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Background The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. Methods We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. Results Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. Conclusions Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Trypanosoma and Leishmania parasites are the etiological agents of various threatening neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination therapy eflornithine–nifurtimox. Nevertheless, due to drug resistance issues and the exitance of animal reservoirs, the development of new NTD treatments is still required. For this purpose, we explored the combined targeting of two key folate enzymes, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). We formerly showed that the TbDHFR inhibitor cycloguanil (CYC) also targets TbPTR1, although with reduced affinity. Here, we explored a small library of CYC analogues to understand how their substitution pattern affects the inhibition of both TbPTR1 and TbDHFR. Some novel structural features responsible for an improved, but preferential, ability of CYC analogues to target TbPTR1 were disclosed. Furthermore, we showed that the known drug pyrimethamine (PYR) effectively targets both enzymes, also unveiling its binding mode to TbPTR1. The structural comparison between PYR and CYC binding modes to TbPTR1 and TbDHFR provided key insights for the future design of dual inhibitors for HAT therapy.

Atovaquone-proguanil (ATV-PG) plus amodiaquine (AQ) has been considered as a potential replacement for sulfadoxine-pyrimethamine plus AQ for seasonal malaria chemoprevention in African children. This randomized, double-blind, placebo-controlled, parallel group study assessed the safety, tolerability, and pharmacokinetics (PKs) of ATV-PG plus AQ in healthy adult males and females of Black sub-Saharan African origin. Participants were randomized to four treatment groups: ATV-PG/AQ (n = 8), ATV-PG/placebo (n = 12), AQ/placebo (n = 12), and placebo/placebo (n = 12). Treatments were administered orally once daily for 3 days (days 1-3) at daily doses of ATV-PQ 1000/400 mg and AQ 612 mg. Co-administration of ATV-PG/AQ had no clinically relevant effect on PK parameters for ATV, PG, the PG metabolite cycloguanil, AQ, or the AQ metabolite N-desethyl-amodiaquine. Adverse events occurred in 8 of 8 (100%) of participants receiving ATV-PG/AQ, 11 of 12 (91.7%) receiving ATV-PG, 11 of 12 (91.7%) receiving AQ, and 3 of 12 (25%) receiving placebo. The safety and tolerability profiles of ATV-PG and AQ were consistent with previous reports. In the ATV-PG/AQ group, 2 of 8 participants experienced extrapyramidal adverse effects (EPAEs) on day 3, both psychiatric and physical, which appeared unrelated to drug plasma PKs or cytochrome P450 2C8 phenotype. Although rare cases are reported with AQ administration, the high incidence of EPAE was unexpected in this small study. Owing to the unanticipated increased frequency of EPAE observed, the combination of ATV-PQ plus AQ is not recommended for further evaluation in prophylaxis of malaria in African children.

Available antimalarial drugs have been associated with numerous side effects, which include skin rashes and myelo-suppression. Therefore, it is of interest to explore compounds from natural source having drug-like properties without side effect. This study focuses on the screening of compounds from Cannabis sativa against malaria Plasmodium falciparum dihydrofolate reductase for antimalarial properties using Glide (Schrodinger maestro 2018-1). The result showed that phytochemicals from Cannabis sativa binds with a higher affinity and lower free energy than the standard ligand with isovitexin and vitexin having a glide score of -11.485 and -10.601 respectively, sophoroside has a glide score of -9.711 which is lower than the cycloguanil (co-crystallized ligand) having a glide score of -6.908. This result gives new perception to the use of Cannabis sativa as antimicrobial agent.

The role of organic cation transporter 1 (OCT1) in humans is gaining attention as data emerges regarding its role in physiology, drug exposure, and drug response. OCT1 variants with decreased in vitro function correlate well with altered exposure of multiple OCT1 substrates in variant carriers. In the current research, we investigate mechanisms behind activity of OCT1 variants in vitro by generating cell lines expressing known OCT1 variants and quantifying membrane OCT1 protein expression with corresponding OCT1 activity and kinetics. Oct knockout mice have provided additional insight into the role of Oct1 in the liver and have reproduced effects of altered OCT1 activity observed in the clinic. To assess the complex effect of Oct1 depletion on pharmacokinetics of prodrug proguanil and its active moiety cycloguanil, both of which are OCT1 substrates, Oct1/2−/− mice were used. Decreased membrane expression of OCT1 was demonstrated for all variant cell lines, although activity was substrate-dependent, as reported previously. Lack of change in activity for OCT1*2 resulted in increased intrinsic activity per pmol of OCT1 protein, particularly for sumatriptan but also for proguanil and cycloguanil. Similar to that reported in humans with decreased OCT1 function, systemic exposure of proguanil was minimally affected in Oct1/2−/− mice. However, proguanil liver partitioning and exposure decreased. Cycloguanil exposure decreased following proguanil administration in Oct1/2−/− mice, as did the systemic metabolite:parent ratio. When administered directly, systemic exposure of cycloguanil decreased slightly; however liver partitioning and exposure were decreased in Oct1/2−/− mice. Unexpectedly, following proguanil administration, the metabolite ratio in the liver changed only minimally, and liver partitioning of cycloguanil was affected in Oct1/2−/− mice to a lesser extent following proguanil administration than direct administration of cycloguanil. In conclusion, these in vitro and in vivo data offer additional complexity in understanding mechanisms of OCT1 variant activity as well as the effects of these variants in vivo. From cell lines, it is apparent that intrinsic activity is not directly related to OCT1 membrane expression. Additionally, in situations with a more complicated role of OCT1 in drug pharmacokinetics there is difficulty translating in vivo impact simply from intrinsic activity from cellular data.

