Ex vivo and In vitro antiplasmodial activities of approved drugs predicted to have antimalarial activities using chemogenomics and drug repositioning approach

High malaria mortality coupled with increased emergence of resistant multi-drug resistant strains of Plasmodium parasite, warrants the development of new and effective antimalarial drugs. However, drug design and discovery are costly and time-consuming with many active antimalarial compounds failing to get approved due to safety reasons. To address these challenges, the current study aimed at testing the antiplasmodial activities of approved drugs that were predicted using a target-similarity approach. This approach is based on the fact that if an approved drug used to treat another disease targets a protein similar to Plasmodium falciparum protein, then the drug will have a comparable effect on P. falciparum. In a previous study, in vitro antiplasmodial activities of 10 approved drugs was reported of the total 28 approved drugs. In this study, six out of 18 drugs that were previously not tested, namely epirubicin, irinotecan, venlafaxine, palbociclib, pelitinib, and PD153035 were tested for antiplasmodial activity. The drug susceptibility in vitro assays against five P. falciparum reference strains (D6, 3D7, W2, DD2, and F32 ART) and ex vivo assays against fresh clinical isolates were done using the malaria SYBR Green I assay. Standard antimalarial drugs were included as controls. Epirubicin and irinotecan showed excellent antiplasmodial ex vivo activity against field isolates with mean IC50 values of 0.044 ± 0.033 μM and 0.085 ± 0.055 μM, respectively. Similar activity was observed against W2 strain where epirubicin had an IC50 value of 0.004 ± 0.0009 μM, palbociclib 0.056 ± 0.006 μM, and pelinitib 0.057 ± 0.013 μM. For the DD2 strain, epirubicin, irinotecan and PD 153035 displayed potent antiplasmodial activity (IC50 < 1 μM). Epirubicin and irinotecan showed potent antiplasmodial activities (IC50 < 1 μM) against DD2, D6, 3D7, and F32 ART strains and field isolates. This shows the potential use of these drugs as antimalarials. All the tested drugs showed antiplasmodial activities with IC50 values below 20 μM, which suggests that our target similarity-based strategy is successful at predicting antiplasmodial activity of compounds thereby circumventing challenges in antimalarial drug discovery.

High malaria mortality coupled with increased emergence of resistant multi-drug resistant strains of Plasmodium parasite, warrants the development of new and effective antimalarial drugs. However, drug design and discovery are costly and time-consuming with many active antimalarial compounds failing to get approved due to safety reasons. To address these challenges, the current study aimed at testing the antiplasmodial activities of approved drugs that were predicted using a target-similarity approach. This approach is based on the fact that if an approved drug used to treat another disease targets a protein similar to Plasmodium falciparum protein, then the drug will have a comparable effect on P. falciparum. In a previous study, in vitro antiplasmodial activities of 10 approved drugs was reported of the total 28 approved drugs. In this study, six out of 18 drugs that were previously not tested, namely epirubicin, irinotecan, venlafaxine, palbociclib, pelitinib, and PD153035 were tested for antiplasmodial activity. The drug susceptibility in vitro assays against five P. falciparum reference strains (D6, 3D7, W2, DD2, and F32 ART) and ex vivo assays against fresh clinical isolates were done using the malaria SYBR Green I assay. Standard antimalarial drugs were included as controls. Epirubicin and irinotecan showed excellent antiplasmodial ex vivo activity against field isolates with mean IC 50 values of 0.044 ± 0.033 μM and 0.085 ± 0.055 μM, respectively. Similar activity was observed against W2 strain where epirubicin had an IC 50 value of 0.004 ± 0.0009 μM, palbociclib 0.056 ± 0.006 μM, and pelinitib 0.057 ± 0.013 μM. For the DD2 strain, epirubicin, irinotecan and PD 153035 displayed potent antiplasmodial activity (IC 50 < 1 μM). Epirubicin and irinotecan showed potent antiplasmodial activities (IC 50 < 1 μM) against DD2, D6, 3D7, and F32 ART strains and field isolates. This shows the potential use of these drugs as antimalarials. All the tested drugs showed antiplasmodial activities with IC 50 values below 20 μM, which suggests that our target similarity-based strategy is successful at predicting antiplasmodial activity of compounds thereby circumventing challenges in antimalarial drug discovery.

