Malaria Box Research Articles

An adaptable, fit-for-purpose screening approach with high-throughput capability to determine speed of action and stage specificity of anti-malarial compounds.

A revamped in vitro compound identification and activity profiling approach is required to meet the large unmet need for new anti-malarial drugs to combat parasite drug resistance. Although compound hit identification utilizing high-throughput screening of large compound libraries is well established, the ability to rapidly prioritize such large numbers for further development is limited. Determining the speed of action of anti-malarial drug candidates is a vital component of malaria drug discovery, which currently occurs predominantly in lead optimization and development. This is due in part to the capacity of current methods which have low throughput due to the complexity and labor intensity of the approaches. Here, we provide an adaptable screening paradigm utilizing automated high content imaging, including the development of an automated schizont maturation assay, which collectively can identify anti-malarial compounds, classify activity into fast and slow acting, and provide an indication of the parasite stage specificity, with high-throughput capability. By frontloading these critical biological parameters much earlier in the drug discovery pipeline, it has the potential to reduce lead compound attrition rates later in the development process. The capability of the approach in its alternative formats is demonstrated using three Medicines for Malaria Venture open access compound "boxes," namely Pathogen Box (malaria set-125 compounds), Global Health Priority Box [Malaria Box 2 (80 compounds) and zoonotic neglected diseases (80 compounds)], and the Pandemic Response Box (400 compounds). From a total of 685 compounds tested, 79 were identified as having fast ring-stage-specific activity comparable to that of artemisinin and therefore of high priority for further consideration and development.

Identification and characterization of malaria box compounds possessing inhibition effect on the SARS-CoV2 spike protein

Identification and characterization of malaria box compounds possessing inhibition effect on the SARS-CoV2 spike protein

The Malaria Box molecules: a source for targeting the RBD and NTD cryptic pocket of the spike glycoprotein in SARS-CoV-2.

SARS-CoV-2, responsible for COVID-19, has led to over 500 million infections and more than 6 million deaths globally. There have been limited effective treatments available. The study aims to find a drug that can prevent the virus from entering host cells by targeting specific sites on the virus's spike protein. We examined 13,397 compounds from the Malaria Box library against two specific sites on the spike protein: the receptor-binding domain (RBD) and a predicted cryptic pocket. Using virtual screening, molecular docking, molecular dynamics, and MMPBSA techniques, they evaluated the stability of two compounds. TCMDC-124223 showed high stability and binding energy in the RBD, while TCMDC-133766 had better binding energy in the cryptic pocket. The study also identified that the interacting residues are conserved, which is crucial for addressing various virus variants. The findings provide insights into the potential of small molecules as drugs against the spike protein.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

In Vitro Activity and Atom Pair Fingerprint Analysis of Potent Hits from Malaria Box against Staphylococcus aureus Isolated from Cows with Clinical Mastitis

Staphylococcus aureus (S. aureus) is one of the most prevalent contagious bacterial pathogens causing clinical bovine mastitis. In the present study, plate (A), which consists of 80 compounds (40 drug-like and 40 probe-like), was assessed from the Malaria Box against the in vitro growth of S. aureus isolated from cows naturally infected with S. aureus. Anti-Staphylococcus aureus medications that are newer and more potent than those that have been used previously were discovered. Bioinformatics analysis for the potent identified hits was performed. Three compounds—two probe-like and one drug-like—showed strong inhibitory action against S. aureus growth in vitro. Among these hits, MMV665941 exhibited the highest minimum inhibitory concentration value of 0.0078. Combination therapy was carried out using the most potent anti-S. aureus medication, MMV665941, and the traditionally used anti-S. aureus drug, amoxicillin/clavulanate (Augmentin 625®). The atom pair fingerprint (APfp) analysis revealed that MMV665941 and Augmentin showed maximum structural similarity. Based on the results obtained, it can be concluded that the Medicines for Malaria Venture Box has a wealth of potential anti-S. aureus candidates.

Evaluation of the Inhibitory Effect of Moringa oleifera Leaves Methanolic Extract against In Vitro Growth of Several Babesia Species and Theileria equi and the In Vivo Growth of Babesia microti.