Background The drug combination atovaquone–proguanil, is recommended for treatment of uncomplicated falciparum malaria in France. Despite high efficacy, atovaquone–proguanil treatment failures have been reported. Resistance to cycloguanil, the active metabolite of proguanil, is conferred by multiple mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and resistance to atovaquone by single mutation on codon 268 of the cytochrome b gene (pfcytb). Case presentation A 47-year-old female, native from Congo and resident in France, was admitted in hospital for uncomplicated falciparum malaria with parasitaemia of 0.5%, after travelling in Congo (Brazzaville and Pointe Noire). She was treated with atovaquone–proguanil (250 mg/100 mg) 4 tablets daily for 3 consecutive days. On day 5 after admission she was released home. However, many weeks after this episode, without having left France, she again experienced fever and intense weakness. On day 39 after the beginning of treatment, she consulted for fever, arthralgia, myalgia, photophobia, and blurred vision. She was hospitalized for uncomplicated falciparum malaria with a parasitaemia of 0.375% and treated effectively by piperaquine–artenimol (320 mg/40 mg) 3 tablets daily for 3 consecutive days. Resistance to atovaquone–proguanil was suspected. The Y268C mutation was detected in all of the isolates tested (D39, D42, D47). The genotyping of the pfdhfr gene showed a triple mutation (N51I, C59R, S108N) involved in cycloguanil resistance. Conclusion This is the first observation of a late clinical failure of atovaquone–proguanil treatment of P. falciparum uncomplicated malaria associated with pfcytb 268 mutation in a traveller returning from Congo. These data confirm that the Y268C mutation is associated with delayed recrudescence 4 weeks or more after initial treatment. Although atovaquone–proguanil treatment failures remain rare, an increased surveillance is required. It is essential to declare and publish all well-documented cases of treatment failures because it is the only way to evaluate the level of resistance to atovaquone.

Malarija je oduvijek bila glavni uzrok smrti u tropskim krajevima, a antimalarijski lijekovi odigrali su ključnu ulogu u kontroli njezina širenja primjenom u liječenju pacijenata zaraženih plazmodijskim parazitima i prilikom kontrole njihovog prijenosa. S druge strane, antimalarijski lijekovi mogu imati i nepoželjne učinke i nuspojave koje ponekad mogu biti ozbiljne. Atovakvon (ATO) i progvanil hidroklorid (PROG) fiksna je kombinacija lijekova koja se koristi za prevenciju i liječenje malarije uzrokovane Plasmodium falciparum. Kako su sigurni i učinkoviti antimalarijski lijekovi potrebni u liječenju i profilaksi malarije, ovo istraživanje je provedeno kako bi se istražio njihov mogući citogenotoksični učinak te mehanizam odgovoran za njihovo djelovanje na ne-ciljnim ljudskim stanicama, limfocitima iz periferne krvi. Dvije različite koncentracije ATO i PROG, bilo zasebno ili u kombinaciji, korištene su sa i bez S9 frakcije. Korištene koncentracije bile su one koje su pronađene u ljudskoj plazmi kada su ATO i PROG primijenjeni u profilaksi (2950/130 ng/mL) i u liječenju malarije (11800/520 ng/mL). Mogući učinci na staničnoj razini i na molekulu DNK procijenjeni su testovima stanične vijabilnosti i alkalnim komet testom, dok je mogući mehanizam djelovanja procijenjen formamidopirimidin-DNK glikozilazom (Fpg)-modificiranim komet testom, uz mjerenje razine malondialdehida i glutationa kao biomarkera oksidacijskog stresa. Prema dobivenim rezultatima pojedinačni tretman ATO nije pokazao citogenotoksični učinak, dok je pojedinačni tretman PROG pokazao statistički značajan citogenotoksični potencijal. Kombinacija ATO/PROG također je pokazala statistički značajan citogenotoksični potencijal prema ljudskim limfocitima bez utjecaja na parametre oksidacijskog stresa, što pokazuje da oksidacijski stres nije uključen u njihov mehanizam djelovanja. S obzirom da je najveći dio štetnih učinaka bio induciran dodatkom S9 frakcije, pretpostavlja se da je glavni metabolit PROG, ciklogvanil, imao najveći utjecaj na citogenotoksičnost. U konačnici, dobiveni rezultati pokazuju da je kombinacija ATO/PROG relativno sigurna sa stanovišta citogenotoksičnosti, naročito ako se koristi za profilaksu. Ipak, potrebna su daljnja citogenetička istraživanja i redovito praćenje pacijenata kako bi se smanjio rizik od nuspojava, osobito među čestim putnicima. Malaria has always been a major cause of death in the tropics, and antimalarial drugs have played a key role in controlling its spread through the treatment of patients infected with plasmodial parasites and the control of its transmissibility. On the other hand, antimalarial drugs may exert adverse effects and side-effects that can sometimes be serious. Atovaquone (ATO) and proguanil hydrochloride (PROG) is the fixed combination for the prevention and treatment of Plasmodium falciparum malaria. As safe and effective antimalarial drugs are needed in both the treatment and the prophylaxis of malaria, this study was performed in order to investigate their possible cytogenotoxic potential towards human lymphocytes, as non-target cells, and the possible mechanism responsible for it. Two different concentrations of ATO and PROG, either alone or in combination, were used with and without S9 fraction. The concentrations used were those found in human plasma when a fixed-dose combination of ATO and PROG was used: 2950/130 ng/mL after prophylactic treatment and 11800/520 ng/mL after treatment of malaria, respectively. Possible cellular and DNA-damaging effects were evaluated by cell viability and alkaline comet assays, while oxidative stress potential was evaluated by formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assay, in addition to assessing malondialdehyde and glutathione levels as biomarkers of oxidative stress. Based on the obtained results, single exposure to ATO did not display cytogenotoxic effect while PROG showed week but significant cytogenotoxic potential. The ATO/PROG combination also displayed weak but significant cytogenotoxic potential towards human lymphocytes with no impact on oxidative stress parameters, suggesting that oxidative stress is not implicated in their mechanism of action. Given that the key portion of the damaging effects was induced after S9 metabolic activation, it is to presume that the principal metabolite of PROG, cycloguanil, had the greatest impact on their cytogenotoxic potential. The obtained results indicate that the ATO/PROG combination is relatively safe for the consumption from the aspect of cytogenotoxicity, especially if used for prophylaxis. Nevertheless, further cytogenetic research and regular patient monitoring are warranted to minimize the risk of adverse events especially among frequent travellers.

