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Malaria, AIDS (acquired immunodeficiency syndrome) and Tuberculosis, according to the United Nations, are the most devastating diseases affecting individuals living on under 2 dollars a day; accounting annually for 5.6 million deaths and the total loss of 166 million disability-adjusted life years (DALYs). There is also a constant battle for UN recognition of neglected tropical diseases, which also affect millions of people each year, an estimated total of 56.6 million DALYs. Infection of any one of these devastating diseases combined with a neglected tropical disease will have a significant detrimental effect on the health of individuals and in turn exacerbate Africa and Asia's socioeconomic crises, keeping a greater number of individuals below the poverty line.
Intestinal nematodes are helminths and account for the top three most common types of human parasitic infections, these include Ascariasis, Trichuriasis and Hookworm (HAT) accounting for an estimated 2371.5 million infections globally and causing 534,000 annual deaths. Infection results in negative implications for human nutrition, metabolism and growth, as well as putting significant pressures on social factors such as a school attendances and potential earnings.
It has been suggested that helminth infection plays a role in the pathogenesis of the global HIV-1 (human immunodeficiency virus-1) pandemic, with infection further compromising the host immune system, having a significant effect on health and hindering current therapeutic intervention. The result of helminth infection sees a dominant Th2 profile with raised levels of IgE coupled with a reduction in CD4+ T cell count. This could potentially account for the correlation between helminth infected individuals with profoundly affected immune systems and the rapid onset of AIDS documented in subtropical regions of Africa and Asia.
The most concerning problem with the widespread HAT infections is the limited number of therapeutic interventions available. If left untreated, helmithiasis sees recurrent chronic inflammatory disorders including some striking conditions such as lymphatic filariasis causing elephantiasis and blindness. The more common widespread effects of HAT infection results in unrelenting insidious conditions including malnutrition, fatigue, poor cognitive development, as well as putting a substantial burden on the rural communities following livestock infection . Even though HAT infections are the most prevalent amongst poverty stricken regions of the world, only two classes of anthelmintics have been given approval by the world health organisation (WHO). These are benzimidizoles, (e.g. mendazole, ) used to treat soil-transmitted helminth infections and the nicotinic acetylcholine receptor (nAChR) agonists (e.g. pyrantel, ) predominantly used in the deworming of domesticated animals.
With such a restricted array of anthelmintics currently available, the push for the development of drugs within these classes is high, especially as only one drug has been allocated for mass drug administration (MDA), albendazole, Fig. S1d. Widespread use of available drugs has unsurprisingly led to strong resistance and the drive to develop new nAChR agonists and benzimidizoles against human helminth infection is urgent. With the total number of infections rising annually, research into new anthelmintics is a global therapeutic necessity.
Current anthelmintics work by selectively inhibiting nematode motor control at the autonomic ganglia; either through the excitation of nACh receptors causing spastic paralysis or by agonising inhibitory GABA receptors resulting in flaccid paralysis of the nematode musculature. The outcome of helminth paralysis sees the detachment of the worm from the host intestinal epithelium allowing for the natural expulsion of the parasite.
Alternatively, other deworming agents have their mechanism of action intracellularly through direct inhibition of microtubule polymerisation as a result of binding selectively to tubulin in helminth intestinal cells. As a consequence, nematodes are unable to uptake glucose effectively, systematically decreasing their ability to synthesis ATP. A reduction in energy eventually immobilises the helminth and sees its release from the intestinal epithelium.
The nicotinic acetylcholine receptor
The nAChR is pentameric in structure, consisting of an ion pore surrounded by five arranged protein subunits (2a, 1ß, 1?and 1d) . Binding of acetylcholine in all nAChR types has been confined to two adjacent alpha subunits, with the relationship of the surrounding subunits also potentially contributing to protein-receptor interaction .
A universal method of approaching questions in neurobiology and the associated activity of pharmacological agents is to use the model nematode Caenorhabditis elegans. (C. elegans). The reason for using the C. elegans model is primarily due to its physiological similarity to other parasites within the phyla. Its extensively researched genome and associated molecular pathways are well documented, providing a sound biochemical foundation for studying unknown pharmacological compounds . nAChRs are found in abundance in the neuromusculature of C. elegans, the nature of these nicotinic receptors present in the muscle are in fact unique to nematodes, this was demonstrated following the notable experiments of Tornoe et.al (1996). These experiments consisted of the expression of mouse muscle nAChR in Xenopus oocytes, showing that bipinnatin B (an analogue of lophotoxin and irreversible inhibitor of nAChRs) was able to block mouse nAChR's, yet bore no response to the nAChRs of C.elegans. This may be due to the conserved tyrosine 190 residue present in a subunit of vertebrate nAChRs causing lophotoxin sensitivity and selectivity . Even though close homology exists between the vertebrate and invertebrate nAChRs, they do vary in their response to inhibitors, which implies a degree of selectivity to specific compounds.
Characterisation of the C.elegans nervous system has allowed for the elucidation of the particular wiring associated with nematode locomotion . The nervous system is constructed from 302 neurones and forms approximately 7000 synapses, 2000 of these form neuromuscular junctions, the basic architecture of the locomotory circuit . Sinusoidal movement is common to all nematodes, with this form of movement created through the contraction and relaxation of opposing musculature within the body of the nematode, this is maintained through a connected excitatory and inhibitory circuit in the contralateral muscle tissue .
A simple wiring diagram of the nematode neuromuscular system, blue arrows represent an inhibitory stimulus initiating from both the ventral and dorsal nerves. Red arrows signify cholinergic motor neurones, controlling muscle wall contraction. Muscular relaxation is mediated by GABA innervation from the dorsal D-type neurons, which occurs simultaneously with the release of ACh, excitatory neurotransmitter acting on the opposing muscle. The two nematodes models, C.elegans and A.Suum diverged over 500 million years ago, yet the specific wiring of their nervous systems are highly conserved . This diagram therefore represents the neuromuscular circuitry of both nematodes and provides us with a good physiological foundation for this pharmacological investigation. VD- Ventral D-type neuron, DD-Dorsal D-type neuron. (image adapted from: Jorgensen, E.M. GABA (August 31, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.14.1)
Excitation is mediated by acetylcholine (ACh) and its action at the nAChRs sees contraction whilst the inhibitory receptors respond to ?- aminobutyric acid (GABA). GABA receptors are expressed on 26 of the 302 neurones within the nematode, with GABA being released along the ventral nerve from D-type neurones, necessary for relaxation and reposture in order to change direction of locomotion . The simultaneous inhibitory and excitatory neuropeptide vesicle exocytosis at the NMJ and forms the 'command circuit' and is the driving force behind locomotion. The net result of command circuitry activation sees innervation of the contralateral muscle along one side of the worm, and relaxation of the musculature on the opposing side. This propagates along the body, from the tail to the head and generates a series of identical body bends and sequential movement through the immediate environment.
The classical nicotinic agonist anthelmintics, pyrantel and levamisole are collectively known as L-type nAChR agonists which selectively gate nACh cation channels causing hypercontractive paralysis followed by relaxation and subsequent lethality in wildtype (wt) nematodes . Resistance to these anthelmintic agents has led to genetic screening and electrophysiological experiments on C. elegans, which has allowed for the identification of a series of genes encoding for levamisole sensitive nAChR subunits: genes lev1 and unc-29 encode non a nAChR subunits, whilst unc-38 and unc-63 encode a corresponding a subunit, critical for levamisole and ACh binding . A detailed examination of the nematode nAChR demonstrates conserved sequences consisting of 4 transmembrane domains and two flanking cysteine residues present on the alpha subunit. This configuration is unique to nematodes, with no vertebrate counterpart .
The unc gene phenotypes demonstrate the highest levels of resistance toward levamisole . With the use of [3H]meta-aminolevamisole, the specific binding of nAChR agonists to the resistant receptor subunits have previously demonstrated strong binding impairment, resulting in only moderately poor motor coordination of resistant C.elegans strains. Incurred resistance will see an increase in both helminth infection and associated fatalities and will have a detrimental effect on individuals suffering from other regional diseases.
