Quinolones substituted at the 2- and 3-positions with biaryl and diphenylether groups have been investigated for their antimalarial potential. ELQ-300, with a 3-position diphenyl ether, is at an advanced stage of preclinical development. Here, we synthesize the 2-position isomer of ELQ-300, i.e., HLQ-102, and describe synthetic procedures for preparing it that avoid the use of expensive catalysts and afford access to substituted quinolones bearing substituents in the 2-position as well as the benzenoid ring and with the critical 3-position CH3 group. We profile HLQ analogs for their antimalarial activity along with pharmacokinetics of the selected lead molecule. Cross-resistance patterns indicate that, like its predecessor, HLQ-102 targets the Qi site of the parasite cytochrome bc1 complex. This finding suggests the existence of two separate troughs in the target protein capable of accommodating such large structural features regardless of whether it is placed at the 2- or 3-positon of the quinolone ring.

Antimalarial therapeutics ideally should target all three major Plasmodium life cycle stages. Here we present an acridone antimalarial chemotype that is potent against blood, liver, and mosquito stages of malaria parasites. Attributes of lead candidate T111 include potent in vitro activity against cultured parasites, ex vivo activity against clinical isolates, oral single-dose cure in an asexual blood-stage rodent model, inhibition of sexual blood-stage parasites, activity against relapsing parasites in non-human primate liver cells, prevention of parasite development in mosquitoes, and synergy in combination with tafenoquine against blood- and liver-stage parasites. Analysis of parasites selected for resistance to T111 suggests inhibition of the mitochondrial electron transport chain, with a mechanism distinct from that of other antimalarials in use or under development. The safety profile of T111, including toxicology evaluations in rats, absence of hemolytic toxicity, and low genotoxicity and cardiotoxicity potential, demonstrates a favorable therapeutic index. Overall, T111 emerges as a promising candidate for treatment and prevention of malaria, with potential for single-dose cure of bloodstream infections, radical cure of liver infections, and interruption of transmission to mosquitoes.

Plasmodium falciparum, the deadliest causative agent of malaria, continues to evade eradication efforts through widespread drug resistance. The recent development of ADC-028, a 4-aminoquinoline antimalarial with excellent activity and pharmacokinetic properties, prompted the investigation of bisquinoline analogs featuring similar structural motifs. Here, we describe a structure-activity relationship study that guided the optimization of compounds with key features, including the 4-anilinoquinoline core and diverse bridging linkers. Several analogs exhibited potent in vitro activity (IC50 < 20 nM) against both drug-sensitive and multidrug-resistant P. falciparum strains, while maintaining favorable cytotoxicity profiles. Among them, 25 demonstrated improved intrinsic metabolic stability (t1/2 = 121 min) and potent in vivo efficacy (ED50 = 0.32 mg/kg/day), achieving complete curative protection at a reduced dose compared to ADC-028. While 25 showed moderately reduced oral bioavailability (F = 43%) and a shorter half-life (T1/2 = 27.2 h) relative to ADC-028, its enhanced in vivo efficacy underscores its therapeutic potential. This work highlights a promising path forward in developing antimalarials that retain the efficacy of legacy compounds while overcoming modern resistance mechanisms.

The apicomplexan mitochondrial electron transport chain is essential for parasite survival and displays a divergent subunit composition. Here we report cryo-electron microscopy structures of an apicomplexan III2-IV supercomplex and of the drug target complex III2. The supercomplex structure reveals how clade-specific subunits form an apicomplexan-conserved III2-IV interface with a unique, kinked architecture, suggesting that supercomplexes evolved independently in different eukaryotic lineages. A knockout resulting in supercomplex disassembly challenges the proposed role of III2-IV in electron transfer efficiency as suggested for mammals. Nevertheless, knockout analysis indicates that III2-IV is critical for parasite fitness. The complexes from the model parasite Toxoplasma gondii were inhibited with the antimalarial atovaquone, revealing interactions underpinning species specificity. They were also inhibited with endochin-like quinolone (ELQ)-300, an inhibitor in late-stage preclinical development. Notably, in the apicomplexan binding site, ELQ-300 is flipped compared with related compounds in the mammalian enzyme. On the basis of the binding modes and parasite-specific interactions discovered, we designed more potent ELQs with subnanomolar activity against T. gondii. Our findings reveal critical evolutionary differences in the role of supercomplexes in mitochondrial biology and provide insight into cytochrome b inhibition, informing future drug discovery.

