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The Proteasome As a Target: How Not Tidying up Can Have Toxic Consequences for Parasitic Protozoa Elizabeth A

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The Proteasome As a Target: How Not Tidying up Can Have Toxic Consequences for Parasitic Protozoa Elizabeth A COMMENTARY COMMENTARY The proteasome as a target: How not tidying up can have toxic consequences for parasitic protozoa Elizabeth A. Winzelera,1 and Sabine Ottiliea With modern drug discovery technologies, there are good pharmacokinetics, and is now being progressed opportunities to discover drugs that are uniquely toward human clinical trials. suited for treatment of specific parasitic diseases and In addition to its potential to radically improve the have few of the liabilities of older, historical medicines. treatment options for VL, another noteworthy feature In PNAS, Wyllie et al. (1) used state-of-the-art methods of the work is the elegant and thorough approach that to identify a clinical candidate for leishmaniasis and to was used to determine the mechanism of action of discover its mechanism of action. GSK3494245 in parasites. Suspecting that the mech- Leishmaniasis is a neglected parasitic infectious anism of action might be shared across closely related disease that can have different clinical manifestations parasites, Wyllie et al. (1) first tested a compound depending on the parasite species. Visceral leishman- from the series (compound 7) against a genome- iasis (VL) (also known as kala-azar or black fever), which wide Trypanosoma brucei RNA interference (RNAi) li- is caused by Leishmania donovani and Leishmania brary (5). This RNAi library consists of ∼750,000 clones, infantum, is the most severe form and marked by a each transformed with one RNAi construct under the swollen spleen. It is a significant cause of morbidity control of a tetracycline-inducible promoter and cov- in areas where the insect vector, the sandfly, is present ers >99% of the ∼7,500 nonredundant T. brucei gene and causes up to 20,000 to 65,000 annual deaths (ref. set (5). To identify parasite knockdown clones that 2andhttps://www.who.int/en/news-room/fact-sheets/ showed increased resistance to the compound, the au- detail/leishmaniasis). Treatments for VL are suboptimal thors isolated DNA from the library before and after and consist of a variety of highly toxic historical mole- tetracycline induction in the presence of compound cules that would likely not be licensed if developed 7. They then created samples for next-generation se- today. In addition, many require long treatment times quencing by amplifying DNA fragments containing (up to 30 d), some require hospitalization, most are RNAi cassette-insert junctions in semispecific PCR reac- not orally bioavailable, and resistance to some is sus- tions in a process called RNAi target sequencing (RIT- pected. Miltefosine, a repurposed breast cancer med- seq) (6). Sequencing these samples showed that some ication, is one of the best treatment options but still of the parasites that were resistant to compound 7 bore requires long dosing regimens and shows variable ef- interfering RNAs that mapped to the protein degradation ficacy against different parasites (reviewed in ref. 3). pathway. There is currently no vaccine. Because resistance does not necessarily reveal With a goal of finding better treatment alterna- a target (genetically knocking down a true target tives, Wyllie et al. (1) first took a set of compounds that should theoretically render parasites more sensi- was active against another parasite, Trypanosoma tive to an inhibitor, rather than resistant), Wyllie cruzi, the etiological agent of Chagas’ disease and a et al. (1) next created drug-resistant parasite lines cousin of Leishmania spp., and then retested these using in vitro evolution. Because the interpretation compounds in a laborious, high-content-imaging in- of whole-genome sequencing data for Leishmania tracellular macrophage replication assay against L. parasites is thought to be messier than for other donovani (4), identifying a weakly active compound parasites, and because of previous publications that showed promise. With several modifications, they showing that the proteasome is a druggable target were able to turn this screening hit into a compound in Leishmania (7), Wyllie et al. examined the genome that was suitable for target identification studies and, sequence of candidate genes in the proteasome ultimately, into a clinical candidate (GSK3494245, also pathway. Selective sequencing revealed that all re- described as compound 8) that could be tested in a sistant mutants bore homozygous mutations within mouse model of VL. This orally bioavailable compound the genes encoding the β4andβ5 subunits of the worked well in a mouse model of VL. Furthermore, parasite proteasome. To confirm the proteasome as GSK3494245 demonstrates a desirable safety profile, the target, the team next overexpressed the subunits aDepartment of Pediatrics, University of California San Diego School of Medicine, La Jolla, CA 92093 Author contributions: E.A.W. and S.O. wrote the paper. The authors declare no conflict of interest. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). See companion article on page 9318 in issue 19 of volume 116. 1To whom correspondence should be addressed. Email: [email protected]. Published online May 13, 2019. 10198–10200 | PNAS | May 21, 2019 | vol. 116 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1904694116 Downloaded by guest on September 28, 2021 Parasites Structure-guided drug discovery A O N N Target NH Discovery O O Phenotypic Screen Initial optimization Clinical candidate B Caspase-like activity 1 1 Trypsin-like 2 2 6 3 5 4 20S alpha 7 7 3 beta 3 proteasome ring 6 3 6 ring 6 5 4 4 4 5 5 6 3 5 4 Chymotrypsin-like 6 3 5 4 C O O GSK3494245 H N OH O DDD01305143 N N B N H N Compound 8 H H N O O OH N O O NH H MPI-1 O O PfIC50 = 120 nM µ PfIC50 = 3.27 nM HepG2 LD50 = 6.7 M Analog 18 N N HepG2 CC50 = 1240 nM F N O Intramacrophage N O N O IC = 1.6 uM H 50 N L. donovani EC50 = 14.6 nM O N N THP-w EC >50 µM H H 50 O N O PKS21004 PfIC50 = 4.6 nM NH HepG2 CC50 = 3670 nM O N N N O N H O N N H N S N N F HN H O GNF6702 NH O H O N L. donovani EC50 = 18 nM µ Macrophage CC50 > 50 M WLW-vs PfIC50 = 290 nM µ HFF CC50 = 12.8 M Fig. 1. (A) Strategy for finding high-value treatments for neglected diseases. Compounds are first discovered using phenotypic screens, and active compounds are then subjected to several rounds of improvement. The target of active compounds is next identified using methods such as in vitro evolution and whole-genome analysis or using over-/underexpression libraries (5). Knowledge of the target can then lead to better leads with high specificity. (B) Diagram of the 20S proteasome subunit showing the position of β subunits that catalyze protein degradation and which are inhibited by GSK3494245 (compound 8). (C) Structures of various inhibitors that show specificity for parasite proteasomes over human ones. Compounds are described in detail elsewhere: GSK3494245 (1), HT1171 (11), MPI-1 (13), analog 18 (17), GNF6702 (7), PKS21004 (15), and WLW- vs (14). GNF6702 and GSK3494245 are active against Leishmania spp., while others act against the human malaria parasite P. falciparum or Mycobacteria tuberculosis. THP-w, HepG2s, macrophages, Vero76, and human foreskin fibroblasts (HFF) are mammalian cells used in toxicity tests. Amounts that give a 50% reduction in viability in whole-cell dose–response assays include effective concentration (EC50), lethal dose (LD50), and cytotoxicity concentration (CC50). Winzeler and Ottilie PNAS | May 21, 2019 | vol. 116 | no. 21 | 10199 Downloaded by guest on September 28, 2021 and showed that overexpression conferred resistance, as did achieved and that the old dogma that pathogens cannot share editing the point mutations into the genome. targets with humans is untrue. Another recent scientific advance that has allowed the discov- An open question is whether resistance will appear readily ery of better treatments for neglected parasitic diseases has been during treatment, given that Wyllie et al. (1) were able to create the development of high-resolution cryoelectron microscopy parasite lines that showed 100X resistance. It is also not entirely (cryo-EM). This powerful method can be used to solve structures clear if the mutated genes would have been as easily identified if of macromolecular complexes such as the proteasome, allowing a the proteasome were not a known target for trypanosomes (see detailed understanding of how compounds bind. To further refs. 7 and 16). On the other hand, work in Plasmodium spp. has confirm on-target activity, Wyllie et al. (1) used cryo-EM to show shown that proteasome mutations can readily be discovered with- that compound 8 bound the Leishmania tarentolae 20S protea- out prior knowledge using in vitro evolution and whole-genome some in a previously undiscovered inhibitor site that lies between analysis methods (13, 15), and the proteasome appears to be a the β4 and β5 proteasome subunits. The mutations suggested that high-value target for malaria as well (Fig. 1). Malaria PIs synergize GSK3494245 would inhibit the chymotrypsin-like activity of the with artemisinin derivatives, which are recommended for the β5 subunit, and this was confirmed in biochemical assays. treatment of malaria, and this could make them even more attrac- tive candidates for drug development (14). One improvement In PNAS, Wyllie et al. used state-of-the-art between the compounds identified for VL and those which have methods to identify a clinical candidate for activity in malaria parasites may be the cost of goods as well as oral bioavailability. Compound 8 can be made with fewer than leishmaniasis and to discover its mechanism six synthetic steps and should thus be affordable. Another in- of action. teresting point is the structural differences between different PIs that have been discovered for different parasites (Fig.
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