Aims of the investigation
In the following study, we aim to investigate a series of new anthelmintics agents whose mode of action is at the nAChR. This neuropharmacological investigation has been designed to confirm the anthelmintic properties of each compound and help elucidate their mechanism of action, testing the hypothesis that each of the unknown compounds are acting as nACh agonists at the dorsal and ventral neurones. In studying the C.elegans model, we will be able to identify and observe the actions of these novel drugs on existing nAChR subunit targets. C.elegans specimens will be put through locomotion assays and recovery assays in order to determine their general function on the nematode neuromuscular circuitry. These agents could have the potential to break the cycle of anthelmintic resistance currently being seen in populations of high anthelmintic administration. Ultimately the discovery of a potent, sustainable and resistant free nicotinic agonist may help alleviate the burden of helminth infections and associated diseases including 'The Big Three', AIDS, Tuberculosis and Malaria, all of which are rife in less economically developed populations.
Materials and Methods
Elegans strain culturing
C.elegans were raised at 26°C under standard laboratory conditions, on NGM (nematode growth medium) plates seeded with E.Coli (OP50) as their only food source. Composition of NGM (500ml): 1.5g NaCl, 10g Agar, 1.25g Peptone and 487.5ml distilled water. We used the N2 wild-type (Bristol variety) animals. All handling was subject to standard procedures .
The three compounds analysed have the following experimental names: MB408, MB442, SH66 (unknown compounds). They were each prepared from a powdered form and dissolved in clean distilled water in order to generate a 1M stock concentration. Serial dilutions of each of the stock solutions were performed in order to obtain an acceptable range of drug concentrations . Each of the unknown compounds was provided to Prof. Lindy Holden-Dye for laboratory analysis, for the purpose of this investigation.
The control anthelmintic levamisole hydrochloride (levamisole cat. No. L9756) was provided by Sigma-Aldrich and was prepared as a 1M stock concentration. Levamisole is soluble in distilled water at 50mg/ml, producing a clear, colourless solution. Levamisole was kept refrigerated between 2- 10°C when not in use experimentally. The chemical structure of levamisole can be seen in figure S1. C.
Primary locomotion assays were conducted for the control anthelmintic levamisole. In order to identify an IC50 value for the drug, a stock solution of levamisole was prepared and 200µl of a range of levamisole concentrations was applied to Escherichia coli (E.coli OP50) seeded plates, the E.coli represents a standard food source for nematodes.
Precise concentrations were made up and added to the seeded plates as follows; 0.1M,0.2M,0.5M,0.8M,1M. 200µl of distilled H2O was used as a standardised control, applied to the seeded plates in an identical fashion . Each plate was incubated for exactly 30 minutes before N2 C.elegans strains were added to the levamisole laced and control plates.
Approximate L4 stage adult hermaphrodites were selected and applied to seeded plates, being picked out individually using a wire 'eyelash' from the animal culture plate. Incubation of the nematodes was carried out at a room temperature of 26°C for exactly 60 minutes, allowing the animals enough time to consume the applied compound. Temperature and pH levels were maintained throughout the entire investigation.
Animals were individually transferred to a drug free seeded plate and left to acclimatise to the new environment for 60 seconds. Nematode locomotion was then quantified through body bend counts, counting was performed for 60 seconds. A body bend was standardised across all assays, being a single, mid-body movement resulting in the tail moving maximally from the left to the right (and vice versa) which enables forward or backward locomotion of the animal, as documented by Miller et.al . Body bend counts following intoxication of various drug concentrations was therefore indicative of dose dependent inhibition of the nematode musculature.
Each drug concentration and control was repeated three times with a representative selection of eight L4 nematodes taken for 60 second body bend counts. Nematode movements were visualised with a dissecting light microscope.
This procedure was then repeated for each of the unknown compounds: MB408, MB442 and SH66. For each of these assayed compounds, both 0.4M concentration of levamisole and H2O was tested alongside representing the known anthelmintic control and standard control. 0.4M was chosen, being the identified IC50, half maximal inhibitory concentrationof the compound .
From the locomotion assay described above, the calculated IC50 values of each compound was used as a representative concentration in drug recovery assessment. Selection of animals, incubation periods of the compounds and C.elegans intoxication followed an identical protocol to the one mentioned above.
