title: Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development.
abstract: Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90.
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom judith.green@crick.ac.uk tony.holder@crick.ac.uk.
authors.forename: Judith L
authors.initials: JL
authors.lastname: Green
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Moon
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Whalley
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Bowyer
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Wallace
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, India.
authors.forename: Ankit
authors.initials: A
authors.lastname: Rochani
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, India.
authors.forename: Rishi K
authors.initials: RK
authors.lastname: Nageshan
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Howell
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom.
authors.forename: Munira
authors.initials: M
authors.lastname: Grainger
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: Hayley M
authors.initials: HM
authors.lastname: Jones
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: Keith H
authors.initials: KH
authors.lastname: Ansell
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Chapman
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: Debra L
authors.initials: DL
authors.lastname: Taylor
authors.affiliation: Centre for Therapeutics Discovery, MRC Technology, London, United Kingdom.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Osborne
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Baker
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, India.
authors.forename: Utpal
authors.initials: U
authors.lastname: Tatu
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom judith.green@crick.ac.uk tony.holder@crick.ac.uk.
authors.forename: Anthony A
authors.identifier: http://orcid.org/0000-0002-8490-6058
authors.initials: AA
authors.lastname: Holder
mesh_terms: D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D062105:Molecular Docking Simulation; D058990:Molecular Targeted Therapy / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011724:Pyridazines / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D018841:HSP90 Heat-Shock Proteins; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D015800:Protozoan Proteins; D011724:Pyridazines; D011494:Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases