Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G

ABSTRACT We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we...

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Autores principales:	Abhisheka Bansal, Kayode K. Ojo, Jianbing Mu, Dustin J. Maly, Wesley C. Van Voorhis, Louis H. Miller
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/77983dde2f9846ecaed7044b4deeda32
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spelling	oai:doaj.org-article:77983dde2f9846ecaed7044b4deeda322021-11-15T15:50:16ZReduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G10.1128/mBio.02011-162150-7511https://doaj.org/article/77983dde2f9846ecaed7044b4deeda322016-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02011-16https://doaj.org/toc/2150-7511ABSTRACT We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes. IMPORTANCE Protein kinases of Plasmodium falciparum are being actively pursued as drug targets to treat malaria. However, compensatory mechanisms may reverse the drug activity against a kinase. In this study, we show that replacement of the wild-type threonine gatekeeper residue with methionine reduces the transphosphorylation activity of CDPK1. Mutant parasites with methionine gatekeeper residue compensate the reduced activity of CDPK1 through the action of PKG possibly by upregulation of CDPK6 or some other kinase. This study highlights that targeting one enzyme may lead to changes in transcript expression of other kinases that compensate for its function and may select for mutants that are less dependent on the target enzyme activity. Thus, inhibiting two kinases is a better strategy to protect the antimalarial activity of each, similar to artemisinin combination therapy or malarone (atovaquone and proguanil).Abhisheka BansalKayode K. OjoJianbing MuDustin J. MalyWesley C. Van VoorhisLouis H. MillerAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 6 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Abhisheka Bansal Kayode K. Ojo Jianbing Mu Dustin J. Maly Wesley C. Van Voorhis Louis H. Miller Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G
description	ABSTRACT We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes. IMPORTANCE Protein kinases of Plasmodium falciparum are being actively pursued as drug targets to treat malaria. However, compensatory mechanisms may reverse the drug activity against a kinase. In this study, we show that replacement of the wild-type threonine gatekeeper residue with methionine reduces the transphosphorylation activity of CDPK1. Mutant parasites with methionine gatekeeper residue compensate the reduced activity of CDPK1 through the action of PKG possibly by upregulation of CDPK6 or some other kinase. This study highlights that targeting one enzyme may lead to changes in transcript expression of other kinases that compensate for its function and may select for mutants that are less dependent on the target enzyme activity. Thus, inhibiting two kinases is a better strategy to protect the antimalarial activity of each, similar to artemisinin combination therapy or malarone (atovaquone and proguanil).
format	article
author	Abhisheka Bansal Kayode K. Ojo Jianbing Mu Dustin J. Maly Wesley C. Van Voorhis Louis H. Miller
author_facet	Abhisheka Bansal Kayode K. Ojo Jianbing Mu Dustin J. Maly Wesley C. Van Voorhis Louis H. Miller
author_sort	Abhisheka Bansal
title	Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G
title_short	Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G
title_full	Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G
title_fullStr	Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G
title_full_unstemmed	Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G
title_sort	reduced activity of mutant calcium-dependent protein kinase 1 is compensated in <named-content content-type="genus-species">plasmodium falciparum</named-content> through the action of protein kinase g
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/77983dde2f9846ecaed7044b4deeda32
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Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in <named-content content-type="genus-species">Plasmodium falciparum</named-content> through the Action of Protein Kinase G

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