Naegleria fowleri is a free-living amoeba causing primary amoebic meningoencephalitis, a rapid-onset brain infection in humans with over 97% mortality rate. Despite some progress in the treatment of the disease, there is no single, proven, evidence-based treatment with a high probability of cure. Here we report the chemical library screening and experimental identification of four new compounds with amoebicidal effects against N. fowleri. The chemical library was screened by molecular docking against a homology model of sterol Δ8-Δ7 isomerase (NfERG2). Thirty top-ranking hits were then tested in a cell-based assay for antiproliferative/amoebicidal activities. Eight chemicals exhibited nearly 100% inhibition of N. fowleri at 50 μM, with the EC50 values ranging from 6 to 25 μM. A cell toxicity assay using human HEK-293 cells was also performed. Four of the compounds preferentially kill amoeba cells with no apparent human cell toxicities. These compounds fall into two distinct chemical scaffolds with druglike properties.
The leaf essential oils of twenty-three species of Lauraceae from Monteverde, Costa Rica, have been screened for inhibition of the cysteine protease cruzain. Of these, nine showed promising cruzain inhibitory activity (IC 50 < 100 μg/mL), six showed marginal activity (IC 50 , 100–500 μg/mL), and eight were inactive (IC 50 > 500 μg/mL). The cruzain inhibitory activities of the essential oils can be attributed to active sesquiterpenoid components as well as synergistic effects between two or more components. The sesquiterpenes α-copaene, β-caryophyllene, α-humulene, and germacrene D are active (IC 50 ~5–30 μg/mL) alone, but also show increased activity in combination with other essential oil components.
A series of oxyguanidine analogues of the cysteine protease inhibitor WRR-483 were synthesized and evaluated against cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi. Kinetic analyses of these analogues indicated that they have comparable potency to previously prepared vinyl sulfone cruzain inhibitors. Co-crystal structures of the oxyguanidine analogues WRR-666 (4) and WRR-669 (7) bound to cruzain demonstrated different binding interactions with the cysteine protease, depending on the aryl moiety of the P1' inhibitor subunit. Specifically, these data demonstrate that WRR-669 is bound noncovalently in the crystal structure. This represents a rare example of noncovalent inhibition of a cysteine protease by a vinyl sulfone inhibitor.
The crude chloroform bark extract of an undescribed species of Salacia from Monteverde, Costa Rica, showed cruzain inhibitory activity (IC 50 = 11.4 ± 0.6 μg/mL). Activity-directed chromatographic separation led to isolation of four active friedelane triterpenoids, 25,28-dihydroxyfriedelin, canophyllol, tingenone, and the novel 29-hydroxyfriedelan-3-on-28-al.
Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 μM. There was no toxicity to any of the host cell lines at 10–100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.
We have identified an aromatic amino acid hydroxylase gene from the nematode C. elegans that likely encodes the worm phenylalanine hydroxylase (PheH). The predicted amino acid sequence is most similar to that of other PheH and TrpH proteins. Reporter gene fusions and staining with an antibody to mammalian PheH indicate the gene is expressed in hypodermal cells. A fusion protein expressed in bacteria can convert phenylalanine to tyrosine, and, to a lesser extent, tryptophan to 5-hydroxytryptophan. We hypothesize that the protein is necessary to produce additional tyrosine for protein crosslinking in the nematode cuticle.
Cruzain, an essential cysteine protease
of the parasitic protozoan, Trypanosoma cruzi, is an important drug target for
Chagas disease. We describe here a new series of reversible but time-dependent
inhibitors of cruzain, composed of a dipeptide scaffold appended to
vinyl heterocycles meant to provide replacements for the irreversible
reactive “warheads” of vinyl sulfone inactivators of
cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing
Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine,
and vinyl-2-(N-methyl)-pyridine groups conferred
reversible, time-dependent inhibition of cruzain (Ki* = 0.1–0.4 μM). These cruzain inhibitors
exhibited moderate to excellent selectivity versus human cathepsins
B, L, and S and showed no apparent toxicity to human cells but were
effective in cell cultures of Trypanosoma brucei brucei (EC50 = 1–15 μM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC50 = 5–8 μM). PVHIs represent a new class of cruzain
inhibitors that could progress to viable candidate compounds to treat
Chagas disease and human sleeping sickness.
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