In silico and In vitro Investigations of Antihelmintic Activities of some Selected Approved Drugs

The search for alternative methods to mitigate the drawbacks associated with wet laboratory drug discovery has been a major challenge in drug design and has limited the options available in the fight against most Neglected Tropical Diseases (NTDs) such as helminthiasis. We investigated the binding affinities of some approved drugs to Ascaris suum mitochondrial rhodoquinol fumarate reductase enzyme (MRFR),an essential enzyme for ascaris survival, and the possibility of repurposing these drugs as antihelmintic agents using in silico molecular docking and in vitro paralysis and mortality times of fifteen selected front runners.Two hundred approved drugs were selected from ZINC® database based on bioactivity scores while MRFR(PDB code, 3vra) was obtained from the Protein Data Bank (PDB). Both were prepared using AutoDock tools v.1.5.6 and Chimerav.1.9.The docking protocol was validated by computationally reproducing the binding of atpenin to MRFR. The selected approved drugs and the receptor were docked using AutoDockVina v. 4.0. The docking results were analyzed using PyMoL v. 1.4.1.The paralysis and mortality times of the identified frontrunners against Pheretima posthuma were determined in vitro and synergistic testings were done by the checkerboard method. Fifteen drugs had binding free energies between −7.825 to 11.025 kcal/mol while four of these drugs (mefloquine, doxycycline, mepacrine and proguanil) emerged as major frontrunners by both in silico and in vitro assessments. The paralysis and mortality times of the four drugs were between 0.33-0.50 hr as against 1.80-2.36 hr for albendazole. They were therefore predicted to have ability to affect MRFR in the same manner as atpenin hence, suggestive of potential antihelmintic activity. In vivo investigation of these frontrunner drugs is strongly recommended. Author Summary For decades, intestinal worm infections have been a major public health problem particularly in the tropical regions of the world with infection figures put at 5.3b [16]. This disease is often associated with poor hygiene and sanitation hence is predominant in the resource-poor nations with physical infrastructural deficit [8]. Children between the ages of 2-10 years are the most vulnerable by reason of their hand habits with morbidity leading to compromise of physical and cognitive development while several sufferers have had to live disability-adjusted life years as a consequence [12]. Efforts at implementing improved hygiene and mass deworming campaigns have not delivered the intended outcomes with many reports coming from clinicians regarding gradual development of resistance to the standard treatments [9]. Given the huge cost and time invested in traditional drug discovery and the rarity of this disease in regions of the world with the resources for research, no new drugs have been developed in the past two decades [18]. The advent of computational techniques in drug discovery has come to mitigate these drawbacks. This study exploited this technique to full effect and through laboratory assays and clinical investigation of selected approved drugs, mepacrine and doxycycline were identified as potential antihelmintic agents fit for combination therapy.


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The fortuitous nature, enormous cost and huge time invested in traditional drug 83 discovery and development is a major factor that has fueled the inertia in most 84 pharmaceutical companies to engage in research into new chemical entities. This is 85 a consequence of the unpredictability of these researches hence results often do not 86 justify the effort. 87 This scenario has mostly affected the neglected tropical and orphan disease 88 domains. It is so due to the limited number of sufferers and the demography that 89 stratifies them to the resource-poor nations [13]. 90 As a fall out of this, pharmaceutical majors located in regions with the technology 91 and wherewithal do not find it attractive to direct research into these areas due to 92 marginal profit prospects. 93 Helminthiasis, a major Neglected Tropical Disease (NTD) has long been identified by 94 the World Health Organization (WHO) as a disease with very high morbidity rate and 95 cognitive deficit especially in school-aged children [20,3]. The prevalence is mostly 96 confined to the tropical and subtropical regions where there still exists infrastructural 97 deficit; poor sanitation; use of untreated fecal matter as fertilizers and subsisting bare 98 soil defecation [1]. 99 In addition, no new antihelmintic agents have been added to the list in the past 100 decade with gradual development of resistance to the standard treatments being 101 reported [17,15]. 102 Drug repurposing has recorded major milestones in several treatment landscapes 103 hence this technique has been considered a veritable tool to achieve this objective in 104 antihelmintic sphere. in this study. Prominent among such studies is the work of Uzochukwu,et al.,[18] 117 which revealed the potentials of some approved drugs as antihelmintic agents.

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This study investigated the antihelmintic potential of some approved drugs using 120 Ascaris suum mitochondrial rhodoquinol fumarate reductase enzyme as target. The 121 in vitro paralysis and mortality times of the frontrunners using the ascaris surrogate,

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On the basis of bioactivity scores similarity of the approved drugs to atpenin, the in-127 house drug database was sorted and one hundred drugs were selected. And the 128 docking protocol was validated by superimposition of the experimental atpenin A5 on 129 the atpenin from docking which showed a near perfect fit (Fig 1). The post docking analysis yielded two hundred drugs(including their isomers) with 135 binding energies within the range of atpenin (-7.825 kcal/mol).

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Out of this list, fifteen drugs with the closest binding energies to atpenin were 137 selected as frontrunners.

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This result is presented in Table 1.  Four out of the fifteen frontrunner drugs were found to exploit the same binding 144 pocket and interacted with the same amino acid residues within the receptor site     The synergistic testing produced the greatest activity (optimal fixed dose) at the 6:4 181 ratio of doxycycline and mepacrine (Fig 6 and 7). This combination produced is usually considered the one with the best interaction and affinity therefore is predicted 191 to have the best activity. The binding energies generated from docking predicted 192 potential antihelmintic activity of some of the approved drugs that were docked.

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The four major frontrunners, mefloquine, doxycycline, proguanil and mepacrine showed 194 better binding affinities than the experimental antihelmintic compound, atpenin as well as 195 the reference antihelmintic drugs, the benzoimidazoles.

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The expectation is that these four drugs with binding energies between -11.0 to -8.1 198 kcal/mol will have activities that would surpass that of atpenin. Their exploitation of same 199 binding pocket and interaction with same amino acid residues at the receptor site is Entomology Department of Nnamdi Azikiwe University, Awka.

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The drugs used for the bioassay were obtained from registered pharmacies in 307 Nigeria and a few from Boots, United Kingdom.

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Patent holders brands were used or brands from reputable manufacturers and seven 309 assay points were chosen between 0.078 to 5.0 mg/ml and were prepared as stock