Developing novel anthelmintics from plant cysteine proteinases

Abstract

Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock.

Figure 1

The effect of varying concentrations of enzyme activity in papaya latex (A) and in papain (B) on the motility of Heligmosomoides bakeri in vitro. Standard errors are given for the control (0 μM) groups and in selected treated groups only, to retain clarity. For the full statistical analysis of these and related data, see Stepek et al. [107]. Reprinted with permission.

Figure 2

A factorial experiment to confirm that the effect on H. bakeri is mediated by cysteine proteinase activity. The worms were incubated in the presence/absence of papaya latex, presence/absence of cysteine, and presence/absence of the specific CP inhibitor E-64. The only treatment to show accelerated loss of motility was when worms were incubated with papaya latex, in the presence of cysteine but absence of E-64. This treatment is highlighted in bold. For the full statistical analysis of these and related data, see Stepek et al. [75]. Reprinted with permission.

Figure 3

Scanning electron micrographs of Heligmosomoides bakeri adult worms exposed to papaya latex and ficin in vitro. Clear evidence of damage to the cuticle can be seen from 30 min in 200 μM crude papaya latex and ficin, 30 μM. Note the transverse wrinkling leading to signs of shedding of the cuticle after 90–120 min. In contrast, worms incubated in Hanks's saline showed no sign of cuticular damage, even after 120 min incubation. Scale bar = 10 μm. For SEM preparation adult specimens of Heligmosomoides bakeri were fixed in glutaraldehyde, postfixed in osmium tetroxide, critical point dried and gold coated before examining in a Jeol JSM 840 scanning electron microscope. For further details see Stepek et al [107]. Reprinted with permission.

Figure 4

Transmission electron micrographs of the cuticle of adult Heligmosomoides bakeri exposed to crude papaya latex (CPL) (200 μM). After 10 minutes exposure the cuticle starts to exhibit swelling and wrinkling of the surface with the appearance of electron dense material and collapse of the cretes. Following 30 minutes of exposure to papaya latex much of the structure of the cuticle has been lost and detachment of the cuticle from the underlying hypodermis is apparent in places. For TEM preparation adult specimens of Heligmosomoides bakeri were fixed in glutaraladehyde, postfixed in osmium tetroxide, dehydrated in ethanol embedded in epoxy resin and sectioned. The sections were stained in lead citrate and uranyl acetate and examined in a Jeol 1010 transmission electron microscope.

Figure 5

Scanning electron microscopy of the surface of Rodentolepis microstoma incubated with crude papaya latex (CPL) in vitro. The micrographs were taken at equivalent points along the worm surface, near the mid-point. The digestion of the tegumental surface was evident by 30 mins when the worms were incubated with 25 μM papaya latex. Lesions attributable to the activity of the CPs in papaya latex are highlighted by the arrows and the damage increased substantially between 30 and 60 mins after exposure. The tegument remained intact, with no visible damage, even after 2 hours, on incubation with Hanks' saline + 16 mM cysteine (not shown). Scale bar = 100 μm. (see reference [60]). Reprinted with permission.

Figure 6

The effect of treatment with papaya latex on faecal egg counts in mice infected with 200 L3 of H. bakeri (A) or 100 eggs of T. muris (B). In some cases, the error bars do not show because they are very tight and obscured by the mean data point. In (A) treatment with papaya latex (133 nmol active cysteine proteinase/mouse/day) or water was initiated on day 18 and continued daily until day 24 [75]. In (B) the first dose of papaya latex (337 nmol active cysteine proteinase/mouse/day) or water was given on day 48 and continued daily until day 54 [76]. In both figures, the days of treatment are shown by vertical arrows on the abscissa. Reprinted with permission.

Figure 7

Dose response with papaya latex in mice infected with H. bakeri. Mice were infected with 200 H. bakeri and given varying doses of papaya latex corresponding to the nmol amounts of active enzyme illustrated in the figure. Worms were counted 25 days post infection. The log₁₀ ED₅₀ was calculated as 1.8 (95% CL = 1.72–1.89), corresponding to 67nmoles of active cysteine proteinase. The log₁₀ ED₉₅ was 2.4 (95% CL = 2.14–2.67), corresponding to 133nmoles of active enzyme. For full details and comprehensive analysis see, Stepek et al. [75]. Reprinted with permission.

Figure 8

Evidence that the in vivo effect of papaya latex on H. bakeri is mediated through cysteine proteinases. Groups of mice infected with 200 L3 of H. bakeri were treated with water, E-64 alone, papaya latex (135 nmoles active enzyme) or papaya latex pre-incubated with E-64 (0.64 nM) for 15 minutes prior to oral delivery to mice. Only untreated papaya latex caused a significant reduction in worm burdens. For full analysis and further details, see Stepek et al [75]. Reprinted with permission.