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Rocaglates As Dual-Targeting Agents for Experimental Cerebral Malaria

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Rocaglates As Dual-Targeting Agents for Experimental Cerebral Malaria Rocaglates as dual-targeting agents for experimental cerebral malaria David Langlaisa,b,1, Regina Cencica,b, Neda Moradina,b, James M. Kennedya,b, Kodjo Ayic, Lauren E. Brownd, Ian Crandalle, Michael J. Tarrya, Martin Schmeinga, Kevin C. Kainc, John A. Porco Jr.d, Jerry Pelletiera,b, and Philippe Grosa,b,1 aDepartment of Biochemistry, McGill University, Montreal, QC, H3G 0B1 Canada; bMcGill University Research Center on Complex Traits, McGill University, Montreal, QC, H3G 0B1 Canada; cDepartment of Medicine, Toronto General Hospital-University Health Network, University of Toronto, Toronto, ON, M5G 2C4 Canada; dDepartment of Chemistry, Center for Molecular Discovery, Boston University, Boston, MA 02215; and eLeslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, M5S 3M2 Canada Edited by David A. Fidock, Columbia University, New York, NY, and accepted by Editorial Board Member Carl F. Nathan January 19, 2018 (received for review July 24, 2017) Cerebral malaria (CM) is a severe and rapidly progressing complica- rent WHO-approved standard of care for uncomplicated malaria. tion of infection by Plasmodium parasites that is associated with high ACTs include Art (artesunate, dihydro-Art, artemether, and rates of mortality and morbidity. Treatment options are currently arteether) in combination with other drugs with pharmacokinetic few, and intervention with artemisinin (Art) has limited efficacy, a profiles showing longer circulation half-lives, such as mefloquine, problem that is compounded by the emergence of resistance to Art in lumefantrine, amodiaquine, piperaquine, and sulfadoxine/pyri- Plasmodium parasites. Rocaglates are a class of natural products methamine (15, 16). In contrast, treatment options for CM are derived from plants of the Aglaia genus that have been shown to few and are effective only when infection is detected early before interfere with eukaryotic initiation factor 4A (eIF4A), ultimately block- irreversible neurological symptoms (5–7). Administration of i.v. ing initiation of protein synthesis. Here, we show that the rocaglate artesunate is the WHO-approved first-line therapy, but efficacy CR-1-31B perturbs association of Plasmodium falciparum eIF4A is limited, with 18–30% of CM cases still being fatal in large (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and thera- randomized clinical trials in children and adults (17–19). Because peutic antiplasmodial activity in vivo in mouse models of infection of the lack of equipotent drug alternatives, the recent emergence with Plasmodium berghei (CM) and Plasmodium chabaudi (blood- and spread of Art resistance in different areas of Southeast Asia is a stage malaria), and can also block replication of different clinical iso- major threat to global health (8, 12). Although the mechanisms of lates of P. falciparum in human erythrocytes infected ex vivo, includ- Art resistance in Plasmodium are being characterized (20–24), it is ing drug-resistant P. falciparum isolates. In vivo, a single dosing of unclear that novel treatment options based on this knowledge will CR-1-31B in P. berghei-infected animals is sufficient to provide pro- be immediately forthcoming. Thus, there is a great urgency to de- tection against lethality. CR-1-31B is shown to dampen expression of velop new therapeutic approaches to overcome Art resistance in the the early proinflammatory response in myeloid cells in vitro and damp- malarial parasite. Likewise, increasing effectiveness of Art-based ens the inflammatory response in vivo in P. berghei-infected mice. The therapies may improve the clinical outcome of CM, the most se- dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the vere and most difficult-to-treat complication of malaria. inflammatory response in myeloid cells should prove extremely valu- Recent studies have pointed to initiation of protein synthesis as able for therapeutic intervention in human cases of CM. a novel pharmacological target in P. falciparum for the develop- ment of effective antimalarial drugs (25–27). Considering the Plasmodium | cerebral malaria | severe malaria | protein translation | dual target Significance alaria is a severe threat to global health, with an estimated Severe complications of malaria, such as anemia and cerebral 200–300 million cases annually and 445,000 deaths in 2016; M malaria (CM) (neuroinflammation), are responsible for ∼25% of it accounts for ∼25% of pediatric deaths in endemic areas of infant mortality in certain regions of Africa. Notwithstanding Africa (1–3). Malaria is also a significant threat to immunologically current effective antiplasmodial treatment, drug resistance is on naive travelers visiting or working in these areas (4). It is caused by the increase in the Plasmodium parasites, and therapy to treat infection with members of the Plasmodium parasite family, with CM is nonexistent. Here, we show that rocaglates derived from a clinical symptoms occurring at the blood stage, when Plasmodium natural