Biology subjects

Pengon, J.

Publications and source records attributed to Pengon, J..

4 recordsLinked to original sources

bioRxiv2021

New insights into antimalarial chemopreventive activity of antifolates

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, the thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, have yet to be achieved. Using a transgenic P. berghei harboring quadruple mutant dhfr from P. falciparum (Pb::Pfdhfr-4M), we demonstrate that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not a new class of antifolate designed to overcome the resistance such as P218. Detailed investigation to pin-point stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data shows that P218 also inhibited the late hepatic stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg, P218 prevented infection from up to 25,000 pyrimentamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates, as a chemopreventive therapeutic which could lead to a more efficient and cost effective treatment regime.

microbiology↗

bioRxiv2020

Transgenic pyrimethamine resistant Plasmodium berghei as a model for in vivo anti-DHFR drug testing

Inhibitors for Plasmodium falciparum dihydrofolate reductase (DHFR) form an important class of antimalarial drugs widely used for malaria treatment, but have been compromised by development of resistance to the drugs. Mutations in DHFR are the main contributing factors to the resistance. Although new, rationally designed antifolates active against resistant P. falciparum, such as P218, have been developed, the activity against the quadruple mutant P. falciparum (V1/S) has only been demonstrated in vitro, and in vivo activity has only been shown in SCID mice. A convenient in vivo model for antifolate testing is desirable. In this study, the endogenous P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) gene was successfully replaced by quadruple dhfr-ts mutant gene from P. falciparum (N51I+C59R+S108N+I164L). The transgenic parasite gained resistance to pyrimethamine but not to other class of antimalarial drugs. While 30 mg/kg of pyrimethamine could not inhibit the transgenic parasite, P218 could inhibit the transgenic parasite with the ED50 of 0.11{+/-}0.02 mg/kg, a level similar to the P. falciparum in SCID mice model. These results demonstrated the validity of our model and showed that P218 was very potent against quadruple Pfdhfr-ts mutant parasite, in vivo.

microbiology↗

bioRxiv2020

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 Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Research 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.

microbiology↗

bioRxiv2020

Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus

Vector control, the most efficient tool to reduce mosquito-borne disease transmission, has been compromised by the rise of insecticide resistance. Recent studies suggest the potential of mosquito-associated microbiota as a source for new biocontrol agents or new insecticidal chemotypes. In this study, we identified a strain of Serratia marcescens that has larvicidal activity against Anopheles dirus, an important malaria vector in Southeast Asia. This bacterium secretes heat-labile larvicidal macromolecules when cultured under static condition at 25{degrees}C but not 37{degrees}C. Two major protein bands of approximately 55 kDa and 110 kDa were present in spent medium cultured at 25{degrees}C but not at 37{degrees}C. The Liquid Chromatography-Mass Spectrometry (LC-MS) analyses of these two protein bands identified several proteases and chitinases that were previously reported for insecticidal properties against agricultural insect pests. The treatment with protease and chitinase inhibitors led to a reduction in larvicidal activity, confirming that these two groups of enzymes are responsible for the macromolecules toxicity. Taken together, our results suggest a potential use of these enzymes in the development of larvicidal agents against Anopheles mosquitoes.

microbiology↗