Background
Malaria is one of the leading causes of morbidity and mortality from infectious diseases. A recent report by World Health Organization (WHO) estimated that 3.2 billion people are at risk of contracting the disease. In 2015 alone, there were an estimated 214 million cases of malaria worldwide with 438,000 deaths. Ninety per cent of the deaths occurred in WHO African Region where children are the main victims [
1].
Multidimensional intervention in malaria control over the last decade has significantly reduced the incidence and mortality caused by malaria worldwide. These include the provision of insecticide-treated bed nets, indoor residual spraying, rapid diagnostic tests, and a supply of effective artemisinin-based combination therapy (ACT). As a result, many countries have initiated malaria elimination programmes. However, the emergence of
Plasmodium falciparum strains that are resistant to artemisinin in Southeast Asian countries is posing a huge challenge to future malaria control and elimination efforts. The possibility of dissemination of the resistant strains to Africa is projected to have potentially catastrophic outcomes [
1]. This calls for the urgent search for new and effective anti-malarial drugs.
Currently, ACT is the most widely used treatment for uncomplicated malaria. Since artemisinin is a fast-acting drug with a short half-life, it should be combined with another drug with longer half-life in order to effectively clear the parasite and prevent the emergence of drug-resistant strains. Several partner drugs have been used as a component of ACT. Unfortunately, treatment failures in the partner drugs have emerged, threatening to curb the positive achievements gained to date in the fight against malaria and exposing artemisinin [
2‐
4]. This necessitates the development of new partner drugs to use in ACT in places where resistance to artemisinin has not developed.
Heat shock proteins (Hsps) are the major chaperone proteins found in all life forms, ranging from prokaryotes to higher organisms, such as plants and mammals. Hsps are both constitutive and stress-inducible [
5,
6]. Environmental factors, such as an abrupt change in temperature, upregulates the expression of Hsps [
7,
8].
The life cycle of parasites such as
Plasmodium,
Leishmania and
Trypanosoma involves poikilothermic insect vectors and homoeothermic mammalian hosts. These parasites are exposed to a sudden change in temperature of up to 10 °C during the transition from the insect-stage to mammalian-stage of the parasites, and have evolutionarily developed molecular chaperones to withstand the drastic change in temperature [
9,
10]: for example, about 2% of the genes of
P. falciparum code for proteins that serve as molecular chaperones [
9]. Su and Wellems [
11] showed that transcription of
P. falciparum heat shock protein 90 (PfHsp90) increases up to three- and four-fold as a result of in vitro cultivation of the parasite at 39 and 41 °C, respectively.
Depending on their molecular size, Hsps are classified as small heat shock proteins (sHsps), Hsp40, Hsp60, Hsp70, Hsp90, and Hsp110. Hsp90 is one of the most abundant cytosolic proteins of a eukaryotic cell. The N-terminal domain of Hsp90 has an ATP binding pocket responsible for its ATPase activity [
12,
13]. Hsps play a crucial role in the normal metabolic activities of cells. By facilitating the proper folding of proteins, Hsps are involved in intracellular protein trafficking, gene expression, cell cycle, as well as cell differentiation [
5,
9,
14].
The crucial role of Hsp90 in chaperoning several important cellular functions and the structural differences in the ATP-binding domain of human and parasite Hsp90 make it a potentially viable drug target against several parasitic infectious diseases [
15,
16]. Molecular characterization of the PfHsp90 protein from clinical isolates of
P. falciparum collected from patients in diverse geographical regions has shown that the ATP-binding domain of PfHsp90 is highly conserved among the isolates [
17] thus reducing the likelihood of resistance emerging. Hsp90 is an essential protein in eukaryotic systems and not compatible with viability if knocked out. That is, mutations in the ATP binding domain of PfHsp90 make the protein inactive, negatively affecting important biological functions and incur the parasite too much fitness cost [
18‐
20]. Interestingly, it has been shown that PfHsp90 may be associated with a
P. falciparum chloroquine resistance transporter (PfCRT) protein from a chloroquine-resistant parasite strain. Immunoprecipitation experiments showed that PfHsp90 complex co-immunoprecipitated with PfCRT from
P. falciparum W2 strain. Moreover, the use of a PfHsp90 inhibitor, PU-H71, resulted in loss of PfCRT protein [
21]. It is postulated that targeting PfHsp90 in chloroquine-resistant
P. falciparum strain could reverse the resistance and render the parasite chloroquine sensitive again. This effect may not be restricted to a single drug class given the broad range of chaperone activity that Hsp90 regulates.
