Jim Reynoldson

The binding kinetics of several benzimidazole compounds were determined with recombinant tubulin from benzimidazole-sensitive and -insensitive organisms. This study utilised the naturally occurring high efficacy of the benzimidazoles for the parasitic protozoa Giardia duodenalis and Encephalitozoon intestinalis, and low efficacy with Cryptosporidium parvum. Direct kinetic analysis of the benzimidazole-β-tubulin interaction was performed using a fluorescence-based quenching method to determine the apparent association (k on) and dissociation (k off) rate constants from which the affinity constant (K a) was calculated. The binding kinetics were determined with recombinant α- and β-tubulin from the parasitic protozoa with several benzimidazole R 2-carbamate analogues. The affinity constant for the binding of several benzimidazoles with β-tubulin from benzimidazole-sensitive protozoa was found to be significantly greater than binding to β-tubulin from benzimidazole-insensitive protozoa. Additionally, the high affinity of several benzimidazole derivatives (albendazole, fenbendazole, mebendazole) for monomeric β-tubulin and heterodimeric αβ-tubulin from benzimidazole-sensitive protozoa was also clearly demonstrated. The affinity constants determined with β-tubulin from G. duodenalis and E. intestinalis also supported the observed in vitro efficacy of these compounds. The binding characteristics of the benzimidazoles with the highest in vitro efficacy (albendazole, fenbendazole, mebendazole) was reflected in their high association and slow dissociation rates with the β-tubulin monomer or dimer from benzimidazole-sensitive protozoa compared with insensitive ones. Benzimidazole-bound αβ-tubulin heterodimers also had a significantly lower rate of microtubule assembly compared with benzimidazole-free αβ-heterodimers. The incorporation of benzimidazole-bound αβ-heterodimers into assembling microtubules was shown to arrest polymerisation in vitro although the addition of benzimidazole compounds to assembled microtubules did not result in depolymerisation. These findings indicate that a benzimidazole-β-tubulin cap may be formed at the growing end of the microtubule and this cap prevents elongation of the microtubule.

The α- and β-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.

This review focuses on chemotherapies used against the parasite, Cryptosporidium parvum, the causative agent of cryptosporidiosis. Populations at risk from severe morbidity or mortality from cryptosporidiosis are discussed with particular reference to those infected with HIV. The review then examines chemotherapies used in the clinical setting, as well as a number of in vitro and in vivo experimental studies. It begins with a discussion of the targets within Cryptosporidium that have been the foci of past treatments and then examines novel target sites that may present an exploitable alternative. Some of the novel target sites discussed include the recently discovered apicomplexan plastid and its associated pathways. Lastly, the review examines tubulin as a potential anticryptosporidial target in view of the fact that it has been exploited successfully for almost 50 years for the treatment of helminthiasis. The review concludes with a five-year outlook on the future of anticryptosporidial drug design.

More than 200 compounds have been tested for activity against Cryptosporidium parvum, both in vitro and in vivo, there is still no effective treatment. Previous studies have revealed the anticryptosporidial effect of the tubulin specific herbicides, the dinitroanilines. The in vitro activities of two members of this class of compounds, oryzalin and trifluralin have been demonstrated against Cryptosporidium. Recent studies exposed IC50 values for oryzalin and trifluralin against Cryptosporidium of 750 and 800 nM, respectively. This chapter presents a study, the aim of which on-going study is to examine tubulin as an effective target both in vivo and ex vivo.

A quantitative-PCR (Q-PCR) method that uses an in vitro culturing system for Cryptosporidium parvum has been developed. This sensitive method allows standardization of an in vitro culturing system and its development for quantitative assessment using PCR. This chapter presents a study in which this system was assessed against an established counting method which is widely used to enumerate parasites, particularly following exposure to anti-parasitic compounds. There are several sources of variability inherent in, in vitro culturing systems which could result in an inaccurate final amount of DNA being detected per culture well. These can be summarized as cumulative effects because of variability in the in vitro system and the DNA extraction and quantification method. Analysis of the variability in this in vitro culturing system using Q-PCR indicates that it is a consistent and reliable system which offers higher sensitivity and specificity when compared with counting methods as well as providing a vast improvement in sample.