Dihydrofolate reductase (DHFR) is an important enzyme for de novo synthesis of nucleotides in Plasmodium falciparum and it is essential for cell proliferation. DHFR is a well known antimalarial target for drugs like cycloguanil and pyrimethamine which target its inhibition for their pharmacological actions. However, the clinical efficacies of these antimalarial drugs have been compromising due to multiple mutations occurring in DHFR that lead to drug resistance. In this background, we have designed 22 s -triazine compounds using the best five parameters based 3D-QSAR model built by using genetic function approximation. In-silico designed compounds were further filtered to 6 compounds based upon their ADME properties, docking studies and predicted minimum inhibitory concentrations (MIC). Out of 6 compounds, 3 compounds were synthesized in good yield over 95% and characterized using IR, 1HNMR, 13CNMR and mass spectroscopic techniques. Parasitemia inhibition assay was used to evaluate the antimalarial activity of s -triazine compounds against 3D7 strain of P. falciparum. All the three compounds (7, 13 and 18) showed 30 times higher potency than cycloguanil (standard drug). It was observed that compound 18 was the most active while the compound 13 was the least active. On the closer inspection of physicochemical properties and SAR, it was observed that the presence of electron donating groups, number of hydrogen bond formation, lipophilicity of ligands and coulson charge of nitrogen atom present in the triazine ring enhances the DHFR inhibition significantly. This study will contribute to further endeavours of more potent DHFR inhibitors. Electronic supplementary material The online version of this article (10.1186/s13065-017-0362-5) contains supplementary material, which is available to authorized users.

Background The simian malaria parasite Plasmodium knowlesi is now a well-recognized pathogen of humans in South-East Asia. Clinical infections appear adequately treated with existing drug regimens, but the evidence base for this practice remains weak. The availability of P. knowlesi cultures adapted to continuous propagation in human erythrocytes enables specific studies of in vitro susceptibility of the species to antimalarial agents, and could provide a surrogate system for testing investigational compounds against Plasmodium vivax and other non-Plasmodium falciparum infections that cannot currently be propagated in vitro. Objectives We sought to optimize protocols for in vitro susceptibility testing of P. knowlesi and to contrast outputs with those obtained for P. falciparum under comparable test conditions. Methods Growth monitoring of P. knowlesi in vitro was by DNA quantification using a SYBR Green fluorescent assay or by colorimetric detection of the lactate dehydrogenase enzyme. For comparison, P. falciparum was tested under conditions identical to those used for P. knowlesi. Results The SYBR Green I assay proved the most robust format over one (27 h) or two (54 h) P. knowlesi life cycles. Unexpectedly, P. knowlesi displays significantly greater susceptibility to the dihydrofolate reductase inhibitors pyrimethamine, cycloguanil and trimethoprim than does P. falciparum, but is less susceptible to the selective agents blasticidin and DSM1 used in parasite transfections. Inhibitors of dihydroorotate dehydrogenase also demonstrate lower activity against P. knowlesi. Conclusions The fluorescent assay system validated here identified species-specific P. knowlesi drug susceptibility profiles and can be used for testing investigational compounds for activity against non-P. falciparum malaria.

Malaria is still one of the most serious diseases in tropical regions. This is due in part to the high resistance against available drugs for the inhibition of parasites, Plasmodium, the cause of the disease. New potent compounds with high clinical utility are urgently needed. In this work, we created a novel model using a regression tree to study structure-activity relationships and predict the inhibition constant, Ki of three different antimalarial analogues (Trimethoprim, Pyrimethamine, and Cycloguanil) based on their molecular descriptors. To the best of our knowledge, this work is the first attempt to study the structure-activity relationships of all three analogues combined. The most relevant descriptors and appropriate parameters of the regression tree are harvested using extremely randomized trees. These descriptors are water accessible surface area, Log of the aqueous solubility, total hydrophobic van der Waals surface area, and molecular refractivity. Out of all possible combinations of these selected parameters and descriptors, the tree with the strongest coefficient of determination is selected to be our prediction model. Predicted Ki values from the proposed model show a strong coefficient of determination, R2=0.996, to experimental Ki values. From the structure of the regression tree, compounds with high accessible surface area of all hydrophobic atoms (ASA_H) and low aqueous solubility of inhibitors (Log S) generally possess low Ki values. Our prediction model can also be utilized as a screening test for new antimalarial drug compounds which may reduce the time and expenses for new drug development. New compounds with high predicted Ki should be excluded from further drug development. It is also our inference that a threshold of ASA_H greater than 575.80 and Log S less than or equal to -4.36 is a sufficient condition for a new compound to possess a low Ki.

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil’s action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo., Tina Skinner-Adams et al. show that proguanil and the cyclization-blocked analogue, tBuPG, have potent, anti-plasmodial activity in vitro. This study suggests that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors to suppress the survival of malarial parasites.

Aims Vonoprazan, a new class of potassium-competitive proton pump inhibitors has been found to attenuate the antiplatelet function of clopidogrel in a recent clinical study, despite weak in vitro activity against CYP2C19. To elucidate the mechanism of this interaction, the present study investigated the effects of esomeprazole and vonoprazan on the pharmacokinetics of proguanil, a CYP2C19 substrate. Methods Seven healthy male volunteers (CYP2C19 extensive metabolizers) received a single oral administration of 100 mg proguanil/250 mg atovaquone (control phase), oral esomeprazole (20 mg) for 5 days followed by proguanil/atovaquone (esomeprazole phase) and oral vonoprazan (20 mg) for 5 days followed by proguanil/atovaquone (vonoprazan phase). Concentrations of proguanil and its metabolite, cycloguanil, in plasma and urine in each phase were determined using liquid chromatography-tandem mass spectrometry. Results Coadministration with proton pump inhibitors resulted in increase and decrease in the area under the plasma concentration-time curve (AUC) of proguanil and cycloguanil, respectively, significantly reducing their AUC ratio (cycloguanil/proguanil) to 0.317-fold (95% confidence interval [CI] 0.256-0.379) and 0.507-fold (95% CI 0.409-0.605) in esomeprazole phase and vonoprazan phase, respectively. Esomeprazole and vonoprazan also significantly reduced the apparent formation clearance (cumulative amount of cycloguanil in urine divided by AUC of proguanil) to 0.324-fold (95% CI 0.212-0.436) and 0.433-fold (95% CI 0.355-0.511), respectively, without significant changes in renal clearance of proguanil and cycloguanil. Conclusions Although further studies are needed, both esomeprazole and vonoprazan potentially inhibit CYP2C19 at clinical doses, suggesting caution in the coadministration of these drugs with CYP2C19 substrates.