product originally identified from plants of the Aglaia merozoites rapidly replicate and lyse erythrocytes (RBCs), leading to species, effectively block blood-stage parasite replication in anemia and high fever (2, 3). Furthermore, parasitized RBCs several mouse models and in infected human RBCs. Importantly, (pRBCs) can adhere to the brain microvasculature, causing local neurological inflammation is mitigated and survival is promoted inflammatory and vascular tissue damage, which, in turn, leads to in experimental CM, highlighting the strong potential of this cerebralmalaria(CM),aseveresyndromethatcanrapidlyevolveto class of compounds to treat complicated malaria. fatal encephalomyopathy and is mainly caused by Plasmodium fal- ciparum (5, 6). Despite antimalarial treatment, CM has a high fatality Author contributions: D.L., J.P., and P.G. designed research; D.L., R.C., N.M., J.M.K., K.A., rate (18–40%) and up to one-third of survivors are afflicted with L.E.B., I.C., M.J.T., M.S., K.C.K., and J.A.P. performed research; D.L., M.S., and J.A.P. con- tributed new reagents/analytic tools; D.L., R.C., N.M., J.M.K., K.A., K.C.K., J.P., and P.G. neurocognitive deficits (7). The malaria problem is further compli- analyzed data; and D.L., K.C.K., J.A.P., J.P., and P.G. wrote the paper. – cated by widespread parasite resistance to antimalarial drugs (8 12), Conflict of interest statement: A provisional patent application based on the technology resistance of the Anopheles vector to insecticides (13), and the ab- described in this work has been filed. sence of an effective antimalaria vaccine despite enormous research This article is a PNAS Direct Submission. D.A.F. is a guest editor invited by the Editorial Board. efforts (14). Published under the PNAS license. Treatment of clinical malaria (uncomplicated disease) relies 1To whom correspondence may be addressed. Email: [email protected] or philippe. entirely on antimalarial drugs, with artemisinin (Art) being the [email protected]. most effective drug currently available (8). To avoid emergence of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Art resistance, Art monotherapy is discouraged and has been 1073/pnas.1713000115/-/DCSupplemental. replaced by Art combination therapy (ACT). ACTs are the cur- Published online February 20, 2018. E2366–E2375 | PNAS | vol. 115 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1713000115 Downloaded by guest on October 2, 2021 functional and structural conservation of components of the expressed as recombinant proteins and purified from Escherichia PNAS PLUS translation initiation machinery, we reasoned that known mam- coli (Fig. 1C), and their ability to interact with [32P]-labeled RNA malian translation initiation inhibitors may display activity against was quantified in the presence and absence of two rocaglates: their Plasmodium counterparts, including antimalarial activity in silvestrol and the hydroxamate CR-1-31B (Fig. 1 D and E). Both vivo. One target of this pathway that has been extensively scruti- eIF4A proteins showed similar behavior, including low-level nized for small-molecule discovery is eukaryotic initiation factor RNA binding under control conditions, which was significantly 4F (eIF4F), a heterotrimeric complex required for ribosome re- stimulated by silvestrol and CR-1-31B (approximately six- to 10- cruitment to capped (m7GpppX; X is any nucleotide) mRNAs fold increase). Clamping of eIF4A onto RNA, especially when in (28). eIF4F is composed of eIF4E, the cap-binding subunit; the context of 5′ leaders, has the potential to block ribosome eIF4A, a DEAD-box RNA helicase required to remodel the scanning and could be one contributor to the inhibition of mRNA 5′ leader region; and eIF4G, responsible for recruiting translation imparted by these compounds. We directly tested the the small ribosomal subunit (and associated factors) to the effect of CR-1-31B on parasite protein synthesis by metabolic mRNA (28). Rocaglates, the most characterized and potent labeling using [35S]-methionine incorporation in PbA pRBCs small molecules found to target eIF4F (29), are a class of natural (Fig. 1 F and G). In these experiments, cycloheximide (CHX) products derived from plants of the Aglaia genus (30) that in- was used as a positive control, while the antimalarial drug terfere with eIF4A activity in two ways. Rocaglates cause eIF4A chloroquine (CQ), which does not target translation, was used as to clamp onto RNA, which, if it occurs in the mRNA 5′ leader a negative control (Fig. 1 F and G). The effect of CR-1-31B on region, may act as a steric barrier to the initiation process, and protein synthesis was monitored directedly by SDS/PAGE of they deplete the eIF4F complex of its eIF4A helicase subunit P. berghei-labeled cell extracts and quantified by scintillation (31–34). The net result is reduced initiation complex formation. counting. CR-1-31B could inhibit P. berghei protein synthesis Since eIF4F discriminates between mRNAs during the initiation potently with an IC50 in the low nanomolar range, proving to be a process, rocaglates, unlike general inhibitors of elongation, exert better inhibitor than CHX (Fig.
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