with a functional competition with (a putatively allosteric) Mg2+ ion. wALADin1 (as well as 3) involves both inhibitory components, while the tricyclic quinoline derivative wALADin2 only acts exclusively by functional competition with Mg2+
and shows improved specificity. The potency of wALADin2 is independent of the substrate concentration, whereas the potency of wALADin1 is high at low 5-ALA concentrations, but decreases upon accumulation of 5-ALA or at high initial 5-ALA concentrations. Thus, biological activity of these two inhibitors in the physiological environment is expected to be different and both compounds may complement one another. Further structural components have been identified as determinants of inhibitory potency and specificity. Finally wALADin-like inhibitors based on other chemotypes have been discovered.
These novel inhibitors mark a significant advancement in the search for species-selective ALAD inhibitors of potential therapeutic utility. The profound characte-rization of structural and functional aspects of benzimimdazoles and other chemo-types along with their inhibitory mechanisms provides the foundation for the design of improved inhibitors with desired nanomolar inhibitory potency and tailored bio-logical activity.
at the end of treatment that were similar those achieved by wALADin1. Impor-tantly L. sigmodontis worms exposed to 3 also featured the ball-like appearance characteristic for wALADin1-treated worms. Viability was significantly reduced by 64% only for the highest concentrations although the trend was sustained also for the lower concentrations. Although the antifilarial effect of 3was quantitatively weaker than that of wALADin1, it displayed a very similar phenotype. In 3, the entire R2 substituent of wALADin1, which comprises about one third of the entire molecule, is missing. Despite this chemical modification, 3has obviously retained the wALAD-inhibitory potency, but it may easily have lost activity on putative se-condary targets with unique strucure-activity profiles of their own. Hence3might display a more ALAD-specific biological profile. It may be expected that it takes a while until the inhibition of ALAD (especially when not complete) translates into heme-deficiency, depending on the turnover rates of preformed heme in the worm, and becomes evident in reduced fitness of the worm. Also accumulation of toxic 5-ALA levels may not occur immediately. Therefore, the antifilarial effect elicited by3may be a more specific result of interference with endobacterial heme biosyn-thesis than the stronger and more rapid effect of wALADin1, that at least at 500 µM likely represents an overlap of activity at ALAD and at a secondary target.
Taken together, the pronounced antifilarial acitivity of wALADin1 and 3, the most potent benzimidazole inhibitors of wALAD, and the Wolbachia-dependent activity of wALADin1 reveal that the specific heme-biosynthesis inhibitors of the wALADin benzimidazole family are a promising novel class of antifilarial agents.
4.5.1 Potential secondary target effects
Potential secondary targets of wALADin1 in Wolbachia or the nematode may be deduced from known targets of chemically related molecules. The most closely related class of bioactive benzimidaziole structures has been reported to target the human kinesin spindle protein (KSP or Hs Eg5) [157, 228]. As inhibition of this protein leads to a misassembly of the mitotic spindle and cell cycle arrest followed by the death of cancer cells, it is an attractive target for the development of anticancer drugs. The recently reported class of hKSP-inhibitors were 2-aminobenzimidazoles with further substituents at the amino group and at benzimidazole atoms N1 and C5, equivalent to the R1 and R3 substituents of wALADin1-benzimidazoles (Fig.
4.2). Although these inhibitors have tolerated a carboxyl group at the R1position (as required by wALADins for inhibitory activity), they had a preference for a primary amide at R1. The R2residue of most KSP inhibitors is a 2-trifluoromethyl benzyl and is thus similar to the 3-trifluoromethyl benzyl of wALADin1. The largest structural difference is in the R2 postion, where KSP inhibitors require an amino-linked aromatic substituent in order to be characterized as active in anin vitroassay system (IC50 values below 20 µM). Nevertheless, at the high concentrations used in the filarial co-culture assay wALADins may have an effect on filarial orthologs of this mitosis-releted protein.