Background Recent artemisinin-combination therapy failures in Cambodia prompted a search for alternatives. Atovaquone-proguanil (AP), a safe, effective treatment for multidrug-resistant Plasmodium falciparum (P.f.), previously demonstrated additive effects in combination with artesunate (AS). Methods Patients with P.f. or mixed-species infection (n = 205) in Anlong Veng (AV; n = 157) and Kratie (KT; n = 48), Cambodia, were randomized open-label 1:1 to a fixed-dose 3-day AP regimen +/-3 days of co-administered artesunate (ASAP). Single low-dose primaquine (PQ, 15 mg) was given on day 1 to prevent gametocyte-mediated transmission. Results Polymerase chain reaction–adjusted adequate clinical and parasitological response at 42 days was 90% for AP (95% confidence interval [CI], 82%–95%) and 92% for ASAP (95% CI, 83%–96%; P = .73). The median parasite clearance time was 72 hours for ASAP in AV vs 56 hours in KT (P < .001) and was no different than AP alone. At 1 week postprimaquine, 7% of the ASAP group carried microscopic gametocytes vs 29% for AP alone (P = .0001). Nearly all P.f. isolates had C580Y K13 propeller artemisinin resistance mutations (AV 99%; KT 88%). Only 1 of 14 treatment failures carried the cytochrome bc1 (Pfcytb) atovaquone resistance mutation, which was not present at baseline. P.f. isolates remained atovaquone sensitive in vitro but cycloguanil resistant, with a triple P.f. dihydrofolate reductase mutation. Conclusions Atovaquone-proguanil remained marginally effective in Cambodia (≥90%) with minimal Pfcytb mutations observed. Treatment failures in the presence of ex vivo atovaquone sensitivity and adequate plasma levels may be attributable to cycloguanil and/or artemisinin resistance. Artesunate co-administration provided little additional blood-stage efficacy but reduced post-treatment gametocyte carriage in combination with AP beyond single low-dose primaquine.

A series of antifolate compounds, i.e. 1-(4-chlorophenyl)-6,6-dimethyl-1,3,5-triazine-2,4-diamine, or cycloguanil analogues, have shown effective inhibiting properties against Plasmodium falciparum dihydrofolate reductase (PfDHFR). In this work, the stereoselectivity of PfDHFR to the R and S enantiomer of cycloguanil analogues was obtained from molecular docking calculations and integrated into QSAR study to obtain a more accurate prediction model. Results indicate that PfDHFR can bind to cycloguanil analogues in the R and S enantiomers. Cycloguanil analogues with alkyl chain substituent prefer the R enantiomer over S because they do not experience steric hindrance with the Phe58 side chain, while cycloguanil analogues with phenol chain substituent prefer the S enantiomer over R because they do not experience steric hindrance with Leu46 and Met55 side chains. Particle swarm optimization and support vector regression were used to select relevant descriptors and generate the effective prediction model, with a high statistical significance level (r2training = 0.941; r2test = 0.884).

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50 values significantly lower than metformin, (range 0.054 and 0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.

The folate biosynthetic pathway and its key enzyme dihydrofolate reductase (DHFR) is a popular target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. Despite its importance, little is known about plant DHFRs, which, like the enzymes from the malarial parasite Plasmodium, are bifunctional, possessing DHFR and thymidylate synthase (TS) domains. Here using genetic knockout lines we confirmed that either DHFR-TS1 or DHFR-TS2 (but not DHFR-TS3) was essential for seed development. Screening mutated Arabidopsis thaliana seeds for resistance to antimalarial DHFR-inhibitor drugs pyrimethamine and cycloguanil identified causal lesions in DHFR-TS1 and DHFR-TS2, respectively, near the predicted substrate-binding site. The different drug resistance profiles for the plants, enabled by the G137D mutation in DHFR-TS1 and the A71V mutation in DHFR-TS2, were consistent with biochemical studies using recombinant proteins and could be explained by structural models. These findings provide a great improvement in our understanding of plant DHFR-TS and suggest how plant-specific inhibitors might be developed, as DHFR is not currently targeted by commercial herbicides.

Development of 2, N6-disubstituted 1,2-dihydro-1,3,5-trizine-4,6-diamines derivatives were carried out by quantitative structure–activity relationship (QSAR) analyses. The nature of the substituent(s) on C-2; the nature of the substituent(s) on the distal aryl ring; as well as the nature and length of the flexible tether between the rings, to find out the structural requirements of their antimalarial activities against cycloguanil resistant (FCR-3) Plasmodium falciparum strain and sensitive to pyrimethamine. The statistically significant best 2D QSAR models for FCR-3, having correlation coefficient (r2) = 0.9821 and cross validated squared correlation coefficient (q2) = 0.6471 were developed by multiple linear regression stepwise (SW–MLR) forward algorithm. The results of the present study may be useful on the designing of more potent analogues as antimalarial agents.

Objective: The aim of the present study is to develop HPLC method for simultaneousquantification of atovaquone and proguanil with its active metabolite cycloguanilin human plasma. Methodology: A specific and accurate highperformance liquid chromatographic method has been developed using Phenyl (150×4.6 mm, 5 µm) column maintained at 18 °C. The separation was achieved using a mobile phase composed of phosphate buffer pH 7.2 and methanol (45:55%). The mobile phase was maintained at flow rate of 0.8 mL/min. The analytes were monitored at 254 nm usingultra-violet detector. The plasma samples extractions was carried out using tert-Butyl Methyl Ether: Dichloromethane (80:20% v/v) mixture. Tramadol was used as an internal standard (ISTD). Result: The developed method was validated as per US FDA guidelines and found to be highly specific, precise and accurate. Moreover, atovaquone, proguanil and cycloguanilwere stable in plasma at various stability conditions. Conclusion: The developed method is simple, economic, and can be used for quantification of said drugs human plasma samples.