Introduction
Worldwide, malaria is a major public health problem with 627,000 deaths out of 241 million cases reported in 2020 [1]. About 95% of the malaria cases reported in 2020 occurred in Sub-Saharan Africa [1]. Limited access to malaria treatment facilities, the emergence of insecticide-resistant vectors [2], and emerging drug-resistant parasites [3] have contributed to the high malaria prevalence. More recently, resistance has been reported to artemisinin-based combination therapy (ACT) in Southeast Asia, where antimalarial drug resistance is most prevalent [2], including resistance to artemether-lumefantrine, which is recommended by the World Health Organization as the first-line treatment for uncomplicated P. falciparum malaria [4]. The trend in drug resistance and parasite evolution necessitates the search for alternative antimalarial drugs with novel modes of action.
Search for antimalarial drugs in the past relied upon testing plants traditionally used as remedies for fever and malaria [5], leading to the discovery of successful antimalarials such as artemisinin from Artemisia annua L. (Asteraceae) and quinine from Cinchona ledgeriana Bern. Moens (Rubiaceae) [6]. Initial antimalarials have often served as scaffolds for newer regimens, minimizing the emergence of resistance. Other molecules such as flavonoids and quinolines have been modified to form analogs with potent antimalarial properties [7,8]. These methods are expensive, time-consuming and many molecules fail to progress to approval due to safety requirements [9]. This therefore calls for alternative methods for antimalarial drug discovery.
Computer-Aided Drug Discovery and Development has been used to reduce the cost, the duration and overcome safety challenges that are involved in traditional antimalarial drug discovery [10,11]. This includes the use of target similarity approach which is based on the principle that if a Plasmodium parasite protein is similar to a putative drug target protein, then the drug in question would have a similar effect on the protein of interest. Previous studies have used the same approach in repositioning drugs against P. falciparum apicoplast and Schistosoma mansoni [12][13][14]. We used a similar strategy to predict approved drugs with activity against P. falciparum [15], although the in vitro antiplasmodial activity of many of the predicted approved drugs remains unknown. Therefore, the current study focused on the antiplasmodial activities of approved drugs with unknown antimalarial activities.
As part of our ongoing studies of identifying antiplasmodial activities of approved drugs that target proteins similar to those present in Plasmodium proteome, this study describes in vitro and ex vivo antiplasmodial activities of six approved drugs that were predicted using a target similarity approach in a previous study [15]. The drugs reported in this study are already approved and therefore overcoming cost, time and safety challenges in antimalarial drug development and discovery. This is likely to therefore hasten discovery of alternative antimalarial drugs.

