Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei.

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs

Prevalence of mutations in the cysteine desulfurase IscS (Pfnfs1) gene in recurrent Plasmodium falciparum infections following artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) treatment in Matayos, Western Kenya

Abstract Background Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya. Methods Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections. Results The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample. Conclusions The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission

Deletion of the PM4 gene significantly attenuates the growth of asexual parasites.

<p><b>(A).</b> Box plots are showing the percentage parasitaemia in mice infected with the PM4_KO, PM7_KO or PM8_KO parasite line relative to the wild-type (WT) parasite line as measured on day four post parasite inoculation in the standard 4-day suppressive test. <b>(B).</b> Violin plots are showing the distribution of the parasites in mice infected with the PM4_KO, PM7_KO or PM8_KO parasite line relative to the wild-type (WT) parasite line as measured in the standard 4-day suppressive test. The PM4_KO parasites acquired a reduced growth rate phenotype (<i>P</i> = 0. 0.003), while the PM7_KO and PM8_KO parasites lines attained an increased growth rate phenotype.</p

Deciphering the targets of retroviral protease inhibitors in <i>Plasmodium berghei</i> - Fig 1

<p><b>PCR genotyping of the knockout (KO) parasites; (A) PM4_KO, (B) PM7_KO, (C) PM8_KO, and the wild-type (WT) control lines.</b> The PCR amplification used three sets of primer pairs; QCR2/GW2 to confirm the presence of the vector, GT/GW1 or GT/GW2 to verify integration of the vector into the parasite genome and QCR1/QCR2 to confirm deletion of the specific genes. The QCR1, QCR2 and GT1 are vector specific primers while GW2 and GW1 primers are standard primers.</p

Deciphering the targets of retroviral protease inhibitors in <i>Plasmodium berghei</i> - Fig 5

<p>(A) The crystallographic structure of PM4 protein in complex with the lopinavir as visualized using PyMOL. (B) 2D LP-PM4 interaction diagram with the oxygen atoms shown in red, nitrogen atoms shown in blue while hydrogen bonds are shown as olive green dotted line. The interaction plots were generated using <a href="https://www.ebi.ac.uk/thornton-srv/software/LigPlus/download.html" target="_blank">LigPlot+</a>.</p

Both the LP and SQ exhibited high binding affinity to the PM4 protein.

<p>The binding geometries for docking of a single ligand with a single receptor as modelled using the Autodock Vina. The binding energies for LP and SQ to the PM4 were the lowest suggesting better affinity to the protein. The LP and SQ yielded higher binding affinity to both PM7 and PM8 as compared to the known RPIs target; the HIV Aspartic protease (HIV Asp). Both LP and SQ exhibited equal or low binding energy to the essential Ddi1 protein as compared to the HIV Asp.</p

Primer pairs used in PCR amplification of gDNA isolated from PM7_KO parasites.

<p>Primer pairs used in PCR amplification of gDNA isolated from PM7_KO parasites.</p

Primer pairs used in PCR amplification of gDNA isolated from PM8_KO parasites.

<p>Primer pairs used in PCR amplification of gDNA isolated from PM8_KO parasites.</p

Primer pairs used in PCR amplification of gDNA isolated from PM4_KO parasites.

<p>Primer pairs used in PCR amplification of gDNA isolated from PM4_KO parasites.</p

The PM4_KO parasites lost significant susceptibility to both LP and SQ.

<p>Violin plots are showing growth profiles and the distribution of the asexual parasites. (A) The wild-type (WT) parasite line displayed expected growth pattern and the susceptibility level to amodiaquine (AQ), piperaquine (PQ), LP/RT and SQ/RT. (B) The PM4_KO parasite line exhibited a significant reduction in the growth outline and a substantial loss of susceptibility to LP/RT (<i>P</i> = 0.036) and SQ/RT (<i>P</i> = 0.030) but not to AQ or PQ drugs. (C) The PM7_KO and (D) PM8_KO parasite lines displayed rapid growth pattern as compared to the WT parasite line but retained susceptibility to AQ and PQ as well as to the RPIs; LP/RT and SQ/RT.</p