New approaches to develop treatments for ‘silent’ parasitic disease
A single-celled parasite Trichomonas vaginalis (T. vaginalis) causes trichomoniasis, the world’s most common non-viral sexually transmitted disease with an estimated 170-180 million cases worldwide every year. While most infected people do not develop any symptoms, they can spread the infection to others. It can lead to infertility, pelvic inflammatory disease in women, premature rupture of placenta membranes and preterm delivery. In men, it can cause inflammation of the prostate gland, or decrease sperm cell mobility. The ProTeCT project focuses on the trichomonas proteasome, an enzyme critical for the parasite’s survival. Understanding its structure, and the functions of its subunits, can ultimately be useful in designing inhibitors that can lead to a new generation of drugs to treat trichomoniasis. Currently only two drugs exist to treat the disease, but emerging resistance makes it imperative to find alternatives.
Testing inhibitors
Pavla Fajtová, a researcher at the University of California San Diego, tested many inhibitors in search of those that selectively inhibit the trichomonas proteasome. The 3-year project was undertaken with the support of the Marie Skłodowska-Curie Actions programme. Inhibitors that block the proteasome action that causes disease are already being studied for cancers, with three already approved for treatment of multiple myeloma, a type of bone marrow cancer. “The goal was to distinguish the substrate specificity of T. vaginalis and the human proteasome in order to develop inhibitors that target the trichomonas proteasome but won’t be toxic for humans,” she says. A large number of inhibitors were investigated for their selectivity in not targeting the human proteasome. That work took two years from late 2019. “We were lucky as we were working on some COVID proteases too. So our lab stayed open during COVID,” she notes.
Specific inhibitory effects
The project succeeded in finding inhibitor specificity that selectively targets trichomonas. However, to develop even more potent inhibitors of the T. vaginalis proteasome, it is essential to understand the proteasome’s structure and which of its subunits to target. “My goal was to develop tools to detect activity of each subunit of the proteasome,” Fajtová explains. “This information is useful because an inhibitor that binds preferentially to more than one subunit is even more effective against the parasite.” The project designed and synthesised three different substrates, one for each subunit, to examine more closely.
Internal structure and function of subunits
Fajtová then spent time at a structural biology laboratory at the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, in her home country Czechia, learning from their expertise in the baculovirus expression system and the structure of large multi-subunit proteins using cryo-electron microscopy (cryo-EM) techniques. The baculovirus expression system is commonly used to generate recombinant proteins in insect cells at high production levels. Without this, old-school laboratory techniques yield too little material from cultures. “I had the opportunity to learn how to recombinantly express fully active proteasome, which is an absolute game changer. Previously, I had been obtaining proteasomes directly from T. vaginalis parasites, which carried significant limitations in the purity and quantity of protein obtained,” Fajtová explains. “With this advance – that I have never done before – I obtained a sufficiently pure enzyme for cryo-EM and subsequent 3D structure determination,” she adds. “Now we have knowledge of what the structure looks like, the shape of the active subunits and what the inhibitor preferences are. This information will be used for the second-generation design of inhibitors.” Fajtová concludes: “I was surprised that it really works – if you inhibit this enzyme, it's toxic for the parasite. So, I see a future in this research, not just for Trichomonas.” She notes that this approach can also be used for plasmodial parasites, which for example, cause malaria.
Keywords
ProTeCT, Trichomonas vaginalis, sexually transmitted disease, proteasome, inhibitors, multiple myeloma, cryo-EM, parasite, malaria