Three representative nematode samples were used for each compound. With the calculated concentrations of the individual compound IC50 values being: levamisole- 0.4M; MB408- 0.5M and MB442- 1.0M. H2O was used as the control for these assays acting as the primary marker for inhibition comparison.
Each of the nematodes transferred to a drug - free medium following incubation and body bends were counted for 60 seconds. After progressive 30 minute intervals, the body bends of the animals were recorded again, a total drug recovery period of 90 minutes following nematode extraction was therefore documented. From this, the rate of recovery was then calculated for each of the unknown compounds and for the levamisole control.
All tables, dose response and drug recovery graphs were designed and constructed using Microsoft Excel 2007. From these tables and graphs, IC50 values, means and standard deviations of the raw data could be calculated, this was achieved through Microsoft Excel 2007 and the data analysis add on pack 'XLSTAT'.
Statistical analysis of each table and graph was performed, IC50 values were tabulated and standard errors for each data point plotted. A pairwise comparison between groups via a one way analysis of variance (ANOVA) and Tukeys HSD test finalised data analysis, allowing for comparisons between all data obtained. Significance between unknown compound data sets and levamisole control data was added to the locomotion assay graphs. The significance level was set atP<0.05.
Locomotion assay results
Levamisole is a potent anthelmintic with all unknown compounds investigated demonstrating a degree of anthelmintic activity
As there is no previous literature concerning the efficacy and mode of action of these unknown compounds, we incorporated each of the drugs into a series of C.elegans models. In order to analyse their mode of action, we required a control anthelmintic for comparative study. Application of levamisole to the N2 wild-type nematodes demonstrated substantial paralysis and subsequent locomotory inhibition of the animals . Under the conditions of levamisole, observed changes in the behaviour and movement of the nematodes became apparent. Even at low concentrations of 0.1M, the nematode worms coiled up tightly in response the levamisole medium. This coiling is indicative of spastic paralysis, with excitation of cholinergic neurones resulting in muscle wall contraction down one side of the nematode. Paralysis saw a loss of movement, with nematodes not responding to being placed into an E.coli free medium at high levamisole concentrations. At 1M levamisole, nematodes were almost entirely immobilised with all L1 - L3 stage animals tightly coiled, there was also little to no response seen to external stimuli such as touch or vigorous shaking of the agar plate. This response to high concentrations of levamisole, meant we had to establish, in some cases, whether or not the nematode was immobilised or actually dead. If there was no response to direct stimulation, observing the continual pharyngeal pumping confirmed nematode survival.
The dose response curve demonstrates the potency of levamisole acting on the nematodes. Drug action was measured by counting sinusoidal body movements (body bends) after 60 minutes of intoxication. Inhibition was measured by calculating the percentage difference in body bends from the control, H2O. Therefore the graph demonstrates an increase in percentage inhibition as greater concentrations of levamisole is added to the nematodes, culminating in an almost 100% inhibition in movement at a 1M levamisole concentration. IC50 values for each of the drugs investigated can be seen in Table 3. Each data point represents 8 nematodes (n=3; 3 repeats per concentration). Error bars represent the standard error of the mean for three experiments.
Levamisole produced an IC50 value of 0.375M, the concentration of drug required to produce half maximal response in the C.elegans model. Inhibition of locomotion reached 98% at a concentration of 1M yet also produced a 19% inhibition at the lowest recorded concentration, 0.1M. Levamisole represented an anthelmintic drug which was relatively potent and for the purposes of this investigation gave a good comparative IC50 value by which to analyse further results.
The first unknown compound, MB408 was assayed in the same way as levamisole, and was conducted alongside both an H2O control and an approximate levamisole IC50 control (calculated from the control assay). MB408 demonstrated a similar response to levamisole albeit having a less potent effect on nematode locomotion. Figure 3. clearly indicates a strong dose response, the strongest level of inhibition is witnessed after 1M of MB408 is applied to the nematodes, 60% inhibition of sinusoidal movement in response to being moved to a different medium. Unlike levamisole , C.elegans response to direct stimulation demonstrated an escape response, with increased sinusoidal movements away from external stimuli. Coiling of the nematodes also occurred less frequently and only in response to higher concentrations, this is still indicative of spastic paralysis.