The current study evaluated the inhibitory effect of Moringa oleifera leaves methanolic extract (MOL) against the in vitro growth of Babesia bovis (B. bovis), B. caballi, B. bigemina, and Theileria equi (T. equi), as well as in vivo growth of B. microti in mice. Active principles of MOL extract were determined using liquid chromatography mass spectrometry (LC-MS). MOL's anti-piroplasm efficacy was assessed both in vitro and in vivo using the SYBR Green I fluorescence assay. Every 96 hours, the hematological parameters, including red blood cell count (RBCs; 104/UL), hemoglobin content (HGB; g/dl), and hematocrit percent (HCT; %), in the treated mice were monitored using a Celltac MEK6450 automated hematological analyzer. LC-MS of MOL revealed that the most abundant polyphenolic catechism found in the MOL extract was isoquercetin and rutin. MOL inhibited B. bovis, B. caballi, B. bigemina, and T. equi in vitro growth in a dose-dependent way, with IC50 values of 45.29 ± 6.14, 19.16 ± 0.45, 137.49 ± 16.07, and 9.29 ± 0.014 μg/ml, respectively. MOL's in vitro antibabesial activity was enhanced when administrated simultaneously with either diminazene aceturate (DA) or MMV665875 compound from malaria box. In mice infected by B. microti, a combination of MOL and a low dose of DA (12.5 mg·kg-1) resulted in a significant (P < 0.05) reduction in B. microti growth. These findings suggest that MOL is an effective herbal anti-piroplasm therapy, especially when combined with a low dosage of either DA or MMV665875.

Structure-Activity Relationships and Antiplasmodial Potencies of Novel 3,4-Disubstituted 1,2,5-Oxadiazoles.

The 4-substituted 3-amino-1,2,5-oxadiazole 1 from the Malaria Box Project of the Medicines for Malaria Venture foundation shows very promising selectivity and in vitro activity against Plasmodium falciparum. Within the first series of new compounds, various 3-acylamino analogs were prepared. This paper now focuses on the investigation of the importance of the aromatic substituent in ring position 4. A number of new structure-activity relationships were elaborated, showing that antiplasmodial activity and selectivity strongly depend on the substitution pattern of the 4-phenyl moiety. In addition, physicochemical parameters relevant for drug development were calculated (logP and ligand efficiency) or determined experimentally (CYP3A4-inhibition and aqueous solubility). N-[4-(3-ethoxy-4-methoxyphenyl)-1,2,5-oxadiazol-3-yl]-3-methylbenzamide 51 showed high in vitro activity against the chloroquine-sensitive strain NF54 of P. falciparum (PfNF54 IC50 = 0.034 µM), resulting in a very promising selectivity index of 1526.

MMV006087 is a potent Malaria Box compound against Plasmodium falciparum clinical parasites

The emergence of drug-resistant malaria parasites to artemisinin and its partner drugs highlights the need to increase the arsenal of new antimalarials with novel mechanisms of action. To help achieve this aim, this study tested the potency of three Malaria Box compounds (MMV006087, MMV085203, and MMV008956) against five laboratory strains and twenty clinical isolates of Plasmodium falciparum using optimized in vitro growth inhibitory assays. The results were compared to the response from four standard antimalarials-artesunate, chloroquine, mefloquine, and halofantrine. From the results, MMV006087 was the most potent compound with an average IC50 of 22.13 nM compared to MMV085203 (average IC50 of 137.90 nM) and MMV008956 (average IC50 of 262.30 nM). On average, the laboratory strains were also less susceptible to the three Malaria Box compounds (average IC50 of 162.30 nM) compared to the clinical isolates (average IC50 of 135.40 nM). Additionally, MMV006087 was less potent than artesunate but twice more efficacious than chloroquine against the laboratory strains and clinical isolates. The data from this study validate the potency of MMV006087 and MMV085203 as promising antimalarials worthy of further exploration. This study further substantiates the need to include clinical isolates in antimalarial compound screening activities.