The evolutionary relationship between plants and the malarial parasite Plasmodium falciparum is well established and underscored by the P. falciparum apicoplast, an essential chloroplast-like organelle. As a result of this relationship, studies have demonstrated that herbicides active against plants are also active against P. falciparum and thus could act as antimalarial drug leads. Here we show the converse is also true; many antimalarial compounds developed for human use are highly herbicidal. We found that human antimalarial drugs (e.g. sulfadiazine, sulfadoxine, pyrimethamine, cycloguanil) were lethal to the model plant Arabidopsis thaliana at similar concentrations to market herbicides glufosinate and glyphosate. Furthermore, the physicochemical properties of these herbicidal antimalarial compounds were similar to commercially used herbicides. The implications of this finding that many antimalarial compounds are herbicidal proffers two novel applications: (i) using the genetically tractable A. thaliana to reveal mode-of-action for understudied antimalarial drugs, and (ii) co-opting antimalarial compounds as a new source for much needed herbicide lead molecules.

We have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza A and B virus and respiratory syncytial virus (RSV) via targeting of the host dihydrofolate reductase (DHFR) enzyme. Most analogues proved active against influenza B virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (EC50 = 0.060 μM), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. Activity was also observed for two influenza A strains, including a virus with the S31N mutant form of M2 proton channel, which is the most prevalent resistance mutation for amantadine. Importantly, the compounds displayed nanomolar activity against RSV and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (EC50 ∼0.008 μM), far surpassing the potency and safety profile of the licensed drug ribavirin (EC50 = 5.8 μM, SI > 43)., Graphical abstract Image 1, Highlights • 4,6-Diamino-1,2-dihydrotriazines were found active against influenza A and B viruses. • 4,6-Diamino-1,2-dihydrotriazines displayed high antiviral activity against RSV virus. • Their mechanism of action was related to the inhibition of the host (human) DHFR.

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [(3)H]granisetron. Kinetic measurements (kon = 4.0 × 10(4) M(-1) s(-1) ; koff = 0.23 s(-1)) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

SummaryInhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.

Combinations of dapsone with proguanil or chlorproguanil have proved effective in the treatment of chloroquine-resistant falciparum malaria in Africa and for prophylaxis in Asia. These combinations have not been used for treatment in areas with multi-drug-resistant parasites such as in Thailand. Combinations of dapsone (approximately 4 mg/kg) plus ether proguanil (approximately 8 mg/kg; DP regimen; N = 10) or chlorproguanil (approximately 1.4 mg/kg; DC regimen; N = 16) were given once a day for 3 days to adult Thai patients with acute, uncomplicated, falciparum malaria. The two regimens were well tolerated and had no side-effects, but the cure rates, assessed at 28-day follow-up, were only 10% for DP (60% with RI response and 30% with RII) and 14% for DC (29% with RI response and 57% with RII). The mean (S.D.) fever-clearance times in those patients who were cured (S) or whose infections recrudesced (RI response) were 103 (56) h for those given DP and 90 (42) h for 6 those given DC. The corresponding parasite-clearance times were 83 (46) for DP and 53 (21) h for DC. In-vitro susceptibility testing of isolates obtained both before treatment and at recrudescence demonstrated marked resistance to cycloguanil, dapsone, chloroquine and mefloquine. The results demonstrate that short-course treatment with dapsone plus either proguanil or chlorproguanil is ineffective for the treatment of falciparum malaria in Thailand.

OBJECTIVE: To determine the effects of late pregnancy and also oestrogen supplementation on the CYP2C19-mediated biotransformation of proguanil (PG) to its active antifol triazine metabolite cycloguanil (CG). METHODS: Case control study conducted on the NW border of Thailand; a single dose of PG (4 mg/kg) was administered to Karen women in late pregnancy and a single blood and urine sample taken 6 h later. Women were studied in late pregnancy (>36 weeks) and restudied 2 months after delivery. A separate cohort of Karen women newly attending a birth-control clinic were studied before and 3 weeks into their first course of oral contraceptives (OCP: levonorgestrel 0.15 mg and ethinyloestradiol 0.03 mg). Forty-five pregnant women and forty-two healthy OCP users were studied. RESULTS: The results were similar in both groups; pregnancy and OCP use were both associated with reduced formation of cycloguanil (CG). Impaired PG biotransformation was seen in women with the "extensive metaboliser" phenotype (urine PG/CG ratio

1. Plasma and whole blood concentrations of proguanil and its two major metabolites cycloguanil (CG) and 4-chlorophenylbiguanide (CPB) were measured by a sensitive h.p.l.c. technique in nine healthy adult male volunteers after a single oral dose of proguanil 200 mg. 2. Proguanil was absorbed with a median time to peak plasma concentration of 3 h (range 2-4 h). 3. Peak plasma concentrations of proguanil ranged between 150 and 220 (median 170) ng ml-1 compared with 12 to 69 (median 41) ng ml-1 for the active antimalarial metabolite CG, and 3 to 16 (median 11) ng ml-1 for CPB. Peak (mean +/- s.d.) plasma CG concentrations occurred 5.3 +/- 0.9 h and peak CPB concentrations occurred 6.3 +/- 1.4 h after oral administration of proguanil. 4. Whole blood concentrations of proguanil were approximately five times higher, and whole blood CPB concentrations were four times higher than corresponding plasma values, whereas plasma and whole blood concentrations of CG were similar. 5. A triexponential function was fitted to these data; mean (+/- s.d.) values for the AUC were 3046 +/- 313 ng ml-1 h for proguanil, 679 +/- 372 ng ml-1 h for CG and 257 +/- 155 ng ml-1 h for CPB. 6. Plasma and whole blood concentrations of proguanil and its metabolites declined in parallel with terminal elimination half-lives estimated as 16.1 +/- 2.9 h and 15.7 +/- 2.4 h, respectively. Mean residence times in plasma and whole blood were estimated as 21.2 +/- 4.9 and 19.3 +/- 2.4 h.