A typical dose response curve is seen for the unknown compound MB408, the graph demonstrates a very similar response as seen with levamisole action on nematodes. Drug action at varying concentrations was measured as the percentage difference in sinusoidal movements in comparison to the H2O control following 60 minutes of intoxication. Each data point on the graph represents 8 nematode samples (n=3; three repeats per concentration). Error bars have been calculated from the standard error of the mean for each data point. Data comparison was undertaken at each concentration using the ANOVA/Tukey HSD test: *P>0.05 (n/s), **P<0.01
Due to the compound's comparable response to levamisole, this may be indicative of a similar mechanism of action at the nAChR, which will be discussed later. The calculated IC50 value for MB408 was 0.425M, higher than that of levamisole.
Locomotion assays for MB442 again bore positive results with a high level of inhibition seen at the lower concentrations, 0.2M-0.5M with locomotory inhibition decreasing in comparison from 0.5M to 1M assays . This is a very typical dose response pattern, however the potency of the drug was approximately half that of the levamisole control, 0.87M .
A 40% increase in locomotory inhibition is seen when 0.5M of MB442 is administered to the nematodes. Sequential inhibition decreases slightly terminating at 51.7% inhibition when 1M of MB442 is added to the E.coli medium. Drug action has been calculated, from the percentage level of inhibition in comparison to that of the H2O control. The IC50 value of this compound is approximately 50% that of levamisole, 0.87M. Each data point represents 8 nematodes (n=3; three experiments per concentration tested). Vertical error bars have been constructed, calculated from the standard error of the mean for each set of data. Data comparison between the original levamisole control and MB442 was undertaken at each concentration using the ANOVA/Tukey test: *P>0.05 (n/s), **P<0.01.
The final intoxication and dose response assay was conducted with the unknown nicotinic compound 'SH66'. As the chemical formula of the compound is unknown, its structure may be slightly different in comparison the MB class drugs. Results of the assay demonstrated a progressive inhibition, more potent than MB442, as its IC50 value was calculated at 0.71M . From Fig.5 we can see that its dose response is strong with a steady increase in inhibition up to 1M concentration. Physiological responses of nematodes to this compound demonstrated centralised body musculature paralysis, with animals still able to move head and tail regions, demonstrating reduced defects in motility at the extremities, this could be due to fewer nACh receptors present in both the head and tail regions. Raw data can be seen in Appendix D.
Graph depicting SH66 in comparison with levamisole control from fig.2. SH66 demonstrated a strong dose response in comparison to the levamisole control, reaching a 72% level of inhibition from the H2O control sample. An IC50 value of 0.71M was calculated, see table 3. Each data point represents 8 nematodes (n=3; 3 repeats per concentration). Error bars represent the standard error of the mean for the three independent experiments. Data comparison between the levamisole control and SH66 was undertaken at each concentration using the ANOVA/Tukey test: *P>0.05 (n/s), **P<0.01.
The figure provides the reader with a comparison of all the compounds which underwent the locomotion and dose response assays. We can immediately see the strong inhibitory action of levamisole, with the unknown compounds reaching approximately half of its IC50 value. This now gives us a comparative representation of drug potency. IC5Os have been added as a drop down vertical line in red. Each data point represents 8 C.elegans samples (n=3; three experiments per concentrations were conducted). Error bars have been calculated from the standard error of the mean.
Recovery assay results
All compounds investigated demonstrated a strong level of recovery, indicative of a reversible mechanism of action. As the unknown compounds all displayed a degree of inhibitory function on nematode locomotion, we decided that to deduce a mechanism of action we would have to see whether the pharmacological agents were acting as either reversible or irreversible agents at the nACh receptors of the body muscle wall. We designed a series of recovery assays, which were intended to show whether or not the drugs lost their inhibitory function when the intoxicated nematodes were left in a sterile medium over a period of 90 minutes.
The results for these assays can be seen in Appendix E; the rate of recovery for each of the compounds was seen to be very similar , and calculated by taking the percentage inhibition from the H2O control, at the compounds identified IC50 level. This was then recorded over a period of 90 minutes, the rate is therefore the % change in inhibition per hour, figure 7 demonstrates this graphically. We saw slow recovery for each of the unknown compounds, this was comparable to that of the levamisole recovery data which could therefore be indicative of a similar mechanism of action agonising the nACh receptors present on the nematode musculature.