The decline in malaria deaths has recently stalled owing to several factors, including the widespread resistance of Anopheles vectors to the insecticides used in long-lasting insecticide-treated nets (LLINs) 1,2 . One way to mitigate insecticide resistance is to directly kill parasites during their mosquito-stage of development by incorporating antiparasitic compounds into LLINs. This strategy can prevent onward parasite transmission even when insecticides lose efficacy 3,4 . Here, we performed an in vivo screen of compounds against the mosquito stages of Plasmodium falciparum development. Of the 81 compounds tested, which spanned 28 distinct modes of action, 22 were active against early parasite stages in the mosquito midgut lumen, which in turn prevented establishment of infection. Medicinal chemistry was then used to improve antiparasitic activity of the top hits from the screen. We generated several endochin-like quinolones (ELQs) that inhibited the P. falciparum cytochrome bc 1 complex (CytB). Two lead compounds that targeted separate sites in CytB (Q o and Q i ) showed potent, long-lasting and stable activity when incorporated and/or extruded into bed net-like polyethylene films. ELQ activity was fully preserved in insecticide-resistant mosquitoes, and parasites resistant to these compounds had impaired development at the mosquito stage. These data demonstrate the promise of incorporating ELQ compounds into LLINs to counteract insecticide resistance and to reduce malaria transmission.

Building from our previous lead compound T111 (1) possessing activity against both Plasmodium falciparum asexual blood-stage (ABS) and Plasmodium berghei liver-stage (LS) parasites, next-generation antimalarial acridones were systematically designed and synthesized. A large number of newly generated acridones displayed excellent antimalarial activities against both ABS and LS parasites, with feasible safety and metabolic profiles. In a high-throughput hypnozoitocidal assay using Plasmodium cynomolgi, a number of these acridones significantly inhibited schizont and hypnozoite formation in both prophylactic and radical cure-dosing modes. Notably, newer generation acridones substantially mitigated cross-resistance with atovaquone. Representative compound 28 (T229) provided full LS protection and a sustained blood-stage cure for murine P. berghei infection dosed at both 10 and 40 mg/kg/day orally. Furthermore, compound 28 demonstrated a low risk of both genotoxicity and cardiotoxicity and was highly effective against ART-resistant parasites. This study demonstrated the first and robust antirelapse LS activity from a novel acridone family.

A structure activity relationship study was conducted to optimize metabolic stability and in vivo activity of amodiaquine analog antimalarials. Antiplasmodial activity of synthesized amodiachins (ADCs) were evaluated against drug-sensitive and drug-resistant strains of Plasmodium falciparum. Select compounds were tested in Plasmodium yoelli murine models. Structural modifications, including introduction of a piperidine ring and varied N-alkyl substitutions enhanced antiparasitic activity, metabolic stability, and in vivo efficacy. Compound 43 (ADC-028) emerged as a standout candidate, exhibiting nanomolar activity against drug-sensitive and multidrug-resistant P. falciparum, minimal cytotoxicity, and favorable murine microsomal stability (t1/2 = 48.2 min). In vivo, 43 achieved a nonrecrudescence dose at 16 mg/kg/d and a single dose cure at 50 mg/kg. Pharmacokinetic analysis of 43 showed excellent metabolic stability (T1/2 = 84 h) and oral bioavailability (F = 76%). This study identifies 43 as a potential candidate for further evaluation as a novel compound to overcome drug-resistant malaria.

Background/Objectives:B. bigemina is a highly pathogenic and widely distributed tick-borne disease parasite responsible for bovine babesiosis. The development of effective and safe therapies is urgently needed for global disease control. The aim of this study is to compare the effects of endochin-like quinolone (ELQ-316), buparvaquone (BPQ), imidocarb (ID), and the combinations of ID + ELQ-316 and BPQ + ELQ-316, on in vitro survival of B. bigemina.Methods: Parasites at a starting parasitemia level of 2%, were incubated with each single drug and a combination of drugs, ranging from 25 to 1200 nM of concentration over four consecutive days. The inhibitory concentrations, 50% (IC50%) and 99% (IC99%), were estimated. Parasitemia levels were evaluated daily using microscopic examination. Data were statistically compared using the non-parametrical Kruskall-Wallis test. Results: All drugs tested significantly inhibited (p < 0.05) the growth of B. bigemina at 2% parasitemia. The combination of ID + ELQ-316 exhibited a lower mean (IC50%: 9.2; confidence interval 95%: 8.7-9.9) than ID (IC50%: 61.5; confidence interval 95%: 59.54-63.46), ELQ-316 (IC50%: 48.10; confidence interval 95%: 42.76-58.83), BPQ (IC50%: 44.66; confidence interval 95%: 43.56-45.81), and BPQ + ELQ-316 (IC50%: 27.59; confidence interval: N/A). Parasites were no longer viable in cultures treated with the BPQ + ELQ-316 combination, as well as with BPQ alone at a concentration of 1200 nM, on days 2 and 3 of treatment, respectively. Conclusions: BPQ and ID increase the babesiacidal effect of ELQ-316. The efficacy of these combinations deserves to be evaluated in vivo, which could lead to a promising and safer treatment option for B. bigemina.