Chlorproguanil-dapsone is a recently released antimalarial drug in which two long-established compounds are formulated in a fixed combination. Proguanil which has similar pharmacological properties to chlorproguanil is a biguanide that was introduced originally in 1945 and has been widely used in antimalarial prophylaxis ever since. Dapsone a sulphone has been the mainstay of leprosy treatment for over half a century. Like other sulpha drugs dapsone inhibits dihydropteroate synthase in the malaria parasite an initial step in folate biosynthesis. Chlorproguanil and proguanil are metabolised in vivo by the polymorphic CYP2C19 to cyclic triazines (chlorcycloguanil and cycloguanil respectively) which inhibit dihydrofolate reductase in the parasite. The two compounds thus provide sequential blockade of folate synthesis and are highly synergistic. The more widely used antifol-sulpha combination sulphadoxine-pyrimethamine acts in the same way. (excerpt)

The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [ 3 H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC 50 s) consistent with those obtained with P. falciparum . This assay provides a platform to use P. knowlesi in vitro for drug discovery.

The interaction between atovaquone and proguanil has never been studied against liver stage malaria, which is the main target of this drug combination when used for chemoprevention. Using human hepatocytes lacking cytochrome P450 activity, and thus avoiding proguanil metabolizing into potent cycloguanil, we show in vitro that the atovaquone-proguanil combination synergistically inhibits the growth of rodent Plasmodium yoelii parasites. These results provide a pharmacological basis for the high efficacy of atovaquone-proguanil used as malaria chemoprevention.

The importance of genomic variation in the biotransformation of drugs is well known. Only in the last two decades, the big importance of drug membrane transporters and of genomic variation in their genes became apparent. For this reason and because of the importance in drug-drug interactions, the identification of the relevant membrane transporters has meanwhile become an essential part of pre-clinical and early clinical drug development. Particularly for hydrophobic drugs the ATP consuming efflux transporters like the multidrug resistance transporter 1 (MDR1) are highly important. In addition to the efflux transporters, there are influx transport proteins which accelerate the uptake of hydrophilic cationic molecules into the cell. One group of these influx transporters are the organic cation transporters (OCTs). In order to measure substrate concentrations in transport experiments within the cell, the analytical method of the high pressure liquid chromatography (HPLC) is the most commonly used procedure alongside to the still widespread use of radioactive labels. However, not every previously published method can be easily transfered to the existing conditions. The specific challenges of this present work resulted from the small volumes and low concentrations to be analyzed in the transport experiments. The thesis had the following objectives: Firstly, the HPLC methods for quantitative analysis of drugs such as proguanil, cycloguanil, amisulpride and debrisoquine for membrane transport experiments should be optimized and validated. The cellular uptake of proguanil and cycloguanil should be measured in HEK293 cells which overexpress the organic cation transporters OCT1, -2, and -3 and OCTN1 and -N2. The data should be further validated by inhibition of the transport using typical inhibitors of OCTs and drugs relevant as comedication. In addition, the impact on the relevant inherited variants of organic cation transporters should be analyzed. Finally, the transport of proguanil into non-infected erythrocytes should be characterized. This should include inhibition experients with prototypical inhibitors of organic cation transporters and with substances relevant as co-medication. 1. Einleitung 36 Based on validation and optimization of HPLC methods, the concentration of the drugs proguanil, cycloguanil, amisulpride and debrisoquine could be determined quantitatively. The drugs were measured and detected in the low nanogram/ml concentration range. In-vitro experiments showed that the antimalarial drug proguanil and its active metabolite cycloguanil can be transported with different maximum transport velocities and affinities by the electrogenic organic cation transporters OCT1, OCT2 and OCT3. In contrast to them, the organic cation transporter OCTN1 and OCTN2 overexpressed in HEK cells didn´t show any increase in the cellular uptake of proguanil and cycloguanil. Selected inherited variants of OCT1 existing in Caucasian populations with a relatively high frequency and differing in their transport functions with many other drug also showed altered uptake of the drug proguanil and its active metabolite cycloguanil here. Coincubation with other drugs or model substrates in different concentrations showed significant reductions in the transport of proguanil and cycloguanil by OCT1. In particular, transport activity was reduced by quinine, mefloquine and omeprazole. A carrier-mediated transport of the antimalarial drug proguanil through the erythrocyte membrane could also be shown. This process could be inhibited and followed a Michaelis-Menten kinetics. Interestingly, the erythrocyte uptake could be inhibited by the OCTN substrate carnitine, a finding which requires further studies considering that overexpression of OCTN1 and OCTN2 in HEK cells did not increase proguanil or cycloguanil uptake. In conclusion, the results showed that the organic cation transporters OCT1, OCT2 and OCT3 may play a role in the absorption and secretion of proguanil and cycloguanil within the human organism., Die Bedeutung genetischer Variation bei der Biotransformation von Arzneimitteln ist gut bekannt. Es hat sich in den letzten zwei Dekaden gezeigt, dass in den Genen einiger Arzneimittel-Membrantransporter eine erhebliche erbliche Variation besteht. Aus diesem Grunde und wegen der Bedeutung bei Arzneimittel-Interaktionen ist die Erforschung der relevanten Membrantransporter inzwischen zu einem wichtigen Bestandteil der präklinischen und frühen klinischen Arzneimittelentwicklung geworden. Für hydrophobe Arzneistoffe sind Efflux-Transporter wie der Multidrug-Resistance-Proteine 1 (MDR1) von großer Bedeutung. Hinzu kommen für die hydrophilen Arzneistoffe viele Influx-Transporterproteine wie beispielsweise die organischen Kationentransporter (OCTs), die die Aufnahme von organischen kationischen Molekülen in die Zelle beschleunigen. Um bei Transportmessungen Konzentration innerhalb der Zelle zu messen, ist heute neben der immer noch weit verbreiteten Anwendung der radioaktiven Markierungen die Hochdruckflüssigkeitschromatographie (HPLC) das am häufigsten verwendete analytische Verfahren. Jedoch lässt sich nicht jede bereits publizierte Methode problemlos auf die vorhandenen Gegebenheiten und Anforderungen bezüglich der Empfindlichkeit übertragen. Daraus ergaben sich die speziellen Herausforderungen der vorliegenden Arbeit. Im Rahmen der vorliegenden Dissertation wurden folgende Ziele verfolgt: Für Untersuchungen zur Bedeutung organischer Kationentransporter sollte zunächst die Optimierung und Validierung von HPLC-Methoden zur quantitativen Bestimmung der Arzneistoffe Proguanil, Cycloguanil, Amisulprid, Debrisoquin und 4-Hydroxydebrisoquin erfolgen. Die Biochemie der zellulären Aufnahme von Proguanil sowie Cycloguanil sollte geklärt werden. Dies sollte erfolgen anhand von HEK293-Zellen, welche die vermutlich hier relevanten organische Kationentransporter OCT1-3 sowie OCTN1 und OCTN2 exprimierten. Zur weiteren Klärung und Bestätigung sollten auch Experimente mit Inhibition des Transports an den oben erwähnten OCTs durchgeführt werden. Ferner sollten zu Proguanil und Cycloguanil die Auswirkungen der bei Europäern relevanten und natürlicherweise auftretenden Genvarianten und Interaktionen mit weiteren Arzneistoffen auf den 1. Einleitung 34 Membrantransport analysiert werden. Schließlich sollte der Transport von Proguanil durch Erythrozytenmembranen mit anschließender Inhibition des vermeintlichen Transports und der Interaktion mit weiteren Arzneistoffen analysiert werden. Durch die Validierung und Optimierung der HPLC-Methoden konnten die Konzentrationen der Arzneistoffe Proguanil, Cycloguanil, Amisulprid sowie Debrisoquin quantitativ bestimmt werden. Die obigen Arzneistoffe konnten mit der erforderlichen Empfindlichkeit und Präzision gemessen werden Die In-Vitro-Versuche zeigten, dass das Antimalariamittel Proguanil und sein aktiver Metabolit Cycloguanil über die elektrogenen organischen Kationentransporter OCT1, OCT2 und OCT3 mit unterschiedlicher Vmax und KM transportiert werden können. Die organischen Kationentransporter OCTN1 und OCTN2 zeigten hingegen keine Beschleunigung der zellulären Aufnahme der beiden Substanzen. Ausgewählte Polymorphismen des OCT1, die mit einer erhöhten Häufigkeit bei Kaukasiern auftreten und Auswirkungen auf die Aufnahme von anderen Arzneistoffen zeigten, zeigten auch bei Proguanil und Cycloguanil einen veränderten und in der Regel reduzierten Membrantransport. Die gleichzeitige Gabe weiterer Pharmaka (insbesondere Mefloquin, Chinin oder Omeprazolmit unterschiedlichen Konzentrationen führten zu einer Reduktion des Membrantransports von Proguanil und Cycloguanil an OCT1. Der erythrozytäre Transport des Antimalariamittels Proguanil durch die Erythrozytenmembran konnte ebenfalls analysiert werden. Dieser war unter anderem mit L-Carnitin hemmbar und folgt einer Michaelis-Menten-Kinetik. Die Ergebnisse zeigen, dass die organischen Kationentransporter eine bedeutende Rolle bei der Aufnahme und Ausscheidung von Proguanil und Cycloguanil im menschlichen Organismus spielen können.