Table demonstrating the recovery of the nematodes over a period of 90 minutes, following intoxication with the IC50 concentrations for each of the unknown compounds and for the levamisole control. The IC50 value used for levamisole demonstrated a higher level of inhibition that initially observed in the locomotion assays, however, nematode recovery and clearly be seen. Over the period of 90 minutes a steady recovery rate is seen with nematodes slowly increasing locomotion on sterile plates, moving toward a food stimulus. Each data point represents 3 nematodes (n=3, 3 experiments were conducted in total for each compound at IC50 value). Error bars represent the standard deviation from the mean.
Results demonstrating the change in inhibition over time, this is expressed as a rate, animals were first intoxicated with a concentration of drug able to give a good response, this was ascertained from the locomotion assays, the approximate IC50 values. Animals were transferred to an inert medium and rate of recovery calculated. The rate is calculated from the change in inhibition per hour, inhibition is the percentage difference from the H2O control, taken at different time points
The rates of drug recovery, and confirms the reversibility of each of the compounds levamisole has the highest recovery rate, of 68.09, taking a shorter period of time for the drug to dissociate from the receptor. MB442 demonstrated the shortest time for nematode recover, 53.65. These figures are indicative of the affinity for each compound with the nicotinic receptor. We can therefore state that levamisole may be binding with a slightly lower affinity to the L type nAChR, with the other compounds associating with somewhat greater affinity, this could potentially indicate these drugs are occupying alternative binding sites.
From the locomotion assays, we were able to collect the IC50 values for each of the compounds investigated. The IC50 values therefore give us a basis of analysis, and allows for an instant identification of the compounds effectiveness at inducing paralysis within the C.elegans model.
From table 3, levamisole is identified as the most potent agent, followed closely by MB408, which has a similar potency. From figure 3, we see that MB408 was able to produce over 60% inhibition of locomotion at 1M concentration. MB442 was identified as the weakest compound investigated, requiring 0.87M concentration in order to induce a half maximal inhibitory response.
Further analysis of the locomotion assays was then performed, we undertook a one-way comparison ANOVA test/Tukey HSD , for each of the comparable concentrations in order to identify whether there was significant difference between these data. Table 4a-c demonstrates the significance between all three unknown compounds and the levamisole control.
Above are the P values for the ANOVA/Tukeys HSD statistical analysis, we performed a one-way comparison for four independent samples. In experimental terms, we were testing the significance between each of the compounds effects on nematodes at varying concentrations. Each table A-C represents a single comparative concentration, with each compounds effect on the sinusoidal body bend count compared to the other compounds (one way comparison) at the same drug concentration. HSD - the absolute difference between any two sample means required for significance at the designated level. HSD.05 for the .05 level and HSD.01 for the .01 level; P>.05 indicates no significant difference detected between the variances of the samples; n/s - not significant. Null hypothesis: there is no significant difference between the inhibitory action of the drugs at identical concentrations
The above tables report an increasing significant difference between the compounds with the levamisole control as the concentrations of the drugs increase. In the tables demonstrating a significant difference between compounds, we reject the null hypothesis, and therefore there is a statistical difference in the amount of sinusoidal body bends counted between the two compounds of the same concentration.
We have demonstrated that the difference in dose responses at these three concentrations is associated with the higher efficacy of levamisole in comparison to the unknown compounds . At a 1M concentration, the comparative P values for MB408, SH66 and MB442 with levamisole are all <0.01 , indicative of a strong significant difference in data with levamisole depicting a strong, well behaved dose response and the unknown compounds reaching approximately half of its 1M inhibitory action. Comparing the three unknown compounds to one another, we see at high concentrations a significant difference between the data recorded for MB442 and SH66, with MB443 producing the lowest rate of 1M inhibition, 51.74% . However, as no significant difference was identified between several of the compound concentration analyses and the recovery rates of the unknown compounds were very similar , we can note the relevance of this in terms of the compound mechanism of action; this may be a strong indicator of both analogous action on the nAChR and chemical structure.
