Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets

John M. Bennett, Sunil K. Narwal, Stephanie Kabeche, Daniel Abegg, Fiona Hackett, Tomas Yeo, Veronica L. Li, Ryan K. Muir, Franco F. Faucher, Scott Lovell, Michael J. Blackman, Alexander Adibekian, Ellen Yeh, David A. Fidock, Matthew Bogyo

Posted on: 2 February 2024

Preprint posted on 11 January 2024

Lessons from nature for antimalarial agent discovery: @ChemBio_John and colleagues (from @mbogyo and @fidocklab) develop serine hydrolase inhibitors targeting plasmodium parasites. Preprint highlighted by @goh_zhanghe.

Selected by Zhang-He Goh

Background of the preprint

Malaria is a highly transmissible disease afflicting hundreds of millions of people worldwide. It is caused by various Plasmodium parasites. One of these parasites, Plasmodium falciparum, is implicated in about half of all malaria cases. Worse still, it causes one of the most dangerous forms of malaria. Currently, resistance to antimalarial treatments targeting P. falciparum is rapidly growing. Given malaria’s widespread disease burden and its threat to global public health, there is an urgent need to develop antimalarial treatments with alternative mechanisms of action.

The search for effective antimalarial agents targeting P. falciparum has been greatly inspired by natural product discovery. In a series of highly collaborative efforts, the Bogyo and Fidock groups have ascribed the antimalarial activity of one such product, Salinipostin A (SalA), to its ability to inhibit various serine hydrolases.1 Serine hydrolases play key roles in lipid metabolism and in maintaining P. falciparum’s ability to remodel its plasma membrane throughout its life cycle in the host’s red blood cells.

The upshot of SalA having multiple targets is that resistance to SalA is unlikely to develop. However, the synthesis and isolation of SalA is too challenging for SalA to be a viable therapeutic option in the treatment of malaria. Therefore, the Bogyo and Fidock groups aimed to develop a class of antimalarial agents that would be easier to make. To this end, Bennett and co-workers developed a series of mixed alkyl/aryl phosphonates, tested their structure-activity relationship, and characterised their activity on both purified enzymes and on P. falciparum parasites (Figure 1).


Figure 1. Summary of this work by Bennett and co-workers.



Key findings of this preprint

(A) Synthesis of phosphonates

Given that the key difficulty in the synthesis of SalA and its analogues lies in the synthesis of the bicyclic enolphosphate group, Bennett and co-workers first developed a synthetic strategy so that they could rapidly access a series of mixed alkyl/aryl phosphonates. The preprint authors then ran a series of biochemical assays to test these mixed alkyl/aryl phosphonates against a serine hydrolase target of SalA named PfMAGL. These experiments proved that the alkyl/aryl phosphonates were potent inhibitors of PfMAGL and showed that electron-withdrawing substituents on the phosphonate phenols were beneficial for inhibitory activity while electron-donating and sterically bulky substituents were not. Unfortunately, the authors also found that these compounds did not exhibit potent antiparasitic properties over 72 hours.

In medicinal chemistry, the structure of a compound is closely linked to its pharmacological activity. Therefore, the authors next synthesised a second series of analogues which more closely resemble SalA with the goal of better preserving its antimalarial activity. This strategy was successful, as further experiments led Bennett and co-workers to identify an analogue, labelled compound 22 in the preprint, as a potent inhibitor of parasite growth. Compound 22 exhibited excellent inhibitory activity in both biochemical and parasitic assays; it also exhibited minimal mammalian cell toxicity, so the authors focussed on compound 22 in subsequent experiments.

Bennett and co-workers performed chemoproteomic experiments to confirm the targets of the analogues that they had synthesised. To do this, the authors made activity-based probe versions of these analogues, including that of compound 22 (labelled compound 22-alk in the preprint). The authors found that compound 22 inhibited multiple serine hydrolases including PfMAGL, a known target of SalA. In fact, the antimalarial activity of compound 22 did not arise from the inhibition of PfMAGL alone; other serine hydrolases including AbH112 were implicated as well.


(B) Phenotypic analyses

Next, Bennett and co-workers performed phenotypic analyses, which showed that compound 22 blocks P. falciparum’s life cycle at a different stage compared to both SalA and the pan-lipase inhibitor Orlistat. This suggested that compound 22 has a different mechanism of action compared to both SalA and Orlistat. Specifically, compound 22 blocks the parasites’ transition from the late ring to the early trophozoite stage. In contrast, both SalA and Orlistat block the parasites’ progression at the late schizont stage.


(C) Antimalarial resistance experiments

Finally, the authors performed cross-resistance experiments between compound 22 and SalA. Interestingly, despite their distinct mechanisms of action, antimalarial resistance against compound 22 implicated mutations similar to those which conferred antimalarial resistance against SalA. These findings led the authors to postulate that compound 22 and SalA may share a common genetic mechanism of resistance. In these experiments on resistance, compound 22 had a minimum inoculum of resistance of 1 x 109, leading the authors to conclude that it is less prone to induce resistance than other antimalarials.

Given that the authors previously identified AbH112 as a possible target, they also examined the role of AbH112 in parasite growth. Interestingly, they found that conditionally knocking down the expression of AbH112 did not significantly affect the growth or development of P. falciparum, nor did it affect its susceptibility to compound 22.




