SignificanceProtozoal proteasome is a validated target for antimalarial drug development, but species selectivity of reported inhibitors is suboptimal. Here we identify inhibitors with improved selectivity for malaria proteasome β5 subunit over each active subunit of human proteasomes. These compounds kill the parasite in each stage of its life cycle. They interact synergistically with a β2 inhibitor and with artemisinin. Resistance to the β5 inhibitor arose through a point mutation in the nonproteolytic β6 subunit. The same mutation made the mutant strain more sensitive to a β2 inhibitor and less fit to withstand irradiation. These findings reveal complex interplay among proteasome subunits and introduce the prospect that combined inhibition of β2 and β5 subunits can afford synergy and thwart resistance.
Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively, an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells, and to a lesser extent in activated human T cells. Reversible, β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells.
The Plasmodium proteasome (Pf20S) emerged as a target for antimalarials. Pf20S inhibitors are active at multiple stages of the parasite life cycle and synergize with artemisinins, suggesting that Pf20S inhibitors have potential to be prophylactic, therapeutic, transmission blocking as well as useful for combination therapy. We recently reported asparagine ethylenediamines (AsnEDAs) as immunoproteasome inhibitors and modified AsnEDAs as selective Pf20S inhibitors. Here we report further structure-activity relationship study of AsnEDAs for selective inhibition of Pf20S over human proteasomes. Additionally, we show a new mutation that conferred resistance to AsnEDAs and collateral sensitivity to an inhibitor of the Pf20S β2 subunit, same as previously identified resistant mutation. This resistance could be overcome through the use of structureguided inhibitor design. Collateral sensitivity to inhibitors among respective proteasome subunits *
Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages—erythrocytic, gametocyte, liver, and gamete activation stages—indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission‐blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time‐dependent inhibition of the β5 subunit of the Pf20S, kills artemisinin‐sensitive and artemisinin‐resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum‐infected mice.
With
over 200 million cases and close to half a million deaths
each year, malaria is a threat to global health, particularly in developing
countries. Plasmodium falciparum, the
parasite that causes the most severe form of the disease, has developed
resistance to all antimalarial drugs. Resistance to the first-line
antimalarial artemisinin and to artemisinin combination therapies
is widespread in Southeast Asia and is emerging in sub-Saharan Africa.
The P. falciparum proteasome is an
attractive antimalarial target because its inhibition kills the parasite
at multiple stages of its life cycle and restores artemisinin sensitivity
in parasites that have become resistant through mutation in Kelch
K13. Here, we detail our efforts to develop noncovalent, macrocyclic
peptide malaria proteasome inhibitors, guided by structural analysis
and pharmacokinetic properties, leading to a potent, species-selective,
metabolically stable inhibitor.
Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders nonreplicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure−activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.
The
immunoproteasome (i-20S) has emerged as a therapeutic target
for autoimmune and inflammatory disorders and hematological malignancies.
Inhibition of the chymotryptic β5i subunit of i-20S inhibits
T cell activation, B cell proliferation, and dendritic cell differentiation
in vitro and suppresses immune responses in animal models of autoimmune
disorders and allograft rejection. However, cytotoxicity to immune
cells has accompanied the use of covalently reactive β5i inhibitors,
whose activity against the constitutive proteasome (c-20S) is cumulative
with the time of exposure. Herein, we report a structure–activity
relationship study of a class of noncovalent proteasome inhibitors
with picomolar potencies and 1000-fold selectivity for i-20S over
c-20S. Furthermore, these inhibitors are specific for β5i over
the other five active subunits of i-20S and c-20S, providing useful
tools to study the functions of β5i in immune responses. The
potency of these compounds in inhibiting human T cell activation suggests
that they may have therapeutic potential.
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