Structural insights into Plasmodium PPIases

et al. 2023

Preprint

Plasmodium falciparum accounts for the majority of over 600’000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its anti-proliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action – with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.

Section: Introductionmentioning

confidence: 64%

Genetic validation of PfFKBP35 as an antimalarial drug target

et al. 2023

Preprint

“…Pf FKBP35 emerged as a promising antimalarial drug target ( Bharatham et al, 2011 ) and considerable efforts were made to define interactions of this immunophilin with small molecules in the last decade ( Harikishore et al, 2013b ; Harikishore et al, 2013a ; Rajan and Yoon, 2022 ; Monaghan et al, 2017 ). Several studies conclusively demonstrated that FK506 – the most well-known ligand of mammalian FKBPs ( Van Duyne et al, 1991 ) – binds to Pf FKBP35 ( Yoon et al, 2007 ; Bianchin et al, 2015 ; Kotaka et al, 2008 ) and kills asexual blood stage parasites with a half-maximal inhibitory concentration (IC50) in the low micromolar range at 1.9 µM ( Bell et al, 1994 ).…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with these observations, in vitro enzyme activity assays showed that FK506 inhibits the PPIase activity of recombinant Pf FKBP35 in a dose-dependent manner ( Monaghan and Bell, 2005 ). Previous research aimed at exploiting structural differences between human and parasite-encoded FKBPs to circumvent the immunosuppressive activity of drug-bound Hs FKBP12 ( MacDonald and Boyd, 2015a ; Harikishore et al, 2013b ; Harikishore et al, 2013a ; Rajan and Yoon, 2022 ). These efforts include investigation of non-covalent FKBP inhibitors such as adamantyl derivatives, macrocycles, the small molecule D44 ( MacDonald and Boyd, 2015a ; Harikishore et al, 2013b ; Harikishore et al, 2013a ; MacDonald and Boyd, 2015b ; Deepa and Thirumeignanam, 2020 ) and the synthetic ligand for FKBP (SLF) as well as derivatives thereof designed to covalently bind Pf FKBP35 ( Atack et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Genetic validation of PfFKBP35 as an antimalarial drug target

et al. 2023

eLife

Plasmodium falciparum accounts for the majority of over 600’000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed-death phenotype that is characterized by defective ribosome homeostasis and stalled protein translation. We furthermore show that FK506, unlike the role of this drug in model organisms, exerts its anti-proliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action – with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.

“…Pf FKBP35 emerged as a promising antimalarial drug target (4) and considerable efforts were made to define interactions of this immunophilin with small molecules in the last decade (24–26, 43). Several studies conclusively demonstrated that FK506 – the most well-known ligand of mammalian FKBPs (44) – binds to Pf FKBP35 (15, 21, 22) and kills asexual blood stage parasites with a half-maximal inhibitory concentration (IC50) in the low micromolar range at 1.9 µM (3).…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with these observations, in vitro enzyme activity assays showed that FK506 inhibits the PPIase activity of recombinant Pf FKBP35 in a dose-dependent manner (5). Previous research aimed at exploiting structural differences between human and parasite-encoded FKBPs to circumvent the immunosuppressive activity of drug-bound Hs FKBP12 (28–31). These efforts include investigation of non-covalent FKBP inhibitors such as adamantyl derivatives, macrocycles, the small molecule D44 (FKBP35 (28-FKBP35 (30, FKBP35 (32, FKBP35 (33) and the synthetic ligand for FKBP (SLF) as well as derivatives thereof designed to covalently bind Pf FKBP35 (34).…”

Section: Introductionmentioning

confidence: 99%

Genetic validation ofPfFKBP35 as an antimalarial drug target

et al. 2022

Preprint

Plasmodium falciparumis the most virulent malaria parasite and accounts for the majority of over 600'000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged, posing a serious threat to disease control efforts. The need for drugs with alternative modes of action is thus undoubted. The parasite's FKBP protein (PfFKBP35) has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506. Whilst there is considerable interest in targetingPfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing. As a consequence, the function ofPfFKBP35 in live parasites remains elusive. Here, we report the generation of inducible knock-out, knock-down and over-expression cell lines and show that blood stage parasites requirePfFKBP35 for maintaining ribosome function. LimitingPfFKBP35 levels are lethal toP. falciparumand result in a delayed-death phenotype that is characterized by defective ribosome homeostasis and protein translation. We furthermore show that, while FK506 is indeed binding toPfFKBP35, the drug exerts it parasiticidal activity in aPfFKBP35-independent manner and, using cellular thermal shift assays, we identify FK506-targets beyondPfFKBP35. In addition to revealing first functional insights on the role ofPfFKBP35 inP. falciparumparasites, our results show that current efforts to developPfFKBP35-targeting drugs need to be reconsidered urgently.