Sulfadoxine-pyrimethamine (SP) is used as malaria chemoprophylaxis for pregnant women and children in Ghana. Plasmodium falciparum resistance to SP is linked to mutations in the dihydropteroate synthase gene (pfdhps), dihydrofolate reductase gene (pfdhfr) and amplification of GTP cyclohydrolase 1 (pfgch1) gene. The pfgch1 duplication is associated with pfdhfr L164, a crucial mutant for high level pyrimethamine resistance which is rare in Ghana. The presence of amplified pfgch1 in Ghanaian isolates could be an indicator of the evolution of the L164 mutant. This study therefore determined the pfgch1 copy number variations and SP resistance mutations in clinical isolates from Ghana. One hundred and ninety-two (192) blood samples collected from children aged ≤14 years with uncomplicated malaria in 2013-14 and 2015-16 were used. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the pfgch1 copy number and nested PCR-Sanger sequencing used to detect mutations in pfdhps and pfdhfr genes. Twelve parasites (6.3%) harbored double copies of the pfgch1 gene out of the 192 samples. Of the 12, 75% had the pfdhfr I51-R59-N108, 92% had the pfdhps G437 mutant, 8% had the pfdhps E540 and 67% had the SP resistance haplotype IRNG. No L164 was detected in samples with amplified pfgch1. The rare T108 mutant associated with cycloguanil resistance showed predominance (60%) over N108 in the 2015-16 isolates. The observation of parasites with increased copy number of pfgch1 gene is indicative of the future evolution of the rare quadruple pfdhfr mutant, I51-R59-N108-L164, in Ghanaian parasites. Mutant pfdhps isolates also had increased gch1 copy number suggestive that it may also facilitate sulphadoxine resistance. The selection of parasites with pfgch1 gene amplification will enhance the sustenance and persistence of parasites with SP resistance in the country. Policy makers need to begin the search for a replacement chemoprophylaxis drug for malaria vulnerable groups in Ghana.

A molecular docking study was carried out on 28 compounds belonging to 2,4-diaminoquinazoline and 2,4-diaminopteridine analogs using Glide, FlexX and GOLD programs and the X-ray crystallographic structures of the quadruple mutant (1J3K:pdb) and wild type (1J3I:pdb) Plasmodium falciparum dihydrofolate reductase enzyme. The experimental conformation the bound ligand WR99210 was precisely reproduced by the docking procedures as demonstrated by low (

A three-dimensional quantitative structure–activity relationship (3D-QSAR) study was carried out on cycloguanil derivatives which are reported as growth inhibitors of Plasmodium falciparum clone (T9/94 RC17) which harbors A16V + S108T mutant dihydrofolate reductase (DHFR) enzyme. Comparative of Molecular Field Analysis (CoMFA) and Comparative of Molecular Similarity Indices Analysis (CoMSIA) were carried out to investigate the structural requirements for the activities of these compounds and to derive predictive models that may be used for designing novel PfDHFR enzyme inhibitors. The global minimum energy (within the search space) conformation of the most active compound (38) was obtained by using simulated annealing, and was subsequently used as a template to build the structures of the rest molecules used in the study. The CoMFA model gave statistically significant results with r cv 2 and r ncv 2 values of 0.654 and 0.951, respectively. The combination of steric, electrostatic and hydrophobic fields resulted in the best CoMSIA model with r cv 2 and r ncv 2 values of 0.669 and 0.907, respectively. The predictive abilities of the CoMFA and CoMSIA models were also evaluated using test compounds which gave r pred 2 values of 0.735 and 0.557, respectively. The results of bootstrapping analyses also confirmed that the generated models are robust and reliable. The models were graphically interpreted using CoMFA and CoMSIA contour plots. The structural regions responsible for the differences in anti-plasmodial activities were analyzed with respect to their electrostatic, steric and hydrophobic nature. The results obtained from this study could be used for rational design of potent inhibitors against A16V + S108T mutant PfDHFR enzyme.

