Background
Since primitive times, malaria, a mosquito-borne infectious disease, has remained the leading cause of mortality from any parasitic disease around the world. It has been estimated that
Plasmodium spp., the causative agent of malaria, infected 228 million people, accounting for about half a million deaths in 2019, mostly affecting poor people living in tropical and sub-tropical regions of the world [
1]. Unlike other
Plasmodium species,
Plasmodium falciparum causes the severe form of malaria and poses higher risk of death due to associated neurological, renal or cardiological complications [
2]. To reduce the number of malaria-related cases and mortality, most of the malaria control programmes besides using other control measures, rely heavily on killing the malarial parasite by anti-malarial chemotherapy [
3]. Quinine, a plant derived chemical extracted from the bark of the Cinchona tree, was used to treat malaria from as early as the 1600 s and is still used as second-line therapy for the management of uncomplicated malaria in Africa when the first-line drug either fails or is not available [
4‐
7]. With the range of adverse side effects experienced by the use of quinine, the search for a safe and most effective anti-malarial medicine led to the discovery of chloroquine, followed by many synthetic anti-malarials, e.g., sulfadoxine-pyrimethamine, amodiaquine and mefloquine [
5,
6,
8,
9]. Despite their early success in therapeutic management of malaria, their overuse contributed to the development and spread of resistance against these drugs by the malarial parasite. Undoubtedly, the unregulated use of these drugs as monotherapy further accelerated their failing therapeutic efficacy [
6,
9‐
11].
At present, the most potent and successful drug available for the treatment of both severe and uncomplicated malaria is artemisinin, which was derived from the Qinghao plant (
Artemisia annua) in the 1970s [
5]. Artemisinin is frequently used in combination with a partner anti-malarial drug to overcome its pharmacokinetic limitations (such as poor bioavailability, low solubility in water and a relatively shorter half-life in vivo (~ 2.5 h)) and to protect its efficacy against parasite resistance for a longer period of time [
5]. Presently, five recommended artemisinin-based combinations include combinations of artemisinin derivates, such as artemisinin, dihydroartemisinin, artemether, artesunate, with lumefantrine, mefloquine, amodiaquine, sulfadoxine/pyrimethamine, piperaquine, and chlorproguanil/dapsone [
5]. Artemisinins are particularly active against the ring and mature trophozoite form of asexual life-cycle stage of parasites persisting within infected red blood cells. However, a sub-population of ring form of the parasite may tolerate artemisinin by becoming temporarily dormant or sequestered to return after a few days or weeks, eventually causing the failure of treatment [
6,
12]. It is for this reason that artemisinin monotherapy is not preferred and is recommended to be used only with longer-acting partner anti-malarial drug that would kill the surviving dormant form of the parasite [
11,
13]. Although artemisinin-based combination therapy (ACT) had a marked effect on malaria cases globally, the appearance of artemisinin-resistant cases in Southeast Asia, especially in the eastern Greater Mekong Sub-region, is cause for concern [
14‐
17]. By relying so heavily upon the use of ACT, eventually this valuable anti-malarial drug will become ineffective, given the history of resistance development in the parasite to most anti-malarials. Bringing safe and new anti-malarial drug candidates with diverse chemical structures and mechanism of action into clinical trials is critical to combat emerging anti-malarial drug resistance in the parasite.
Over time, new concepts in organic synthesis and molecular design, such as fragment-based drug discovery (FBDD), ligand-based drug discovery (LBDD), biology-oriented synthesis (BIOS), and diversity-oriented synthesis (DOS) began to evolve. Compared to traditional drug discovery platforms, these methods have not only expedited the process of bringing new drugs onto the market but also have helped in providing drugs with better specificity towards the target and lesser toxicity. DOS has particularly emerged as a synthetic approach that is used for the design and construction of novel, small molecule libraries containing a high degree of structural and stereo-chemical diversity [
18,
19]. Screening of DOS-derived compound libraries has led to identification of many novel and biologically useful small molecules known for their antibacterial, antifungal, antiparasitic, and anticancer properties [
20‐
25]. Accordingly, the present work aimed to screen and evaluate compounds of DOS library (comprised of 11 indole-based heterocycles which are connected to piperidine or pyrrolidine molecules either through bond fusion or via spiro linkage) for their efficacy in killing both wild-type and artemisinin-resistant strains of
P. falciparum in vitro. These compounds can be segregated into 4 different structures. The initial reaction involved trifluoroacetic acid (TFA) catalyzed condensation reactions of tryptamines with various aldehydes/ ketones to afford various library molecules (Experimental procedure section, Additional file
1). Few of them were further transformed to newer compounds by
N-bromosuccinimide (NBS)-mediated oxidation or ring contraction (Experimental procedure section, Additional file
1).
The study’s screening assay identified that 1-aryltetrahyro-β-carboline class of compounds possess significant anti-plasmodial activity. Further screening of two best compounds, i.e., 2 and 3, showed their potential to kill both wild-type and artemisinin-resistant strains. Also shown was that compound 3 can induce significant reactive oxygen species (ROS) generation in malaria parasites, providing insight into the mechanism of compound 3-induced parasite death.
Discussion
Despite availability of a wide variety of anti-malarial drugs, including quinine, chloroquine, sulfadoxine, atovaquone, primaquine, artemisinin, and their derivatives, increasing cases of drug resistance in malaria parasites and transmission of resistant parasites in new areas is throwing a challenge for worldwide efforts to eradicate malaria [
32,
33]. There is a need to come up with new anti-malarials with different mechanisms of action. DOS method has led to generation of libraries of small molecules based on complex structure scaffolds and has successfully played a role in the discovery of several bioactive compounds that have either emerged as drug leads or have helped enhance the knowledge of complex biological processes [
34‐
36].
The presence of indole-based heterocycles in many bioactive natural products has prompted scientists to use indole scaffold for developing libraries of a diverse range of compounds that are known to show promising activity against many human diseases, including cancer, microbial infection and parasitic diseases [
37,
38]. This has encouraged synthesis of libraries comprising of molecules inspired from various indole heterocycles and natural products as potential lead candidates against malaria parasites [
39‐
42]. In this study, a library of indole- based spiro and fused small molecules were screened and among all the compounds tested, compounds 2 and 3 exhibited toxicity against all the parasitic stages of IDC with similar levels of lethality for artemisinin-sensitive and artemisinin-resistant strains of
P. falciparum. This suggest that these compounds might have a dissimilar mode of action compared to artemisinin for killing malaria parasite. The compounds showed strong anti-plasmodial activity at a concentration range of 3–5 µM which is in comparable range to other indole-based anti-plasmodial compounds shown in previous studies [
40,
41].
Compound 3 induced oxidative stress by enhancing production of ROS, which could be a possible mechanism of action of this compound in killing malaria parasites. ROS are known to damage DNA, RNA and can oxidize proteins and lipids, leading to death of a cell. Bioactive indole compounds are shown to bind with multiple receptors, and thus can act by multiple mechanisms making them attractive chemical molecules for developing novel therapeutic compounds [
38]. Hit compounds identified in this study can be further refined and developed for use as anti-malarial compounds or can be used as a scaffold to develop artemisinin-based hybrid molecules [
43].
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