What I like about this preprint

Antimalarial treatment is a challenging but important research area—I have written about it twice since joining the preLights community in 2018, and would like to highlight it again with my first preLight of 2024. Key advances in chemical biology in recent years have given researchers multiple tools to A) rapidly synthesise complex molecules that were previously deemed difficult or impossible to produce and B) use them to quickly identify their mechanisms of action. This has allowed us to better identify new biological targets for the treatment of complex diseases.

Indeed, covalent inhibitors are making a comeback. Previously, covalent inhibitors were deemed difficult to work with because researchers were concerned about their lack of selectivity—and by extension, increased toxicity. However, researchers are now much better equipped to invent inhibitors specific for their target.2

In this preprint, Bennett and co-workers demonstrate how covalent inhibition can be useful, even when implicated in multiple mechanisms of action. In trying to identify the exact mechanism of action underpinning their SalA-inspired covalent inhibitors, the authors showed that these inhibitors bind to various proteins that have previously been implicated in antimalarial activity. It would be exciting if these findings could ultimately lead to the invention of agents with improved activity, reduced risks of resistance, and reduced toxicity.




Images created using Microsoft Powerpoint, ChemDraw, and BioRender.



Questions for authors

  1. Could you describe how you designed the SalA analogue library? Specifically, how did you select the phenol leaving groups (as opposed to other leaving groups)? Did you also consider other alkyl substituents, such as cyclic alkyl structures, on the SalA analogues?
  2. Given that SalA and its analogues act through a covalent mechanism, do you think the covalent inhibition is irreversible in all cases? Is it possible that the covalent inhibition is more reversible on some enzymes than others?
  3. In the same vein, how quickly does the covalent inhibition occur? Is the inhibition kinetics of these agents (in this case, the phosphonate group) rapid?




  1. E. Yoo, C. J. Schulze, B. H. Stokes, O. Onguka, T. Yeo, S. Mok, N. F. Gnädig, Y. Zhou, K. Kurita, I. T. Foe, S. M. Terrell, M. J. Boucher, P. Cieplak, K. Kumpornsin, M. C. S. Lee, R. G. Linington, J. Z. Long, A.-C. Uhlemann, E. Weerapana, D. A. Fidock and M. Bogyo, Cell Chemical Biology, 2020, 27, 143-157.e145.
  2. L. Boike, N. J. Henning and D. K. Nomura, Nature Reviews Drug Discovery, 2022, DOI: 10.1038/s41573-022-00542-z.

Tags: activity-based protein profiling, drug resistance, malaria, plasmodium, serine hydrolase


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Author's response

John Bennett shared

  1. Could you describe how you designed the SalA analogue library? Specifically, how did you select the phenol leaving groups (as opposed to other leaving groups)? Did you also consider other alkyl substituents, such as cyclic alkyl structures, on the SalA analogues?

The initial library we synthesized was actually for a different project that did not pan out, we then repurposed the tools for use in Plasmodium after recognizing the similarities of the warhead to the phosphate group in the natural product. We ultimately landed on the long chain design by using the natural product as inspiration as it also includes a long alkyl chain. For the phenolic leaving groups, we looked at what was available commercially and compared that to what was known about the scope of the reactions we were running to pick a set that reasonably represents the available chemical space. We did not consider using other types of alkyl substituents, though we have considered use of the alkyl site as a place to further derivatize our compounds. I think the cyclic alkyl structure would be interesting to explore further and I believe other groups have worked out those syntheses.



  1. Given that SalA and its analogues act through a covalent mechanism, do you think the covalent inhibition is irreversible in all cases? Is it possible that the covalent inhibition is more reversible on some enzymes than others?

Regarding the phosphate electrophile in SalA and our phosphonate-based inhibitors, I think both are irreversible covalent inhibitors of serine hydrolases broadly. Studies have shown that diphenyl phosphonates can have reversible inhibition, but we have no evidence to support that with our compounds nor the natural product. It could be possible that inhibition is more reversible for some enzymes than others, especially with known reversible covalent warheads for serine hydrolases such as boronates or nitriles. I believe that variations in reversibility may be attributed to the active site structure. While most serine hydrolases have a catalytic triad, some instances feature catalytic dyads. This would be a really interesting topic to explore going forward for someone interested in mechanisms of covalent inhibition.



  1. In the same vein, how quickly does the covalent inhibition occur? Is the inhibition kinetics of these agents (in this case, the phosphonate group) rapid?

We have evidence that suggests covalent inhibition occurs quite quickly with our phosphonates. Biochemically, we used 30-minute incubation periods that resulted in inhibition suggesting that our compound react readily with the active site serine, though this may only be true for phosphonates with electron withdrawing groups on their phenol leaving group. Phosphonates which are less activated may need longer preincubation times for inhibition to occur. We also performed an assay with our best compound, 22, where we pulse the parasites with the inhibitor for 1 hour and proceed to measure its growth and we found that there is a shift in potency in this pulse assay, but that inhibition still occurs which further supports that inhibition of important targets is rapid. We did not perform enzymatic assays to calculate inhibition kinetics directly, so that would be an interesting next step to take to fully characterize the potential of the mixed alkyl/aryl phosphonate warhead.

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