Enhancing schistosomiasis drug discovery approaches with optimized proteasome substrates

Abstract Schistosomiasis, a neglected tropical disease infecting over 200 million people globally, has limited therapeutic options. The 20S proteasome is a validated drug target for many parasitic infections, including those caused by Plasmodium and Leishmania , and we have previously demonstrated antischistosomal activity with inhibitors targeting Schistosoma mansoni 20S proteasome (Sm20S). Here, we developed optimized subunit‐specific substrates for Sm20S based on data generated by Multiplex Substrate Profiling by Mass Spectrometry (MSP‐MS). These substrates exhibit 9‐fold or more improved activity compared to traditional human constitutive 20S proteasome (c20S) substrates. The optimized substrates also eliminated the need for extensive Sm20S purification, as robust enzyme activity could be detected in parasite extracts following an ammonium sulfate precipitation step. Finally, we show that the substrate and inhibition profiles for the 20S proteasome from the three medically important schistosome species are similar. This suggests that Sm20S‐focused inhibitor development can be efficiently extrapolated to the other schistosome species, leading to significant time and resource savings.