Knockdown of Anopheles dirus far upstream element-binding protein gene lower oocyst numbers of Plasmodium vivax.

The modulation of gene expression levels of Anopheles dirus on Plasmodium vivax infection at the ookinete and oocyst stages was previously reported. In the present study, several upregulated An. dirus genes were selected based on their high expression levels and subcellular locations to examine their roles in P. vivax infection. Five An. dirus genes-carboxylesterase, cuticular protein RR-2 family, far upstream element-binding protein, kraken, and peptidase212-were knocked down by dsRNA feeding using dsRNA-lacZ as a control. The dsRNA-fed mosquitoes were later challenged by P. vivax-infected blood, and the oocyst numbers were determined. The expression of these five genes was examined in many organs of both male and female mosquitoes. The results showed that the decreased expression level of the far upstream element-binding protein gene could lower the oocyst numbers, whereas the others showed no effect on P. vivax infection. The expression levels of these genes in ovaries were found, and in many organs, they were similar between male and female mosquitoes. The reduction of these five gene expressions did not affect the lifespan of the mosquitoes. In addition, the malaria box compound, MMV000634, demonstrated the lowest binding energy to the far upstream element-binding protein using virtual screening. This protein might be a target to block malaria transmission.

Novel inhibitors of Rickettsia prowazekii methionine aminopeptidase from the Malaria Box

Methionine aminopeptidases (MetAp) are dinuclear metalloenzymes found in both prokaryotes and eukaryotes that catalyze the hydrolysis of the N-terminal methionine residue from nascent proteins, an important post-translational modification, which makes it an attractive target for drug discovery. Rickettsia prowazekii (Rp) is an obligate pathogen and causative agent of epidemic typhus and typhus fever. In our ongoing search for anti-rickettsial agents we screened 400 compounds from the Malaria Box for inhibition of RpMetAp1 and discovered 12 compounds that inhibited the enzyme with IC50 values ranging from 800 nM to 22 μM. These inhibitors are from eleven different chemical series and represent leads that can be used to discover more potent and efficacious anti-rickettsial agents.

New Derivatives of the Multi-Stage Active Malaria Box Compound MMV030666 and Their Antiplasmodial Potencies.

MMV's Malaria Box compound MMV030666 shows multi-stage activity against various strains of Plasmodium falciparum and lacks resistance development. To evaluate the importance of its diarylether partial structure, diarylthioethers and diphenylamines with varying substitution patterns were prepared. A number of evident structure-activity relationships were revealed. Physicochemical and pharmacokinetic parameters were determined experimentally (passive permeability) or calculated. Compared to the lead compound a diarylthioether was more active and less cytotoxic resulting in an excellent selectivity index of 850. In addition, pharmacokinetic and physicochemical parameters were improved.

PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion.

The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture's 'Malaria Box' and 'Pathogen Box'. Recent screens of the 'Malaria Box' and 'Pathogen Box' revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.

Deciphering antiviral efficacy of malaria box compounds against malaria exacerbating viral pathogens- Epstein Barr virus and SARS-CoV-2, an in silico study

In malaria endemic countries, coinfections and cotransmissions of different viral pathogens are widely reported. Prior studies have shown that malaria can trigger the Epstein-Barr virus (EBV) reactivation in the body. Besides, the altered immunity due to malaria could increase susceptibility to acquire co-circulating viruses like SARS-CoV-2 or vice versa during pandemic times. The dual burden of pathogens can deteriorate health by inducing disease severity. There are no or limited antiviral therapies available against EBV and SARS-CoV-2. Exploring the novel antimalarials for checking antiviral efficacy and using them in such cases could be the efficient approach of ‘hitting two birds with one stone’. We investigated the antiviral potency of medicine for a malaria venture’s malaria box containing 400 drug-like or probe-like compounds with experimentally proven antimalarial activity. We utilized a molecular docking approach to screen these compounds against crucial proteins- EBNA1 of EBV and RdRp of SARS-CoV-2 respectively. Based on binding affinity we shortlisted the top three compounds for each protein. Further, for validation of complex stability and binding, the protein–ligand complex is subjected to 100 ns molecular dynamic simulation. All the compounds showed stable binding with respective proteins. Based on binding free energies, involvement of important residues from target sites, and ADMET properties of compounds, the top ligand for each protein is selected. Ligand B (MMV665879) for EBNA1 (ΔGbind = -183.54 kJ/mol) and Ligand E (MMV665918) for RdRp (ΔGbind = -172.23 kJ/mol) could act as potential potent inhibitors. These antimalarial compounds can hence be utilized for further experimental investigation as antivirals against EBV and SARS-CoV-2.