The benzimidazole core of wALADins represents a structural link to classical benzimidazole anthelmintics like albendazole. These anhelmintics target the filarial β-tubulin and cause a destabilization of microtubules [156]. While the lack of a car-boxylic acid function will prevent inhibitory activity of benzimidazole anhelmintics on wALAD (for albendazole this was even experimentally verified, although the data is not shown), nematode microtubules may be a secondary target of wALA-Din inhibitors. Most benzimidazoles like albendazole, flubendazole and mebenda-zole feature a carbamate moiety at the R2 position and differ in their R3 moieties although the active metabolites are often biotransformed molecules [89]. In case of albendazole, both albendazole and its in vivo metabolite albendazole sulfoxide target wormβ-tubulin. Its other major metabolite albendazole sulfone has recently
Figure 4.2: Chemical structures of selected benzimidazole compounds The chemical structures of wALADin1, the KSP inhbitor with highest potency [228] and of selected benzimidazole anthelminthics including, mebendazole, flubendazole, albendazole and its metabolites albendazole sulfoxide and albendazole sulfone are shown.
been shown to have a bacteriostatic effect onWolbachia proliferation inB. malayi and to disrupt binary fission [226]. It may not be ruled out that the Wolbachia-dependent activity of wALADin1 is based, at least in part, on this yet unidentified endobacterial benzimidazole target, although this scenario is highly speculative.
Besides testing of wALADin1 and derivatives in enzymatic assays for secondary target candidates, if available, comparative analysis of the filarial and Wolbachia phenotypes under exposure to wALADin1 and other chemical probes such as al-bendazole sulfone may help unravel secondary targets and effector pathways of wALADin1.
4.5.2 In vivo activity of wALADin1
The proof-of-principle experiments demonstrating the macrofilaricidal effects of wALADin1 and derivatives in the ex vivo co-culture system revealed that the in vitroALAD-inhibitory activity of wALADin1 can be translated into an antifilarial effect in the living worms. However, providing the required high concentrations of
> 100µM in anin vivo infecion model ofL. sigmodontis in BALB/c mice is not a trivial task. Despite continued administration of wALADin1 of up to 4 mg/mouse for up to four weeks, the use of different formulations and administration routes (intraperitoneal injections and intrathoracic injections to the site of infection) no antifilarial effect of wALADin1 treatment was observed. Treatmtent was tolerated well without evident signs of toxicity. Although it may not be ruled out that the filariae have access to host-derived heme sources in their physiologic environment [11] and thus be naturally resistant to inhibition of endobacterial heme biosynthe-sis, it is likely that the concentration of wALADin1 in the biophase is simply too low to reach a biological effect. Possible pharmacokinetic reasons are manifold, but determination of the pharmakokinetic profile and ADME parameters (adsorption, distribution, metabolization and excretion) of wALADin1 in the mouse were beyond the scope of the current study and represent a project of its own. In the mouse, wALADin1 may be rapidly metabolized and excreted or sequestered by promiscuous non-specifically binding proteins of the host such as serum albumin. For the ben-zimidazole anthelminthic albendazole the major pharmakokinetic handicap is poor absorption from the intestinal tract after oral administration [89]. This obstacle was
circumvented in the present study as wALADin1 was administered intraperitoneally or intrathoracically. However, in the human intestinal mucosa and the liver, al-bendazole is rapidly metabolized to alal-bendazole sulfoxide, alal-bendazole sulfone and several further metabolites [182]. Rapid hepatic and mucosal biotransformation has also been reported for other benzimidazole drugs, i.e. flubendazole in sheep [169]. Similar though non-analogous biotransformation of wALADin1 may occur in the mouse liver and yield non-inhibitory metabolites devoid of antifilarial activity.
Pharmacokinetic shortcomings that prevent biologically active concentrations of a compound are, of course, structure bound. ADME properties are determined by defined chemical features and structural elements of a xenobiotic that e.g. represent contact points for biotransformation enzymes. In order to develop drug-like Wol-bachiaALAD inhibitors with a potent antifilarial activityin vivotwo considerations are imperative. First, inhibitory potency must be increased such that lower doses are sufficient to yield bioactive concentrationsin vivo. Second, the pharmacokinetic profile must be enhanced, which may be achieved by the identification of inhibitors based on other chemical scaffolds that retain the biological activity. A first step towards the identification of such improved inhibitors has been undertaken and has led to the identification of the non-benzimidazole based inhibitor wALADin2, which has a slightly increased inhibitory potency and an improved specificity profile in the in vitro assay.