Comparative molecular field analysis (CoMFA) and quantum chemical calculations were performed on cycloguanil (Cyc) derivatives of the wild type and the quadruple mutant (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) of Plasmodium falciparum dihydrofolate reductase (PfDHFR). The represented CoMFA models of wild type ( $$ r_{\text{cv}}^{2} = 0.727 $$ and r 2 = 0.985) and mutant type ( $$ r_{\text{cv}}^{2} = 0.786 $$ and r 2 = 0.979) can describe the differences of the Cyc structural requirements for the two types of PfDHFR enzymes and can be useful to guide the design of new inhibitors. Moreover, the obtained particular interaction energies between the Cyc and the surrounding residues in the binding pocket indicated that Asn108 of mutant enzyme was the cause of Cyc resistance by producing steric clash with p-Cl of Cyc. Consequently, comparing the energy contributions with the potent flexible WR99210 inhibitor, it was found that the key mutant residue, Asn108, demonstrates attractive interaction with this inhibitor and some residues, Leu46, Ile112, Pro113, Phe116, and Leu119, seem to perform as second binding site with WR99210. Therefore, quantum chemical calculations can be useful for investigating residue interactions to clarify the cause of drug resistance. CoMFA steric contour maps of Cyc derivatives against the quadruple mutant PfDHFR. Electrostatic van der Waals surface interaction of Cyc and the key resistance residue Asn108.

A total of 248 Plasmodium falciparum isolates were sampled in travelers with malaria who came to Marseille, France from Comoros to investigate in vitro activities of antimalarial drugs and molecular markers of drug resistance. Of the 248 isolates, 126 were maintained in culture. Of these, 53% were resistant to chloroquine, and 3% had reduced susceptibility to quinine, mefloquine, and atovaquone; 1% had reduced susceptibility to halofantrine and dihydroartemisinin; 7% had reduced susceptibility to monodesethylamodiaquine; 37% had reduced susceptibility to cycloguanil; and none had reduced susceptibility to lumefantrine. Resistance-associated point mutations were screened in 207 isolates. No mutations in the cytochrome b gene were found. Of the 207 isolates, 119 (58%) had a mutation in the P. falciparum dihydrofolate reductase (Pfdhfr) gene at codon 108, 6 (5%) had mutations in both Pfdhfr codon 108 and the P. falciparum dihydropteroate synthase codon 437, and 115 (56%) had the chloroquine resistance–associated K76T mutation in the P. falciparum chloroquine resistance transporter gene. This study represents a unique opportunity to improve surveillance of P. falciparum drug resistance in Comoros with consequences for treatment and chemoprophylaxis guidelines.

With the emergence of drug-resistant vivax malaria, in vitro studies are urgently needed to examine resistance mechanisms and for drug development. Currently, Plasmodium vivax culturing is inadequate for addressing these needs; therefore, surrogate biological systems have been developed. Although these systems are informative, they do not address Plasmodium species-specific mechanisms, such as drug delivery through erythrocytes and parasite membranes. Here, we demonstrate that P. falciparum is an excellent biological system for expression of P. vivax dhfr-ts alleles to assess dihydrofolate reductase (DHFR)-thymidylate synthase interactions with antifolates. Our results show that the P. vivax dhfr-ts quadruple-mutant allele AMRU1, expressed in P. falciparum, provides significant protection against pyrimethamine, cycloguanil, and clocicguanil. Moreover, the PvDHFR quadruple mutant confers greater resistance to cycloguanil, clociguanil, and WR99210 than the PfDHFR quadruple mutant. Modeling of both P. vivax and P. falciparum DHFR quadruple mutants suggests that mutations unique to P. vivax DHFR are responsible for differences seen in parasite susceptibility to antifolates.

Background Sulphadoxine-pyrimethamine (SP) is the recommended drug for intermittent preventive treatment in pregnancy (IPTp) in most African countries, including Zambia. However, malaria is still one of the leading causes of morbidity and mortality in pregnant women despite reports of greater than 50% of women taking at least two doses of SP in IPTp. Studies have shown that resistance to SP is associated with mutations in the dhfr and dhps gene of Plasmodium falciparum. This study examined the prevalence of dhfr and dhps polymorphisms in P. falciparum found in pregnant women of Nchelenge district. Method This cross-sectional study was conducted in 2013 in Nchelenge, a holoendemic area with malaria prevalence estimated at 50% throughout the year. Three rural health centres were randomly selected and a census survey carried out at each health centre. A questionnaire was administered and malaria testing done using RDT and microscopy, with collection of a dried blood spot. A chelex extraction was done to extract parasite DNA from dried blood spots followed by nested PCR and enzyme restriction digestion. Results Of the enrolled participants (n = 375), the median age of the women was 23. The prevalence of malaria by PCR was 22%. The PCR positive samples examined (n = 72) showed a high prevalence of dhfr triple (Asn-108 + Arg-59 + Ile-59) mutant (68%) and dhps double (Gly -437 + Glu-540) mutant (21%). The quintuple haplotype was found in 17% with 2 samples with an additional Gly-581mutation. In addition 6% mutations at Val-16 were found and none found at Thr-108 respectively, these both confer resistance to cycloguanil. Multivariate analysis showed that there was an association between malaria and women aged 30-34 years old p