The discovery of the natural compound, geldanamycin opened a new avenue in the development of drugs targeting Hsp90. Geldanamycin has displayed a strong anti-cancer and anti-malarial effect in vitro. However, a strong hepatotoxic signal precludes its clinical use. As a result, different derivatives of geldanamycin have been developed that have an acceptable level of hepatotoxicity [
22]. In vitro and in vivo experiments have shown that the geldanamycin-derivatives, 17AAG and 17-PEG-Alkyn-GA, are promising anti-malarial compounds targeting PfHsp90 [
23,
24].
In addition to geldanamycin and its derivatives, other small molecules such as purine analogues have been found to effectively inhibit Hsp90 in vitro and in vivo. One such molecule is PU-H71. This molecule has been tested as an anti-cancer drug [
25]. Recently, it has been demonstrated that PU-H71 has a good anti-malarial activity in vitro and in mice [
21]. Harmine, a beta carboline alkaloid, is able to selectively bind PfHsp90 to a greater extent than human Hsp90 (HsHsp90) [
26]. Harmine has been reported as having a specific high-affinity interaction with the ATP-binding domain of PfHsp90 with strong inhibition of
P. falciparum in cell culture systems. In the
P. berghei ANKA infection model in mice, harmine showed a significant reduction in parasitaemia. However, it did not significantly prolong the survival of the infected mice [
17,
26].
In this study, a library of harmine analogues were generated and their ability to bind PfHsp90, inhibit P. falciparum in culture, and kill parasites in the P. berghei infection mice model was tested. It was hypothesized that the harmine analogues have a strong anti-malarial effect both in vitro and in vivo in mice. It has been found that some of the harmine analogues effectively bind to PfHsp90, inhibit the growth of P. falciparum in vitro, significantly reduce parasitaemia in infected BALB/c mice in a dose-dependent manner, and prolong the survival of infected mice. Interestingly, two daily injections with a combination of 100 mg/kg of 21A (one of the harmine analogues) and 10 mg/kg dihydro-artemisinin (DHA) was able to reduce parasitaemia to an undetectable level in infected mice.
Discussion
Heat shock proteins are crucial molecular chaperones that are involved in various central metabolic activities of both prokaryotic and eukaryotic cells. Because Hsps are necessary for normal metabolic activities of a cell and to protect cells from stress conditions, they are conserved in all life forms. Although they are constitutively expressed, the level of expression of Hsps increases when challenged by stressors such as heat or pH changes. Hsp90’s crucial role as an essential chaperone, association with resistance, and high degree of conservation make it an attractive adjunctive drug target [
18,
19,
38,
39].
As a parasite with a life cycle involving poikilothermic insects and homoeothermic humans, P. falciparum has to adapt to the drastic change in temperature during transmission from the insect vector to human host. Plasmodium falciparum Hsp90 protein is the most abundant cytosolic protein and plays an indispensable role in resisting the effect of temperature change that occurs during transmission from insect vectors to humans.
The ATPase activity of Hsp90 is a necessary precursor to its function and interaction with client proteins. Therefore, targeting the Hsp90 ATP-binding using a small molecule is expected to inhibit its chaperoning activity and render the client proteins to degradation via the cytosolic proteasome, which consequently inhibit major metabolic activities of the cell [
40]. Several studies have tested the use of small molecules that bind to and competitively inhibit the ATPase activity of Hsp90 as candidate drugs for different infectious diseases and cancer [
23,
41,
42]. Although the Hsp90 in humans and the malaria parasite have a high degree of homology, there are subtle and potentially significant structural differences that can be exploited when designing selective small-molecule inhibitors [
16]. Moreover, cells from different organisms show variable degree of dependency on chaperone-supported metabolic activities. As a result, it is hypothesized that it is possible to inhibit the activity of Hsp of a pathogen without significant deleterious effects on the Hsps of the host [
5,
17]. It has been shown previously that natural compounds such as harmine and the ATP mimetic PU-H71 exert anti-
Plasmodium activity by targeting PfHsp90 [
21,
26]. In this study, 42 different derivatives of harmine were synthesized and the in vitro and in vivo anti-malarial activities of two of the harmine analogues was tested as single agents and in combination with artemisinin. Unfortunately, 22 harmine analogues displayed auto-fluorescence in bis-ANS assay and were consequently excluded from the study.
