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. 2024 Dec 15:280:116921.
doi: 10.1016/j.ejmech.2024.116921. Epub 2024 Oct 3.

Exploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome b

Jon Kyle Awalt  1 , Wenyin Su  1 , William Nguyen  1 , Katie Loi  2 , Kate E Jarman  1 , Jocelyn S Penington  1 , Saishyam Ramesh  3 , Kate J Fairhurst  4 , Tomas Yeo  4 , Heekuk Park  5 , Anne-Catrin Uhlemann  5 , Bikash Chandra Maity  6 , Nirupam De  6 , Partha Mukherjee  6 , Arnish Chakraborty  6 , Alisje Churchyard  7 , Mufuliat T Famodimu  8 , Michael J Delves  8 , Jake Baum  9 , Nimisha Mittal  10 , Elizabeth A Winzeler  10 , Anthony T Papenfuss  1 , Mrittika Chowdury  11 , Tania F de Koning-Ward  11 , Alexander G Maier  3 , Giel G van Dooren  3 , Delphine Baud  12 , Stephen Brand  12 , David A Fidock  13 , Paul F Jackson  14 , Alan F Cowman  1 , Madeline G Dans  15 , Brad E Sleebs  16
Affiliations

Exploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome b

Jon Kyle Awalt et al. Eur J Med Chem. .

Abstract

Drug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype.

Keywords: Antimalarial; Cytochrome b; Malaria; Mitochondria; Plasmodium.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Structures and activity of carboxamide hits from the screen of the Jumpstarter library and structurally related compounds previously reported [16,17].
Scheme 1
Scheme 1
General synthetic pathway to access derivatives. Reagents and conditions: (a) R2-NH2, NaBH4, AcOH, MeOH, 0 °C, then rt, 2 h, 60–93 %; (b) i. oxalyl chloride, cat. DMF, DCM, rt, 2 h; ii. cyclopropylamine, Et3N, DCM, 0 °C, then rt, 2 h, 87 %; (c) K2CO3, MeCN, reflux, 16 h, 21–62 %; (d) R2-NH2, NaBH(OAc)3, AcOH, DCE, rt, 16 h, 10–54 %; (e) R1-PhCHO, NaBH(OAc)3, AcOH, DCE, rt, 2 h, 13–86 %.
Scheme 2
Scheme 2
Synthesis of N-substituted carboxamide analogs. Reagents and conditions: (a) cyclopropylamine, NaBH4, AcOH, MeOH, DCE, 0 °C, then rt, 24 h, 66–70 %; (b) methyl 4-formylbenzoate, NaBH(OAc)3, AcOH, DCE, rt, 16 h, 66 %; (c) LiOH·H2O, THF, MeOH, H2O, 70 °C, 3 h, 91 %; (d) R–NH2, EDCI, HOBt, DIPEA, DMF, rt, 15 h, 27–71 %.
Fig. 2
Fig. 2
Activity of W466 (1) and W499 (2) against W466- and W499 -resistant populations respectively. W466-resistant population 1 has an A122T cyt b Qo site mutation and populations 2 and 3 have an F264L cyt b Qo site mutation. W499-resistant populations 2 and 3 have a 2- to 5-fold CNV in region that encodes DHODH. EC50 values represent an average of 3 experiments using the LDH assay. Error bars are SD.
Fig. 3
Fig. 3
A homology model of P. falciparum cyt b showing the A122T Qo site mutation (red) found in 3D7 W466-resistant population 1, the F246L Qo site mutation (pink) found in 3D7 W466-resistant populations 2 and 3 and Dd2-Polδ W466-resistant F3 clones (from MIR study), and the G131S Qo site mutation (blue) found in the Dd2 W466-resistant population H3 (from MIR study), relative to the location of ELQ300, 3-oxadiazole quinolone and ATQ P. falciparum resistant mutations (used in Table 12). The homology model of P. falciparum cyt b was created from the X-ray structure of Gallus gallus cyt bc1 (PDB: 3H1I) [28]. TM90-C2B (ATQ resistant) strain cyt b Y268S Qo site mutation is shown in magenta; ELQ300 resistant Dd2 strain cyt b I22L Qi site mutation (orange); 3-oxadiazole quinolone resistant 3D7 strain cyt b V259L Qo site mutation [8] (cyan). Heme molecules (grey), ATQ (green) and ELQ300 (brown) are overlayed using previous structural data [22,32].
Fig. 4
Fig. 4
P. falciparum oxygen consumption rate (OCR) assay. Data represent the means and SDs of 3 technical replicates. Time points and reagent injections were as follows, 1. First 5 timepoints measured the basal level of malate-dependent OCR; 2. Compounds (5 μM) were injected independently (indicated by arrow), and OCR measured for 8-timepoints; 3. TMPD (a cytochrome c electron donor) was injected, and OCR measured for 5 time points; 4. NaN3 (a complex IV inhibitor) was then added, and OCR measured for 5 time points. ATQ = atovaquone and DMS265 is a DHODH inhibitor.
Fig. 5
Fig. 5
A. Representative Giemsa-stained microscopy images showing the asexual stage of arrest on treatment with ATQ, W466 (1) and W499 (2). Other representative images can be found in Fig. S12. B. Flow cytometry of SYBR green-stained infected RBCs. Data points represent the mean of three technical replicates analyzed via flow cytometry. Compounds in these experiments were used at a concentration of 10 times the asexual EC50 value. C. Activity of W466 (1) and W499 (2) in a parasite reduction ratio assay in comparison to antimalarial drugs. Data represent the means and SDs of 3 replicate experiments using P. falciparum 3D7 parasites in a LDH assay. ATQ = atovaquone.
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