All of the unknown compounds investigated demonstrated an unquestionable degree of anthelmintic activity upon the model nematode C.elegans , albeit with a slightly weaker efficacy in comparison to the recognized anthelmintic levamisole. Currently there is no published data on these novel compounds, and this investigation represents a step toward the prospective development of new nAChR agonists, which will help to alleviate the current resistance to nicotinic anthelmintics seen in poverty stricken regions of Africa and Asia .
The C.elegans model
The C.elegans model has provided us with a solid experimental foundation for the analysis of these unknown compounds. From the locomotory assays, we have discovered that MB408, MB442 and SH66 were all able to inhibit locomotion by at least 51% when compared to the H2O control (MB442, appendix C, fig 4. In comparison to levamisole's action upon the nematode model, seen in fig.2, we see a significant level of locomotory inhibition occurring following intoxication. We can therefore put forward the hypothesis that the each of the unknown compounds exhibit a comparable mechanism of action to levamisole, acting upon the nACh receptor, resulting in hypercontractive paralysis (L-type agonism) . This is founded upon the evidence collected; we demonstrated their similar locomotory inhibition, characteristic of levamisole and the reversible nature of the drugs acting upon the nematode receptors .
The unknown drug, MB408, produced the lowest IC50 value and therefore displayed the greatest level of efficacy out of the three compounds tested . Drug-receptor dissociation, represented as the recovery rate showed that MB408 was also the quickest of the unknown compounds. The pharmacological characteristic of MB408 was therefore most like levamisole, displaying a similar affinity for the nAChR and a close efficacy in its inhibition of locomotion. It is therefore likely that this agonist is behaving in a similar fashion to levamisole at the nACh receptors present in the autonomic ganglia, exciting both dorsal and ventral D-type neurons inducing gross contraction of the nematode muscle wall.
SH66 and MB442 also depicted a typical dose response in C.elegans . Although the efficacy was not as high as seen for MB408, both SH66 and MB442 were able to generate an inhibitory percentage difference from control value of 51.7% and 63.4% respectively . Recovery rates for these drugs were also very similar, with both compounds acting as reversible agonists of the nAChR. From the recovery assays, we can deduce that these compounds had a higher binding affinity for the receptor in comparison to levamisole and MB408, however their efficacy was lower, requiring higher concentrations in order to induce a similar contractile response.
Levamisole has its mechanism of action on the alpha subunits of the nACh receptor, acting as a cholinergic agonist. Genes, unc-38 , unc-29, unc-63 and lev-1 encode and contribute to the 'levamisole receptor' which sees levamisole binding specifically, acting as an agonist. The type of muscular contraction witnessed in the C.elegans models following intoxication with 1M levamisole, was identical to past experimentation literature by J.Lewis et.al . Lewis et.al documented signs of muscle contraction which was apparent within seconds of levamisole application, with observed hypercontractive paralysis. They also described how a contracting adult nematode removed from the levamisole medium was able to recover; an attribute we were able to quantify in the recovery assays . We can therefore conclude that complete removal of the nematode following levamisole exposure can result in full recovery.
We have demonstrated the general inhibitory effect on locomotion following application of these drugs to wild type model nematodes in vitro. However the individual results demonstrated a degree of variation in their effects, with some nematode samples being heavily affected at lower concentrations of all of the investigated compounds . The extent of paralysis differs between different nematodes and this may provide a justification as to why there was a degree of variation of inhibition between the MB408, MB442 and SH66 in the locomotion assays. This has been seen for the drug mebendazole and ivermectin, Bernt et.al were able to conclude that a myriad of external conditions, uptake mechanisms and time of drug exposure all contributed to the intoxication and subsequent efficacy of a drug acting within individual nematodes.
Absorption of a compound is a crucial pharmacokinetic attribute and can dictate the efficacy of a drug both in vivo and in vitro. Levamisole works rapidly as a cholinergic partial agonist at nAChR at the autonomic ganglia as it is readily absorbed across the gastro-intestinal tract. Therefore the cuticle of the nematode represents an immediate physical barrier to all compounds. Depending on the size and polarity of the drug administered, absorption rates will vary greatly.
