Transgenic pyrimethamine-resistant P. falciparum reveals transmission blocking potency of P218, a novel antifolate

Antimalarial drug which target more than one life stage of the parasite are valuable tools in the fight against malaria. Previous generation of antifolate drugs are able to inhibit replicative stages of drug-sensitive, but not resistant parasites in humans, and mosquitoes. The lack of reliable gametocyte-producing, antifolate resistant P. falciparum hindrance the development of new antifolate compounds against mosquito stages. We used CRISPR-Cas9 technology to develop transgenic gametocyte producing P. falciparum with quadruple mutations in dhfr gene, using NF54 as a parental strain. The transgenic parasites gained pyrimethamine resistance while maintaining the gametocyte producing activity. In contrast to pyrimethamine that cannot inhibit exflagellation of the quadruple dhfr mutant parasite, the novel antifolate P218 showed a good potency for exflagellation inhibition (exflagellation IC50 10.74 ± 4.22 nM). The exflagellation IC50 was 5.3 times lower than erythrocytic IC50 suggesting that the human to mosquito transmission poses as a strong barrier to prevent P218 resistant parasite among population. This study demonstrates that P218 can be considered as a highly potent tool to prevent the spread of antifolate resistant parasites.Graphical AbstractResearch Highlights- Transgenic gametocyte producing pyrimethamine resistant P. falciparum was generated.- P218 asexual stage IC50 in NF54-4mutPfdhfr was 56.94 ± 15.69 nM.- P218 exflagellation IC50 in NF54-4mutPfdhfr was 10.74 ± 4.22 nM.- P218 exflagellation IC50 in NF54-4mutPfdhfr is 5.3 times lower than erythrocytic IC50.- P218 is an invaluable tool for malaria treatment and transmission control.

The genomic architecture of antimalarial drug resistance

Plasmodium falciparum and Plasmodium vivax, the two protozoan parasite species that cause the majority of cases of human malaria, have developed resistance to nearly all known antimalarials. The ability of malaria parasites to develop resistance is primarily due to the high numbers of parasites in the infected person’s bloodstream during the asexual blood stage of infection in conjunction with the mutability of their genomes. Identifying the genetic mutations that mediate antimalarial resistance has deepened our understanding of how the parasites evade our treatments and reveals molecular markers that can be used to track the emergence of resistance in clinical samples. In this review, we examine known genetic mutations that lead to resistance to the major classes of antimalarial medications: the 4-aminoquinolines (chloroquine, amodiaquine and piperaquine), antifolate drugs, aryl amino-alcohols (quinine, lumefantrine and mefloquine), artemisinin compounds, antibiotics (clindamycin and doxycycline) and a napthoquinone (atovaquone). We discuss how the evolution of antimalarial resistance informs strategies to design the next generation of antimalarial therapies.

Effects of Drug Policy Changes on Evolution of Molecular Markers of Plasmodium falciparum Resistance to Chloroquine, Amodiaquine, and Sulphadoxine-Pyrimethamine in the South West Region of Cameroon

Background. As a result of the spread of parasites resistant to antimalarial drugs, Malaria treatment guidelines in Cameroon evolved from nonartemisinin monotherapy to artemisinin-based combination therapy. The aim of this study was to assess the effect of these therapy changes on the prevalence of molecular markers of resistance from 2003 to 2013 in Mutengene, Cameroon. Methodology. Dry blood samples (collected in 2003–2005 and 2009–2013) were used for parasite DNA extraction. Drug resistance genes were amplified by PCR and hybridized with oligonucleotide probes or subjected to restriction digestion. The prevalence of individual marker polymorphisms and haplotypes was compared in these two study periods using the Chi square test. Results. Alleles conferring resistance to 4-aminoquinolines in the Pfcrt 76T and Pfmdr1 86Y, 184F, and 1246Y genotypes showed a significant reduction of 97.0% to 66.9%, 83.6% to 45.2%, 97.3% to 56.0%, and 3.1% to 0.0%, respectively (P<0.05). No difference was observed in SNPs associated with antifolate drugs resistance 51I, 59R, 108N, or 540E (P>0.05). Haplotype analysis in the Pfmdr1 gene showed a reduction in the YFD from 75.90% to 42.2%, P<0.0001, and an increase in the NYD (2.9% to 30.1%; P<0.0001). Conclusions. The results indicated a gradual return of the 4-aminoquinoline sensitive genotype while the antifolate resistant genotypes increased to saturation.