A method based on the polymerase chain reaction has been developed for differentiating between genotypically and phenotypically distinct strains of Giardia duodenalis and quantifying the amount of initial template of the different genotypes in mixed populations. The assay relies on a sequence-specific probe, labelled with two fluorescent dyes, designed to bind within the small subunit ribosomal (SSU) RNA gene. This target region is amplified by primers specific for either Group 1 or Group 2-type isolates of G. duodenalis and the probe binds within the primer-targeted region. This quantitative method takes advantage of the 5′ nuclease activity of Taq DNA polymerase, which, on encountering a probe bound within the target DNA sequence cleaves it, causing it to become dissociated from the template. When the two fluorescent dyes bound to the probe are in close proximity (when the probe is intact), the interaction of the two dyes prevents the reporter dye from fluorescing. However, during the extension phase of amplification, the activity of the DNA polymerase causes the dyes to become separated and hence the reporter dye increases its fluorescent intensity. This release of fluorescence is directly related to the amplified amount of target template. This assay was developed with the aim of providing a unique method with which to investigate interactions within mixed populations of genetically distinct strains of G. duodenalis.

This paper reports the anti-cryptosporidial effects of, and concomitant amelioration of the histological changes in the gut of neonatal rats with intestinal cryptosporidiosis treated with the dinitroaniline, oryzalin. The ED50 was determined to be 7 mg/kg using twice daily doses administered for 3 consecutive days. A maximum inhibition of 85.5% was achieved at 25 mg/kg and this inhibition remained constant despite increasing the oryzalin dose to 200 mg/kg. Cryptosporidiosis significantly decreased the intestinal villus/crypt (VC) ratio by approximately 50% (duodenum = 2.3, jejunum = 2.5 and ileum = 1.7) when compared to uninfected untreated controls (duodenum = 4.3, jejunum = 5.9 and ileum = 4.5). Treatment with oryzalin doubled the VC ratio in the duodenum, jejunum and ileum following doses of 5 mg, 50 mg and 200 mg/kg respectively. Oryzalin concentrations in the small intestine contents and plasma were determined, using HPLC, at 0.5, 1 and 2 h after dosing. The much greater dose required to return VC ratios to normal in the ileum (200 mg/kg) compared to the duodenum (6.25 mg/kg) appeared to reflect the decreased concentration of the drug in the distal small intestine. Concentrations of oryzalin equivalent to the in vitro IC50 were maintained for 2 h in the first half of the small intestine following a single dose of 100 mg/kg.

A reliable and sensitive quantitative-PCR (Q-PCR) method using an in vitro culturing system for Cryptosporidium parvum has been developed and characterised. This method allows standardisation of an in vitro culturing system and its development for quantitative assessment using PCR. This system was assessed against an established counting method which is widely used to enumerate parasites, particularly following exposure to antiparasitic compounds. There are several sources of variability inherent in in vitro culturing systems which could result in an inaccurate final amount of DNA being detected per culture well. These can be summarised as cumulative effects due to variability in the in vitro system, and the DNA extraction and quantification method. Analysis of the variability in this in vitro culturing system using Q-PCR indicates that it offers higher sensitivity and specificity when compared with counting methods as well as providing a vast improvement in sample throughput and efficiency. Further to this, we have also determined a method for calculating the inhibitory concentration of anticryptosporidial compounds and present a comparison of this method with a counting method and published data. We conclude that this method of quantification could be used as a substitute for haemocytometer methods, particularly, and also antibody-based techniques.

A rodent model of malaria, Plasmodium berghei, was used to assess the antimalarial potential of dinitroaniline herbicides. Trifluralin, pendimethalin, oryzalin, and benfluralin were all active against P. berghei in vitro at, or close to, submicromolar concentrations, with a rank order of potency similar to that against other protozoa. The dinitroanilines did not elicit a cytotoxic effect against a mammalian cell line at concentrations 100-fold higher than those for activity against P. berghei. Neither trifluralin nor oryzalin exhibited any antimalarial activity in vivo after oral administration at the maximum dose tolerated by the host. In a pharmacokinetic study, it was found that the lack of in vivo antimalarial activity was due to poor absorption. Other DNs which have better absorption characteristics than either trifluralin or oryzalin may offer more scope for antimalarial activity in vivo.