. Herein we will summarize the experiments validating riboswitches as drug targets, describe the existing technology for riboswitch drug discovery and discuss the challenges that may face riboswitch drug discoverers.     

Mitochondria as cancer drug targets

Cancer cells are defined by their unlimited replicative potential and resistance to cell death stimuli. It is generally considered that a point of no return in apoptotic cell death is the permeabilisation of the mitochondrial membranes. For this reason, agents that permeabilise cancer cell mitochondria have the potential to circumvent their resistance to apoptotic cell death. Fortunately, the proliferative and bioenergetic differences between normal and cancerous cells provide an opportunity to selectively target cancer cell mitochondria.     

Cyclin‐dependent kinases as therapeutic targets in melanoma

Decades of scientific insights have led to a recent expansion of the therapeutic menu for melanoma. Despite these advances, the current targeted therapies and immune checkpoint agents continue to yield suboptimal response and cure rates. Hitherto, the most effective targeted therapy strategies have centered on effectors in the mitogen‐activated protein kinase (MAPK) pathway. This review focuses on the emerging evidence of combinatorial approaches targeting both MAPK signaling and dysregulations in cell‐cycle checkpoints. We discuss the prospects and limitations of utilizing strategies that promote cellular senescence, such as inhibition of the interphase cyclin‐dependent kinases (CDKs) and highlight the current state of CDK drug discovery in melanoma.     

Validation of spermidine synthase as a drug target in African trypanosomes

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.