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

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.

 

 

Acknowledgements

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?

 

 

References

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

doi: https://doi.org/10.1242/prelights.36398

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