Drugs used for antiplasmodial tests
This work is part of our ongoing study which predicted approved drug targets with antiplasmodial activity. This was done by searching approved drugs with known drug targets that were similar to P. falciparum proteins on three drug databases, namely DrugBank, Therapeutic Target Database (TTD), and STITCH 4.0. This led to the identification of 28 approved drugs with unknown antiplasmodial activity [15]. Antiplasmodial activities of six of the drugs that were not previously tested are hereby reported, including irinotecan, PD153035 HCL, venlafaxine, pelinitib, epirubicin and palbociclib. These test drug compounds were obtained from Pfizer and the University of Potsdam, Germany.
Drugs were dissolved in 99.5% dimethylsulfoxide (DMSO) (Sigma-Aldrich) and then diluted in a complete Roswell Park Memorial Institute (RPMI 1640) cell culture medium. Drugs at serial dilutions of; mefloquine (0.488-250 ng/mL), chloroquine (1.953-1000 ng/ mL) alongside test-approved drugs (24.414-2000 ng/mL) were generated on a 96-well plate with DMSO ≤0.0875% [19]. A modified non-radioactive malaria SYBR Green I assay technique [20] was used to determine the IC 50 s of the approved drugs with suspected antimalarial activities against the five laboratory-adapted strains of P. falciparum (W2, DD2, D6, 3D7, and F32 ART) alongside fresh clinical isolates. Briefly, the parasites with a dose range of reference and test drugs were then added to 96 well plates. The plates were incubated at 37 • C in a gas mixture of 5% CO 2 , 5% O 2 , and 90% N 2 for 72 h. The assay was then terminated by adding lysis buffer containing SYBR Green I dye (1 × final concentration) and then incubated in the dark at room temperature for 24 h. Lysis buffer was prepared as earlier described by Akala et al., 2011 [21]. Relative fluorescence units (RFUs) of each well were used to determine the parasite growth inhibition by use of Tecan Genios Plus (Tecan US, Inc., Durham, NC) set at an excitation wavelength of 485 nm and emission wavelength of 535 nm, the number of flashes was set at 10, the gain set at 60 with and integration time set at 40 μs?

Ex vivo antiplasmodial tests
Fresh blood samples were collected from individuals presenting at Kisumu County Hospital and Kombewa Sub-County Hospital within Western Kenya as described by Akala et al., 2011 [21]. A written informed consent/assent was obtained from each individual before each blood draw. The samples were tested within 6 h of collection. Parasitemia was determined and the presence of P. falciparum was confirmed at the malaria drug resistance lab using microscopic techniques. Samples whose parasitemia exceeded 1% parasitemia were adjusted to 2% hematocrit and 1%. Erythrocytes, RPMI media, and parasites were added to 96 well plates coated with dose ranges of reference and test drugs. The plates were then incubated, the assay was terminated and plates read as described in section 2.2.

Data analysis
The RFUs readings were analyzed using Graph Pad Prism® 8.0 windows software (Graph Pad Software, San Diego, CA, USA) to calculate IC 50 s of the test drugs. More than three separate readings from each sample were presented as mean ± standard deviation (Mean IC 50 ± SD). The single factor Analysis of Variance (ANOVA) was used to determine if there was a significant difference in the means of the test drugs across all P. falciparum strains and field isolates. The Turkey's post-hoc test was also done to determine the difference between the drugs and Dunnett's post-hoc test was done to further check if the means of the approved drugs were different from the control groups, chloroquine (CQ) and mefloquine (MQ). Data was analyzed using GraphPad Prism® 8.0 windows software (GraphPad Software, San Diego, CA, USA).

Ethical approval
The current study was approved by the Scientific and Ethics Review Unit (SERU) of Kenya Medical Research Institute (KEMRI), Nairobi, Kenya, and Walter Reed Army Institute of Research (WRAIR) Institutional Review Board, Silver Spring, MD, under protocols KEMRI/SERU/CCR/0018/3126, WRAIR#2257 for antiplasmodial activities of compound libraries for malaria drug discovery and Table 1 Antiplasmodial activities of approved drugs in mean IC 50 s (μM ± STDEV) against P. falciparum strains (P = 0.337).  Table 1 and Table 2 below summarizes the IC 50 s for all the drugs tested. Mean IC 50 s across different strains of P. falciparum and field isolates is also reported in Table 1. There was no significant difference (P = 0.337) in the mean IC 50 s of the tested drugs across different strains of P. falciparum and the field isolates. Similarly, when the test drugs were compared with the control drugs (CQ and MQ), there was no significant difference.

