ABSTRACT
We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.
Subject(s)
Antimalarials/chemistry , Plasmodium falciparum/enzymology , Proteasome Endopeptidase Complex/chemistry , Proteasome Inhibitors/chemistry , Protozoan Proteins/antagonists & inhibitors , Artemisinins/chemistry , Bortezomib/chemistry , Drug Resistance, Microbial , Humans , Lactones/chemistry , Oligopeptides/chemistry , Protozoan Proteins/chemistryABSTRACT
UNLABELLED: Jumonji domain-containing protein 3 (JMJD3/KDM6B) demethylates lysine 27 on histone H3 (H3K27me3), a repressive epigenetic mark controlling chromatin organization and cellular senescence. To better understand the functional consequences of JMJD3 its expression was investigated in brain tumor cells. Querying patient expression profile databases confirmed JMJD3 overexpression in high-grade glioma. Immunochemical staining of two glioma cell lines, U251 and U87, indicated intrinsic differences in JMJD3 expression levels that were reflected in changes in cell phenotype and variations associated with cellular senescence, including senescence-associated ß-galactosidase (SA-ß-gal) activity and the senescence-associated secretory phenotype (SASP). Overexpressing wild-type JMJD3 (JMJD3wt) activated SASP-associated genes, enhanced SA-ß-gal activity, and induced nuclear blebbing. Conversely, overexpression of a catalytically inactive dominant negative mutant JMJD3 (JMJD3mut) increased proliferation. In addition, a large number of transcripts were identified by RNA-seq as altered in JMJD3 overexpressing cells, including cancer- and inflammation-related transcripts as defined by Ingenuity Pathway Analysis. These results suggest that expression of the SASP in the context of cancer undermines normal tissue homeostasis and contributes to tumorigenesis and tumor progression. These studies are therapeutically relevant because inflammatory cytokines have been linked to homing of neural stem cells and other stem cells to tumor loci. IMPLICATIONS: This glioma study brings together actions of a normal epigenetic mechanism (JMJD3 activity) with dysfunctional activation of senescence-related processes, including secretion of SASP proinflammatory cytokines and stem cell tropism toward tumors.