Drug application to different parasite larval stages also causes substantial variation in the efficacy of a drug, the developmental cycle of the C.elegans sees a progression through four stages, with the life cycle taking place in approximately 3 days at 20°C . Age related changes to the C.elegans may account for variable expression of molecular pathways within cells, this was observed in an investigation of the novel anthelmintic emodepside acting on C.elegans models . It was discovered that emodepside was able to inhibit locomotion less efficiently in the L4 stage adults, variance of drug efficacy acting on different developmental stages may therefore be observed due to cuticle thickness causing changes in permeability, coupled with variation in expression of molecular pathways during the C.elegans lifecycle. It was challenging to identify precise nematode stages, and therefore a careful estimate was made in order to select only fully developed L4 stage adults. We attempted to maintain C.elegans L4 consistency throughout the investigation, as a result, any anomalies could be due to different larval stages enforcing the age related data variance mentioned above.
The integrity of our results was reflected in the calculated execution of our investigation, however, it was the methods by which the sinusoidal body bends were counted which could have produced the greatest degree of inaccuracy. Many authors have noted the amount of human error affecting the quantification of nematode motility . In retrospect, implementation of an automated, computerised 'thrashing assay' may have been more appropriate for this investigation , due to the vast amount of human counting required. However due to both time constraints and budget allowance, this was not possible. It represents a new method for anthelmintic research, one which could be implemented in future investigations.
Further study into these compounds will give us a greater picture as to their precise mechanism of action at the nAChR. We have noted that from this investigation they are all acting as nicotinic agonists, however in order to confirm this, chemical mutagenesis of different genes and C.elegans knock out populations could act as a screening method to identify specific nAChR binding sites for each of the compounds. This is carried out routinely and represents a very powerful approach to anthelmintic research . We will also require testing of all three compounds in parasitic nematodes, C.elegans represents a prevalent model for anthelmintic testing, however if these drugs are to function effectively in vivo, they will need to demonstrate action upon common parasitic model organisms such as A. Suum. The nACh receptors of A. Suum are phylogenetically distinct from that of C.elegans as they do not contain share the same gene products, missing lev-1 and lev-8 yet are still capable of expressing levamisole sensitivity. Their expression within the musculature is also different, they are not solely clustered at the neuromuscular junction, but are present all over the membrane, which may potentially induce a stronger inhibitory effect for A.Suum models. Resistance to anthelmintics has led to detailed research into the stoichiometric arrangements of nACh receptor subunits . Williamson et.al were able to identify and recreate many of the properties of the N- and L-type receptors from the parasitic nematode A.Suum; including their sensitivity to levamisole by expressing just two subunits at different ratios. This demonstrates that altering the expression of level of a single receptor subunit can dramatically alter the efficacy of anthelmintics. This discovery could provide an explanation as to why the unknown compounds tested produced a lower efficacy in comparison to levamisole within the C.elegans models in our investigation, depending on their precise binding site located on the specific nAChR subunits. The idea of receptor subtypes and their dissected stoichoimetries, also enhances rational drug design through the development of parasite specific screening; a promising concept allowing for the identification of precise receptor subtypes for individual parasites. In order to evade current resistance to nicotinic agonists, a combination of drugs may prove beneficial, with two nicotinic agonists binding to two distinct binding sites within the nAChR subunits.
In conclusion we have identified three novel compounds with distinct anthelmintic properties, acting as reversible agonists at the nAChR of the model nematode, C.elegans, demonstrating strong dose responses. We have provided characterisation of the net affects seen with levamisole intoxication and through comparative locomotory assays, identified candidate MB408 as the most potent novel agent from the three investigated and has the potential to be implemented as an anthelmintic agent. In addition, through a series of recovery assays we can put forward the theory that each of the compounds were acting as reversible agonists at the L-type nAChR, binding with slightly higher affinity and lower efficacy than levamisole. It must be noted that if these compounds are acting in the same way to levamisole then they may not be viable anthelmintics where resistance to L-type agonists is prevalent. From this investigation we have provided a foundation by which to study and further elucidate the specific mechanism of action for the anthelmintic candidates MB408, MB442 and SH66.
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