Supramolecular hydrogen-bonding patterns in salts of the antifolate drugs trimethoprim and pyrimethamine

Nine salts of the antifolate drugs trimethoprim and pyrimethamine, namely, trimethoprimium [or 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidin-1-ium] 2,5-dichlorothiophene-3-carboxylate monohydrate (TMPDCTPC, 1:1), C14H19N4O3 +·C5HCl2O2S−, (I), trimethoprimium 3-bromothiophene-2-carboxylate monohydrate, (TMPBTPC, 1:1:1), C14H19N4O3 +·C5H2BrO2S−·H2O, (II), trimethoprimium 3-chlorothiophene-2-carboxylate monohydrate (TMPCTPC, 1:1:1), C14H19N4O3 +·C5H2ClO2S−·H2O, (III), trimethoprimium 5-methylthiophene-2-carboxylate monohydrate (TMPMTPC, 1:1:1), C14H19N4O3 +·C6H5O2S−·H2O, (IV), trimethoprimium anthracene-9-carboxylate sesquihydrate (TMPAC, 2:2:3), C14H19N4O3 +·C15H9O2 −·1.5H2O, (V), pyrimethaminium [or 2,4-diamino-5-(4-chlorophenyl)-6-ethylpyrimidin-1-ium] 2,5-dichlorothiophene-3-carboxylate (PMNDCTPC, 1:1), C12H14ClN4 +·C5HCl2O2S−, (VI), pyrimethaminium 5-bromothiophene-2-carboxylate (PMNBTPC, 1:1), C12H14ClN4 +·C5H2BrO2S−, (VII), pyrimethaminium anthracene-9-carboxylate ethanol monosolvate monohydrate (PMNAC, 1:1:1:1), C12H14ClN4 +·C15H9O2 −·C2H5OH·H2O, (VIII), and bis(pyrimethaminium) naphthalene-1,5-disulfonate (PMNNSA, 2:1), 2C12H14ClN4 +·C10H6O6S2 2−, (IX), have been prepared and characterized by single-crystal X-ray diffraction. In all the crystal structures, the pyrimidine N1 atom is protonated. In salts (I)–(III) and (VI)–(IX), the 2-aminopyrimidinium cation interacts with the corresponding anion via a pair of N—H...O hydrogen bonds, generating the robust R 2 2(8) supramolecular heterosynthon. In salt (IV), instead of forming the R 2 2(8) heterosynthon, the carboxylate group bridges two pyrimidinium cations via N—H...O hydrogen bonds. In salt (V), one of the carboxylate O atoms bridges the N1—H group and a 2-amino H atom of the pyrimidinium cation to form a smaller R 2 1(6) ring instead of the R 2 2(8) ring. In salt (IX), the sulfonate O atoms mimic the role of carboxylate O atoms in forming an R 2 2(8) ring motif. In salts (II)–(IX), the pyrimidinium cation forms base pairs via a pair of N—H...N hydrogen bonds, generating a ring motif [R 2 2(8) homosynthon]. Compounds (II) and (III) are isomorphous. The quadruple DDAA (D = hydrogen-bond donor and A = hydrogen-bond acceptor) array is observed in (I). In salts (II)–(IV) and (VI)–(IX), quadruple DADA arrays are present. In salts (VI) and (VII), both DADA and DDAA arrays co-exist. The crystal structures are further stabilized by π–π stacking interactions [in (I), (V) and (VII)–(IX)], C—H...π interactions [in (IV)–(V) and (VII)–(IX)], C—Br...π interactions [in (II)] and C—Cl...π interactions [in (I), (III) and (VI)]. Cl...O and Cl...Cl halogen-bond interactions are present in (I) and (VI), with distances and angles of 3.0020 (18) and 3.5159 (16) Å, and 165.56 (10) and 154.81 (11)°, respectively.