Antiplasmodial activities
Epirubicin was the most active drug against all tested P. falciparum strains except for the F32 ART malaria parasite strain. Epirubicin (IC 50 = 0.004 μM), pelinitib (IC 50 = 0.057) and palbociclib (IC 50 = 0.057 μM) showed excellent antiplasmodial activity (IC 50 < 1 μM) against W2 P. falciparum strain. PD 153035 had the least activity with an IC 50 value of 1.84 μM against the W2 strain ( Table 1). All drugs displayed a mean IC 50 of less than 6 across all strains and field isolates with the highest mean activity expressed by epirubicin (mean IC 50 = 0.158 μM) and irinotecan (mean IC 50 = 0.468 μM) ( Table 1). All drugs showed higher activity when tested against freshly collected field isolates ex vivo than when tested against cultured adapted strains of P. falciparum in vitro.

Antiplasmodial activities
Antimalarial drug development and discovery from traditional methods such as the screening of plants for antimalarial activity takes a lot of time, it is costly, and some fail to get approval due to safety reasons [9]. This leaves a few compounds in the pipeline for the development as antimalarial drugs. The current study is on the antiplasmodial activities of six drugs approved for treatment of other diseases but with no report on their antimalarial activity. The results provide insight into the potential use of these approved drugs as antimalarials thereby overcoming some of the challenges in antimalarial drug discovery. Antiplasmodial activities of pure compounds of the approved drugs was defined as follows in this study: (i) IC 50 >200 μM inactive; (ii) IC 50 100-200 μM low activity; (iii) IC 50 of 20-100 μM moderate activity; (iv) IC 50 of 1-20 μM good activity; and (v) IC 50 of <1 μM excellent/potent [22].
The drugs displayed no significant difference (P = 0.337) in the terms of their antiplasmodial activities across different strains of P. falciparum and field isolates. Similarly, the Dunnet's post-hoc test revealed that the approved drugs tested did not significantly differ (P > 0.9) in the means of the antiplasmodial activities from control drugs, except for venlafaxine (P = 0.293 compared with CQ and P = 0.156 compared with MQ) and PD 153035 (P = 0.625, compared with MQ). This indicates that the sample drugs' ability to clear P. falciparum parasites is comparable to reference antimalarial drugs. Therefore, the tested drugs generally demonstrate a high malaria parasite clearance across different strains of P. falciparum and freshly collected field isolates. The observed potent antiplasmodial Table 2 Antiplasmodial activities of other standard antimalarial drugs against freshly collected clinical isolates in mean IC 50 s (μM ± STDEV). NS: assay was not successful; values at 50% inhibition concentration means ± standard deviation in micro molar; STEV: standard deviation; HK1: field samples from hospital 1; HK2 field samples from hospital activities (IC 50 < 1 μM) of the test drugs; PD 15393035, epirubicin, and irinotecan against the DD2 strain and PD 15393035, epirubicin and irinotecan, against 3D7 strain is suggestive of their potential as candidate antimalaria drugs. These drugs exhibit high parasite clearance that is comparable to currently used antimalarial drugs. Epirubicin exhibited excellent antiplasmodial activity with a mean IC 50 value of 0.158 μM which was better than that of CQ with a mean IC 50 value of 0.928 μM across all strains of P. falciparum and field isolates. The antiplasmodial activity of epirubicin compares well with other studies [23], especially to Ferreira and colleagues' study that used computational chemogenomics and drug repositioning [23], where epirubicin displayed potent in vitro antiplasmodial activity against three different strains of P. falciparum; 0.111 μM (3D7), 0.099 (DD2) and 0.069 (W2). Similarly, irinotecan exhibited a mean IC 50 value of 0.468 μM when tested against different strains of P. falciparum and fresh field isolates. This value was significantly similar to that of CQ (P=0.292) and MQ (P=0.078). The observed similarity depicts that the drugs have similar therapeutic activity to standard antimalarial drugs and therefore the high inhibition activity against P. falciparum. Similar antiplasmodial activity of approved drugs with unknown antimalarial activity has been observed previously [15].
The ex vivo antiplasmodial activities of the approved drugs with unknown antimalarial activities (Table 1) were slightly lower than the selected standard antimalarial drugs when tested against freshly collected field isolates ( Table 2). There was no significant difference (P = 0.42) between the antiplasmodial activities of the standard antimalarial tested against fresh field isolates from the two hospitals (HK1 and HK2) ( Table 2). Antiplasmodial activities of epirubicin and irinotecan against field isolates were comparable with most of the tested standard antimalarial drugs (Table 1 and Table 2). The observed antiplasmodial activities further indicate a potential use of these two drugs as antimalarials. Considering that the samples tested in this study are approved drugs, therefore they are considered safe. Moreover, additional bioactivity information of the drugs is known which can be related to the reported antiplasmodial activity in this study. These findings will play a key role in hastening the use of these drugs for the treatment of malaria.