Background/Objectives: B. bigemina is a highly pathogenic and widely distributed tick-borne disease parasite responsible for bovine babesiosis. The development of effective and safe therapies is urgently needed for global disease control. The aim of this study was to compare the effects of endochin-like quinolone (ELQ316), buparvaquone (BPQ), imidocarb (ID), and the combinations of ID + ELQ-316 and BPQ + ELQ-316, on in vitro survival of B. bigemina. Methods: Parasites at a starting parasitemia level of 2%, were incubated with each single drug and combination of drugs, ranging from 25 to 1200 nM of concentration over four consecutive days. The inhibitory concentration 50% (IC50%) and 99% (IC99%) were estimated. Parasitemia levels were evaluated daily using microscopic examination. Data were statistically compared using the non-parametrical KruskallWallis test. Results: All drugs tested significantly inhibited (p<0.05) the growth of B. bigemina at 2% parasitemia. The combination of ID + ELQ-316 exhibited lower mean IC50% (9.2); confidence interval 95% (8.7 – 9.9) than ID (IC50%: 61.5; confidence interval 95%: 59.54 - 63.46), ELQ-316 (IC50%: 48.10; confidence interval 95%: 42.76 – 58.83), BPQ (IC50%: 44.66; confidence interval 95%: 43.56 – 45.81), and BPQ + ELQ-316 (IC50%: 27.59; confidence interval: N/A). Parasites were no longer viable in cultures treated with the BPQ + ELQ-316 combination, as well as with BPQ alone at a concentration of 1200 nM, on days 2 and 3 of treatment, respectively.; Conclusions: BPQ and ID increase the babesiacidal effect of ELQ-316. The efficacy of these combinations deserves to be evaluated in vivo, which could lead to a promising and safer treatment option against B. bigemina.

Emergence and spread of parasite resistance to artemisinins, the first-line antimalarial therapy, threaten the malaria eradication policy. To identify therapeutic targets to eliminate artemisinin-resistant parasites, the functioning of the apicoplast and the mitochondrion was studied, focusing on the fatty acid synthesis type II (FASII) pathway in the apicoplast and the electron transfer chain in the mitochondrion. A significant enrichment of the FASII pathway among the up-regulated genes in artemisinin-resistant parasites under dihydroartemisinin treatment was found, in agreement with published transcriptomic data. However, using GC-MS analyzes of fatty acids, we demonstrated for the first time that the FASII pathway is non-functional, ruling out the use of FASII inhibitors to target artemisinin-resistant parasites. Conversely, by assessing the modulation of the oxygen consumption rate, we evidenced that mitochondrial respiration remains functional and flexible in artemisinin-resistant parasites and even at the quiescent stage. Two novel compounds targeting electron transport chain (ELQ300, ELQ400) efficiently killed quiescent artemisinin-resistant parasites. Therefore, mitochondrial respiration represents a key target for the elimination of artemisinin-resistant persistent Plasmodium falciparum parasites.

The overarching premise of this investigation is that injectable, long-acting antimalarial medication would encourage adherence to a dosage regimen for populations at risk of contracting the disease. To advance support for this goal, we have developed oil-based formulations of ELQ-331 (a prodrug of ELQ-300) that perform as long-acting, injectable chemoprophylactics with drug loading as high as 160 mg/ml of ELQ-331. In a pharmacokinetic study performed with rats, a single intramuscular injection of 12.14 mg/kg maintained higher plasma levels than the previously established minimum fully protective plasma concentration (33.25 ng/ml) of ELQ-300 for more than 4 weeks. The formulations were well tolerated by the rats and the tested dose produced no adverse reactions. We believe that by extending the length of time between subsequent injections, these injectable oil-based solutions of ELQ-331 can offer a more accessible, low-cost option for long-acting disease prevention and reduced transmission in malaria-endemic regions and may also be of use to travelers.