Repurposing of the Malaria Box for Babesia microti in mice identifies novel active scaffolds against piroplasmosis

BackgroundAn innovative approach has been introduced for identifying and developing novel potent and safe anti-Babesia and anti-Theileria agents for the control of animal piroplasmosis. In the present study, we evaluated the inhibitory effects of Malaria Box (MBox) compounds (n = 8) against the growth of Babesia microti in mice and conducted bioinformatics analysis between the selected hits and the currently used antibabesial drugs, with far-reaching implications for potent combinations.MethodsA fluorescence assay was used to evaluate the in vivo inhibitory effects of the selected compounds. Bioinformatics analysis was conducted using hierarchical clustering, distance matrix and molecular weight correlation, and PubChem fingerprint. The compounds with in vivo potential efficacy were selected to search for their target in the piroplasm parasites using quantitative PCR (qPCR).ResultsScreening the MBox against the in vivo growth of the B. microti parasite enabled the discovery of potent new antipiroplasm drugs, including MMV396693 and MMV665875. Interestingly, statistically significant (P < 0.05) downregulation of cysteine protease mRNA levels was observed in MMV665875-treated Theileria equi in vitro culture in comparison with untreated cultures. MMV396693/clofazimine and MMV665875/atovaquone (AV) showed maximum structural similarity (MSS) with each other. The distance matrix results indicate promising antibabesial efficacy of combination therapies consisting of either MMV665875 and AV or MMV396693 and imidocarb dipropionate (ID).ConclusionsInhibitory and hematology assay results suggest that MMV396693 and MMV665875 are potent antipiroplasm monotherapies. The structural similarity results indicate that MMV665875 and MMV396693 have a similar mode of action as AV and ID, respectively. Our findings demonstrated that MBox compounds provide a promising lead for the development of new antibabesial therapeutic alternatives.Graphical

Promising antimalarials targeting apicoplast DNA polymerase from Plasmodium falciparum

Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.

The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites.

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs—modifications which are both mediated by parasite‐derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.

Malaria Box Compounds against Anopheles gambiae (Diptera: Culicidae) Carboxypeptidase B Activity to Block Malaria Transmission.

Carboxypeptidase B (CPB) plays an important role in blood digestion in mosquitos, aiding the release of free amino acids. Anopheles CPB is a target to block malaria transmission because it facilitates Plasmodium invasion of the mosquito midgut. Our study aimed to discover inhibitors of Anopheles CPB to prevent Plasmodium development in the mosquito. The Anopheles gambiae cpb (Agcpb) gene without a signal sequence was cloned into the pET28b expression vector. The recombinant AgCPB protein was expressed in E. coli BL21(DE3) within inclusion bodies after induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The protein pellet was dissolved in 6 M urea, purified by affinity chromatography, and dialyzed in reaction buffer. The refolded recombinant AgCPB could digest the hippuryl-arginine substrate similarly to that of the commercial porcine pancreas CPB. The 20 top-scoring malaria box compounds from the virtual-screening results were then chosen for an in vitro inhibition assay against AgCPB. Four of the 20 malaria box compounds could inhibit AgCPB activity. The compound MMV007591 was the most potent inhibitor with an IC50 at 0.066 µM. The results indicate that these candidate compounds may be utilized in drug development against mosquito CPB activity to curb malaria transmission.