Two out of the remaining 20 compounds (17A and 21A) effectively bind to the ATP-binding domain of PfHsp90 with IC
50 value in the low micromolar range. The IC
50 values of 17A and 21A was comparable to that of radicicol, a compound that has been shown to tightly bind Hsp90 [
43].
In vitro susceptibility of 21A showed that the compound has anti-malarial activity against both chloroquine- and artemisinin-resistant
P. falciparum strains with micromolar IC
50 value. Generally, the chloroquine- or artemisinin-resistant strains seem more susceptible to 21A than the chloroquine-sensitive 3D7 strain. Similar effect was seen in the previous study with harmine. That is, chloroquine-resistant W2 strain was almost twice more susceptible to harmine than chloroquine-sensitive 3D7 strain [
17]. It remains to be seen if inhibition of PfHsp90 in chloroquine-resistant strains affects other metabolic activities of the parasite in addition to reversing the resistance trait. The effect of inhibition of PfHsp90 activity on proteins associated with chloroquine- or artemisinin-resistance needs further study.
At a fixed dose of 100 mg/kg administered daily for three consecutive days, both 17A and 21A showed a significant parasitaemia reduction. Unlike 17A, treatment with 21A significantly prolonged the survival time of treated mice. This property was not seen with the parent molecule harmine [
26]. Therefore, 21A was selected for the dose-ranging and combination experiments. 21A showed a dose-dependent activity with the highest parasitaemia reduction at a dose of 100 mg/kg followed by 75 and 50 mg/kg. One day after the last dose of treatment, the group treated with 25 mg/kg did not show significant difference from the vehicle control. Interestingly, 21A showed an additive effect with DHA in vivo in mice. Treatment with a combination of 100 mg/kg 21A and 10 mg/kg DHA resulted in a dramatic reduction of parasitaemia to undetectable levels by microscopy on Giemsa-stained peripheral blood in just two doses. This stands in stark contrast with the result from mice that were treated with DHA alone where only two out of five mice cleared the infection. As such, 21A has promising potential as a partner drug with artemisinin either in combination or co-formulation. This is in line with previous studies that showed the possibility of using Hsp90 inhibitors as adjunctive drugs with the current anti-malarial drugs [
39]. Of note, it is not possible to determine if the combined effect in mice was due to synergy or an alternative effect. Because the parasitaemia in the combination treatment groups was reduced to undetectable levels by microscopy, it was not possible to calculate whether 21A has synergistic effect with DHA in vivo. FACS analysis of DNA-stained RBCs could have been an alternative approach to determine the parasite load in such conditions. However, FACS analysis was not performed due to lack of the required laboratory facility. On the other hand, pharmacokinetic analysis of 21A and DHA in mice was not performed in this study. Pharmacokinetic and drug–drug interaction study between 21A and DHA is warranted to better understand the synergy.
In this study, differences were seen in the parasitaemia of mice that were treated with the same amount of 21A in different experiments. This could be probably due to differences in experimental conditions. One such factor is the difference in the initial parasitaemia before drug administration.
In order to be considered as an effective candidate drug, a compound should not be toxic to the human host. The first step in evaluating toxicity is to investigate the cytotoxicity in cell lines in vitro. In this regard, the cytotoxicity of 17A and 21A was tested in HepG2 and HeLa cells in vitro. Comparing the IC50 value for the cytotoxicity with that of in vitro anti-malarial activity, in HepG2 cells, 17A and 21A had a favourable cytotoxicity selectivity index (SI) of 38 and 25, respectively. In HeLa cells, 17A and 21A had an SI of 259 and 84, respectively. Future studies require the pursuit of a second generation library based on the current data which probably will yield compounds with increased potency against malaria in all stages of the life cycle.
Authors’ contributions
AGB, AF and DRP conceived and designed the study. SE and MA participated in the design of the study and synthesized the harmine analogues. AGB, AF and ANM conducted the in vitro and animal experiments. AGB, AF and DRP analysed the data. AGB and DRP wrote the manuscript. All authors read and approved the final manuscript.