Introduction:B. bovis is an apicomplexan parasite responsible for bovine babesiosis, a tick-borne disease with a worldwide impact. The disease remains inefficiently controlled, and few effective drugs, including imidocarb dipropionate (ID), are currently available in endemic areas. The objective of this study was to evaluate whether buparvaquone (BPQ), a drug currently used to treat cattle infected with the Babesia-related Theileria spp. parasites, could be active against Babesia parasites. Herein, we compared the effect of ID and BPQ on B. bovis growth in vitro erythrocyte culture.Methods:We compared the effect of ID and BPQ on the culture-adapted Texas T2Bo strain of B. bovis. In vitro cultured parasites were incubated with ID and BPQ at two starting parasitemia levels (PPE), 0.2% and 1%. In vitro cultured parasites were treated with ID or BPQ at concentrations ranging from 10 to 300 nM, during 4 consecutive days. Parasitemia levels were daily evaluated using microscopic examination. Data was compared using the independent Student’s t-test.Results and discussion:Both ID and BPQ significantly inhibited (p < 0.05) the growth of B. bovis, regardless of the initial parasitemia used. At 1% parasitemia, BPQ had lower calculated inhibitory concentration 50 (IC50: 50.01) values than ID (IC50: 117.3). No parasites were found in wells with 0.2% starting parasitemia, treated previously with 50 nM of BPQ or ID, after 2 days of culture without drugs. At 1% parasitemia, no parasite survival was detected at 150 nM of BPQ or 300 nM of ID, suggesting that both drugs acted as babesiacidals.Conclusion:Overall, the data suggests that BPQ is effective against B. bovis and shows a residual effect that seems superior to ID, which is currently the first-line drug for treating bovine babesiosis globally.

Schistosomiasis caused by Schistosoma spp. is a disease that causes a considerable health burden to millions of people worldwide. The limited availability of effective drugs on the market and the increased risk of resistance development due to extensive usage, highlight the urgent need for new antischistosomal drugs. Recent studies have shown that robenidine derivatives, containing an aminoguanidine core, exhibit promising activities against Plasmodium falciparum, motivating further investigation into their efficacy against Schistosoma mansoni, due to their similar habitat and the resulting related cellular mechanisms like the heme detoxification pathway. The conducted phenotypic screening of robenidine and 80 derivatives against newly transformed schistosomula and adult Schistosoma mansoni yielded 11 candidates with low EC50 values for newly transformed schistosomula (1.12 - 4.63 μM) and adults (2.78 - 9.47 μM). The structure-activity relationship revealed that electron-withdrawing groups at the phenyl moiety, as well as the presence of methyl groups adjacent to the guanidine moiety, enhanced the activity of derivatives against both stages of Schistosoma mansoni. The two compounds 2,2’-Bis[(3-cyano-4-fluorophenyl)methylene] carbonimidic Dihydrazide Hydrochloride (1) and 2,2’-Bis[(4-difluoromethoxyphenyl) ethylidene] carbonimidic Dihydrazide Hydrochloride (19), were selected for an in vivo study in Schistosoma mansoni-infected mice based on their potency, cytotoxicity, pharmacokinetic-, and physicochemical properties, but failed to reduce the worm burden significantly (worm burden reduction < 60%). Thus, robenidine derivatives require further refinements to obtain higher antischistosomal specificity and in vivo activity.

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc1 complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.

Background/Objectives: Bovine babesiosis is a vector-borne disease transmitted by ticks that causes important losses in livestock worldwide. Recent research performed on the drugs currently used to control bovine babesiosis reported several issues including drug resistance, toxicity impact, and residues in edible tissue, suggesting the need for developing novel effective therapies. The endochin-like quinolones ELQ-316 and buparvaquone (BPQ) act as cytochrome bc1 inhibitors and have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp. and Babesia microti, without showing toxicity in mammals. The objectives of this study are investigating whether ELQ-316, BPQ, and their combination treatment could be effective against Babesia bovis in an in vitro culture model and comparing with imidocarb (ID), the routinely used drug. Methods: In vitro cultured parasites starting at 2% percentage of parasitemia (PPE) were treated with BPQ, ELQ-316, ID, and the combinations of BPQ + ELQ-316 and ID + ELQ-316 at drug concentrations that ranged from 25 to 1200 nM, during four consecutive days. The IC50% and IC99% were reported. Parasitemia levels were evaluated daily using microscopic examination. Data were compared using the non-parametrical Mann–Whitney and Kruskall–Wallis test. Results: All drugs tested, whether used alone or in combination, significantly decreased the survival (p < 0.05) of B. bovis in in vitro cultures. The combination of BPQ + ELQ-316 had the lowest calculated inhibitory concentration 50% (IC50%) values, 31.21 nM (IC95%: 15.06–68.48); followed by BPQ, 77.06 nM (IC95%: 70.16–86.01); ID + ELQ316, 197 nM (IC95%:129.0–311.2); ID, 635.1 nM (IC95%: 280.9–2119); and ELQ316, 654.9 nM (IC95%: 362.3–1411). Conclusions: The results reinforce the higher efficacy of BPQ at affecting B. bovis survival and the potential synergistic effects of its combination with ELQ-316, providing a promising treatment option against B. bovis.