MMV020275 and MMV020490, promising compounds from malaria box for the treatment of equine piroplasmosis

Equine piroplasmosis is a tick-transmitted disease that is considered one of the most serious infectious diseases affecting equines. Searching for novel antipiroplasm drugs remains indispensable due to the emergence and spreading of resistant piroplasm parasites against the limited currently used drugs, diminazene aceturate and imidocarb dipropionate. Therefore, novel drugs with specified targets need to be identified and exploited. The inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds with potent in vitro anti-equine piroplasmosis activity were evaluated against the growth of B. microti in mice in this study. Using a nested PCR assay targeting the B. microti ss-rRNA gene, we investigated the far-reaching impacts of effective combinations to inhibit parasite recrudescence. Using real-time PCR, this study revealed potential targets for the found potent compounds. When used as monotherapy, screening the Malaria Box against the in vivo growth of the B. microti parasite resulted in the discovery of new, potent antipiroplasm medicines, such as MMV020275 and MMV020490. In MMV020275-treated Theileria equi in vitro culture, a statistically significant difference (P<0.05) in the cGMP-dependent protein kinase (PKG) mRNA level was identified as a down-regulation in contrast to non-treated cultures. In conclusion, new potent antipiroplasm drugs, including MMV020275 and MMV020490 are identified. MMV020275 significantly down-regulate the mRNA levels of the PKG gene. Clofazimine enhanced the inhibitory efficacy of MMV compounds which is suggested to use in treatment of animal or human babesiosis in the future.

In vivo antibabesial activity and bioinformatic analysis of compounds derived from the Medicines for Malaria Venture box against Babesia microti

Here, we have evaluated the inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds that exhibited potent in vitro anti-bovine Babesia efficacy against the growth of B. microti in mice and conducted follow-up investigations of the structural similarity between the identified potent MMV compounds and the commonly used antibabesial drugs was performed using atom Pair fingerprints (APfp). Screening the Malaria Box against the in vivo growth of the B. microti parasite helped with the discovery of new, effective anti-bovine Babesia drugs, including MMV667488, MMV007285, and MMV019881. Of note, MMV019881 exhibited the highest anti-B. microti efficacy in vivo among the screened MMV compounds. The APfp results revealed that the maximum structural similarity (MSS) was observed between MMV007285, diminazene aceturate, and imidocarb dipropionate (ID). In the same way, clofazimine (CF) and MMV667488 showed the MSS with either each other based on the analysis. The distance matrix and molecular weight correlation findings highlight the possible potential antibabesial efficacy of MMV667488, ID, and CF when administrated as a combination therapy. In conclusion, in the current study new potent antibabesial drug, MMV019881 was identified. CF and MMV667488 showed the MSS with either each other based on the hierarchical clustering analysis (HCA) and such relation is confirmed by the distance matrix and molecular weight correlation findings. Such combination therapy might have a potential as a novel regime for treating animal or human babesiosis.

Evaluation of the inhibitory effect of Zingiber officinale rhizome on Babesia and Theileria parasites.

The effect of Zingiber officinale rhizome methanolic extract (ZOR) on the in vitro growth of bovine Babesia (B. bovis, B. bigemina, and B. divergens) and equine piroplasm (B. caballi, and Theileria equi) parasites and on the growth of B. microti in mice was evaluated in this study. The possible in vitro synergistic interaction between ZOR and either diminazene aceturate (DA) or potent Medicines for Malaria Venture (MMV) hits from the malaria box was also investigated. In vitro, ZOR reduced the growth of B. bovis, B. bigemina, T. equi, and B. caballi in a dose-dependent manner. B. divergens was the most susceptible parasite to the in vitro inhibitory effect of ZOR. DA and MMV compounds enhanced the in vitro inhibitory antibabesial activity of ZOR. 12.5mg/kg DA when administrated in combination with ZOR in mice exhibited a significant inhibition (P<0.05) in B. microti growth better than those observed after treatment with 25mg/kg DA monotherapy. These findings suggest that ZOR could be a viable medicinal plant for babesiosis treatment, particularly when combined with a modest dose of either DA or powerful anti-B. bigemina MMV hits.