Antiplasmodial activities' relation to druggability index and consurf analysis
In addition to potent antiplasmodial activities, DNA topoisomerase II (the P. falciparum protein) which is similar to DNA topoisomerase 2-alpha (the putative protein target of epirubucin), the targeted protein has a high percentage of conserved amino acids of 61% according to the Consurf analysis (Table 3). This was the highest Consurf analysis percentage among all the P. falciparum proteins that are similar to the proteins of the drugs tested in this study. Protein-protein pairwise alignment was performed to determine conserved amino acids in a protein [23]. This identifies functional amino acid residues shared between P. falciparum proteins and its homologous putative drug targets. Since these amino acids are preserved over many generations, they are believed to play key structural and functional roles in these proteins. By searching on NCBI using a drug target as the query and the P. falciparum homologous target as a subject, protein pairs with > 80% query coverage progressed for Consurf server analysis [24].
Consurf web server uses algorithmic tools to identify functional regions in proteins with a score being calculated using the Bayesian method. Consurf analysis percentage is based on the criteria that; > 80% has high similarity, 50-79% as moderate, and < 50% as low similarity. This indicates that the protein predicted to be targeted by the drug has many amino acids that play a key role in the life of P. falciparum. Therefore, this is suggestive of the high potential of epirubicin as an antimalarial drug.
The Druggability Index of DNA topoisomerase II (the P. falciparum protein) which is similar to the putative protein target of epirubicin was high at 0.8. Druggability index is the ability of a putative target to be therapeutically modulated by drug medication. This shows a likelihood of the P. falciparum protein being modulated by epirubicin drug molecule [25]. The most druggable protein has a score of 1.0 and the least druggable has a score of 0 [26]. This shows that the P. falciparum protein DNA topoisomerase II which has a Druggability Index of 0.9, is highly druggable and is the same target of epirubicin (Table 3). This explains the high antiplasmodial activity of epirubicin against P. falciparum strains and field isolates as reported in this study. The ABC transporter had the lowest Druggability Index of 51% which is considered to be moderate.

Limitations of the Study
The study could perform in vivo antiplasmodial assays using animal models due to financial constraints. This could give a better indication of the clinical use of the drugs as antimalarials since animals are exposed to factors such as diet and the natural environment which is likely to affect the pharmacokinetic and pharmacodynamics activities of the drugs.

Conclusion
Most drugs in this study showed excellent antiplasmodial activities that was comparable (P > 0.05) to standard antimalarial drugs (MQ and CQ) of which, epirubicin displayed the most potent antiplasmodial activity with the highest percentage in the Consurf analysis among the six drugs tested, along with high druggability index. Similarly, irinotecan displayed potent antiplasmodial activity with moderate Consurf percentage and moderate druggability index. Therefore, the two drugs have a high potential use for treatment of P. falciparum malaria which is the most lethal. Furthermore, these findings validate the use of a combination of target similarity approaches to predict approved drugs and ex vivo and in vitro assays to determine their antiplasmodial activities in antimalarial drug discovery. This will circumvent challenges such as safety, cost and time that hinder or delay approval of possible antimalarial drugs from reaching the market. Future in vivo studies should be conducted to confirm the antiplasmodial activity of these drugs and their use in the treatment of malaria using animal models.