Malaria continues to be a serious and debilitating disease. The emergence and spread of high-level resistance to multiple antimalarial drugs by Plasmodium falciparum has brought about an urgent need for new treatments that will be active against multidrug resistant malaria infections. One such treatment, ELQ-331 (MMV-167), an alkoxy carbonate prodrug of 4(1H)-quinolone ELQ-300, is currently in preclinical development with the Medicines for Malaria Venture. Clinical development of ELQ-331 or similar compounds will require the availability of isotopically labeled analogs. Unfortunately, a suitable method for the deuteration of these important compounds was not found in the literature. Here, we describe a facile and scalable method for the deuteration of 4(1H)-quinolone ELQ-300, its alkoxycarbonate prodrug ELQ-331, and their respective N-oxides using deuterated acetic acid.

ELQ-300 is a potent antimalarial drug with activity against blood, liver, and vector stages of the disease. A prodrug, ELQ-331, exhibits reduced crystallinity and improved in vivo efficacy in preclinical testing, and currently, it is in the developmental pipeline for once-a-week dosing for oral prophylaxis against malaria. Because of the high cost of developing a new drug for human use and the high risk of drug failure, it is prudent to have a back-up plan in place. Here we describe ELQ-596, a member of a new subseries of 3-biaryl-ELQs, with enhanced potency in vitro against multidrug-resistant Plasmodium falciparum parasites. ELQ-598, a prodrug of ELQ-596 with diminished crystallinity, is more effective vs murine malaria than its progenitor ELQ-331 by 4- to 10-fold, suggesting that correspondingly lower doses could be used to protect and cure humans of malaria. With a longer bloodstream half-life in mice compared to its progenitor, ELQ-596 highlights a novel series of next-generation ELQs with the potential for once-monthly dosing for protection against malaria infection. Advances in the preparation of 3-biaryl-ELQs are presented along with preliminary results from experiments to explore key structure-activity relationships for drug potency, selectivity, pharmacokinetics, and safety.

Background/Objectives: Bovine babesiosis is a vector-borne disease transmitted by ticks that causes important losses in livestock worldwide. Recent research performed on the drugs currently used to control bovine babesiosis reported several issues including drug resistance, toxicity impact, and residues in edible tissue, suggesting the need for developing novel effective therapies. The endochin-like quinolones ELQ-316 and buparvaquone (BPQ) act as cytochrome bc1 inhibitors and have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp. and Babesia microti, without showing toxicity in mammals. The objectives of this study are investigating whether ELQ-316, BPQ and their combination treatment could be effective against Babesia bovis in an in vitro culture model, and comparing with imidocarb (ID), the routinely used drug. Methods: In vitro cultured parasites at starting 2% percentage of parasitemia (PPE) were treated with BPQ, ELQ-316, ID, and the combinations of BPQ+ ELQ-316 and ID+ ELQ-316 at drug concentrations that ranged from 25 to 1200 nM, during four consecutive days. Parasitemia levels were evaluated daily using microscopic examination. Data was compared using the independent Student's t-test and ANOVA test. Results: All drugs tested, whether used alone or in combination, significantly decreased the survival (p<0.05) of B. bovis in in vitro cultures. The combination of BPQ+ ELQ-316 had the lowest calculated inhibitory concentration 50 (IC50) 31.21 nM (IC95%: 15.06 - 68.48) values, followed by BPQ, 77.06 nM (IC95%: 70.16- 86.01), ID+ELQ316 197 nM (IC95%:129.0 - 311.2), ID 635.1 nM (IC95%: 280.9 – 2119) and ELQ316 654.9 nM (IC95%: 362.3 – 1411). Conclusion: The results reinforce the higher efficacy of BPQ at affecting B. bovis survival, and the potential synergistic effects of its combination with ELQ-316, providing a promising treatment option against B. bovis.