Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design Michael B. Harbuta,1, Bhumit A. Patela, Bryan K. S. Yeungb, Case W. McNamarac, A. Taylor Brightd, Jaime Ballardc, Frantisek Supekc, Todd E. Goldee, Elizabeth A. Winzelerc,f, Thierry T. Diaganab, and Doron C. Greenbauma,2 aDepartment of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104; bNovartis Institute for Tropical Diseases, Singapore 138670; cGenomics Institute of the Novartis Research Foundation, San Diego, CA 92121; dBiomedical Sciences Program, University of California at San Diego, La Jolla, CA 92093; eDepartment of Neuroscience, University of Florida, Gainesville, FL 32610; and fDepartment of Pediatrics, University of California at San Diego, La Jolla, CA 92093 Edited by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved November 15, 2012 (received for review September 17, 2012) Early secretory and endoplasmic reticulum (ER)-localized proteins that redundant protein complexes. During periods of ER stress, ERAD are terminally misfolded or misassembled are degraded by a ubiq- and UPR work together to achieve protein homeostasis within the uitin- and proteasome-mediated process known as ER-associated ER (4–7). degradation (ERAD). Protozoan pathogens, including the causa- P. falciparum lacks conventional transcriptional regulation and tive agents of malaria, toxoplasmosis, trypanosomiasis, and leish- shows little coordinated response to internal or external perturba- maniasis, contain a minimal ERAD network relative to higher tions such as heat stress or drug toxicity (8). Intriguingly, the tran- scription factors that initiate the UPR (IRE1, ATF6) in mammalian eukaryotic cells, and, because of this, we observe that the malaria – parasite Plasmodium falciparum is highly sensitive to the inhibition cells are absent from the genome of P. falciparum (9 11). Lacking of components of this protein quality control system. Inhibitors that any transcriptional response, the down-regulation of translation, identification, and subsequent disposal of misfolded proteins would specifically target a putative protease component of ERAD, signal be the parasite’s major compensatory mechanisms to maintain ER peptide peptidase (SPP), have high selectivity and potency for P. homeostasis during periods of ER stress. falciparum. By using a variety of methodologies, we validate that Here we show through a bioinformatics analysis that the ERAD SPP inhibitors target P. falciparum SPP in parasites, disrupt the pro- pathway of protozoan pathogens, including P. falciparum, is highly ’ tein s ability to facilitate degradation of unstable proteins, and in- simplified relative to mammalian cells, and that P. falciparum is MICROBIOLOGY hibit its proteolytic activity. These compounds also show low therefore vulnerable to small molecules that have been established nanomolar activity against liver-stage malaria parasites and are also to inhibit proteins within the ERAD system. In particular, malaria equipotent against a panel of pathogenic protozoan parasites. Col- parasites within multiple life stages, along with other protozoan lectively, these data suggest ER quality control as a vulnerability of pathogens, are highly sensitive to the inhibition of one of these protozoan parasites, and that SPP inhibition may represent a suit- putative ERAD proteins, signal peptide peptidase (SPP), which able transmission blocking antimalarial strategy and potential pan- we validate to act in this ERAD pathway through a variety of protozoan drug target. techniques, and further suggest that SPP inhibition may be a viable antiparasitic strategy. intramembrane proteolysis | small molecule | target validation Results rotozoan pathogens, including the malaria parasite Plasmodium A Bioinformatics Approach Identifies Minimal ERAD Pathway in Protozoan Pfalciparum, constitute one of the most substantial global public Pathogens, of Which P. falciparum Shows Heightened Susceptibility to health problems faced today. The emergence and spread of drug- Inhibition. A recent analysis of the UPR machinery in protozoan resistant parasites has rendered many of the traditional chemo- parasites revealed a distinct UPR characterized by the absence of therapeutics clinically ineffective in many cases (1). Therefore, the transcriptional regulation and therefore entirely reliant on trans- identification and validation of novel Plasmodium molecular tar- lational attenuation in response to ER stress (12). As a result of gets would greatly facilitate the discovery of new antimalarial drugs. this, Leishmania donovani parasites have heightened sensitization In the pathogenic stage, P. falciparum resides within an eryth- to compounds that promote ER stress, such as DTT (reducing rocyte, which is elaborately remodeled by the parasite to allow the agent) (12). In yeast and mammalian cells, ER stress initiates UPR infected cell to escape immune detection and to facilitate nutrient and ERAD in an intimately coordinated fashion, whereby the uptake and waste disposal in a cell with normally low metabolic induction of one process increases the capacity of the other (5, 7). fi activity. A necessary component of the parasite’s capacity to in- Thus, we reasoned that the modi ed response to ER stress in habit the erythrocyte is the establishment of a unique parasite- protozoan pathogens also likely extends to the ERAD pathway. derived protein secretory network that allows protein trafficking to Our investigation of this hypothesis using standard orthologue destinations beyond the parasite, including a parasitophorous detection tools revealed a striking lack of putative ERAD proteins vacuole and erythrocyte cytosol and plasma membrane (2). in P. falciparum relative to the extensive mammalian network (Fig. The endoplasmic reticulum (ER) is the hub of the secretory 1A and Fig. S1). All functional modules of the ERAD pathway (as pathway, where secretory proteins are folded and targeted for their named in ref. 7), including protein recognition, translocation, respective destination. The ER is sensitive to changes in calcium flux, temperature, and exposure to reducing agents, and, in higher eukaryotes, these stressors elicit transcriptional and translational Author contributions: M.B.H., B.A.P., and D.C.G. designed research; M.B.H. and B.A.P. responses to stabilize already synthesized secretory proteins and performed research; B.K.S.Y., J.B., F.S., T.E.G., E.A.W., T.T.D., and D.C.G. contributed new reagents/analytic tools; M.B.H., B.A.P., C.W.M., A.T.B., E.A.W., and D.C.G. analyzed decrease the load of translocation into the ER, a network collec- data; and M.B.H. and D.C.G. wrote the paper. tively called the unfolded protein response (UPR). In addition to fl the UPR, there exists a coordinated and extensive monitoring The authors declare no con ict of interest. system in the ER to ensure that terminally misfolded proteins or This article is a PNAS Direct Submission. peptides are quickly extracted from this compartment and then Freely available online through the PNAS open access option. degraded via the ubiquitin–proteasome system in the cytosol in a 1Present address: California Institute for Biomedical Research, La Jolla, CA 92037. process known as ER-associated degradation (ERAD) (3). Studies 2To whom correspondence should be addressed. E-mail: [email protected]. in yeast and mammalian cells have shown ERAD to be a complex This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. network that comprises compartmentally restricted and partially 1073/pnas.1216016110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1216016110 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A Ub ligase B Selectivity index OH O ER Stress HRD1 H Substrate Substrate Protein target Inhibitor Pf IC 50 ( M) HepG2 IC 50 ( M) (HepG2/Pf) F N N recognition UBE2G2 N RNF5 extraction H O BIP PDI SEL1 P97 ATX p97 DBeQ 0.31 3.8 12.3 O GRP94 ERDJ5 NPL4 USP13 ERO1 Dislocation PDI 16F16 7.5 2.5 0.33 F LY-411575 SPP UFD1 UBE4B DERLIN1/2 RAD23 DOA1 HSP90 17DMAG 0.061 1.1 18.0 ERAD BiP Ub OH O misfolded Ub Proteasome Epoxomycin 0.0072 0.49 68 H Ub N protein N N Proteasome SPP (Z-LL) 2 1.5 >10 >6.6 H ER cytosol O O Dislocation Substrate Substrate SPP LY-411575 0.10 >10 >100 NITD679 UPR recognition ESTY1/2 Ub ligase extraction ERLEC1 OS9 TRAP gp78 HERP YOD1 PNG1 SPP NITD679 0.065 >10 >100 Translational PERK DERLIN3 SVIP VIMP ERFAD CPVL ERLIN1 TMUB1 SPP NITD731 0.017 >10 >100 regulation eIF2α LONP2 iRhom1 ERLIN2 BRI3BP TRAM1 OH O H H RHBDL4 UBAC2 FAM8A1 UBXD2 O N N Transcription IRE1 VCIP135 N N AUP1 H regulation ATF6 O O O present in P. falciparum absent in P. falciparum NITD731 Fig. 1. P. falciparum has a minimal ERAD pathway and shows heightened susceptibility to inhibition of constituent proteins. (A) A bioinformatics analysis of ERAD in P. falciparum reveals a reduced number of ERAD orthologues in each functional module. Components of ERAD not identified in P. falciparum are sectioned in the shaded boxes, and proteins in white boxes have orthologues identified in P. falciparum.(B) Treatment of P. falciparum and human HepG2 cells with inhibitors of ERAD proteins reveals increased sensitivity of parasites vs. host cells. This is quantified by the selectivity index, which is a ratio of the IC50 for HepG2 cells divided by the IC50 for P. falciparum. ubiquitin ligation, and protein extraction, showed far fewer respectively (Fig. 1B), and not active at all against HepG2 cells,