Function of Leflunomide and Teriflunomide as polyglutamine aggregate inhibitors.

Fuentealba RA, Marasa J, Diamond MI, Piwnica-Worms D, Weihl CC. "Hum Mol Genet. 2011 Nov"

Abstract The emergence and distribute of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the significance of establishing novel antimalarial therapies. Employing a previously produced steady transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a High Throughput Screening assay in 384-well plates.

Assay situations, which includes the proportion of parasitemia and hematocrit, had been optimized. Parameters of assay robustness, such as Z'-value, coefficient variation (CV), and signal-to-background (S/B) ratio, had been determined. The LOPAC(1280) small-compound library was employed to validate this assay.

Our outcomes demonstrated this assay is strong and dependable, having an average Z'-value of >0.7 and CV of < 10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay.


Intracellular protein aggregation is often a frequent pathologic feature in neurodegenerative diseases such as Huntington' disease, amyotrophic lateral sclerosis and Parkinson' disease. Even though progress towards understanding protein aggregation in vitro has been made, small of this understanding has translated to patient treatment.

Moreover, mechanisms controlling aggregate formation and catabolism in cellulo remain inadequately comprehended. One limitation could be the lack of equipment to quantitatively monitor protein aggregation and disaggregation. Here, we developed a protein-aggregation reporter that uses huntingtin exon one that contains 72 glutamines fused towards the N-terminal finish of firefly luciferase (httQ72-Luc).

HttQ72-Luc fails to aggregate unless of course seeded by a non-luciferase-containing polyglutamine (polyQ) protein including Q80-cfp. On co-aggregation, httQ72-luc gets insoluble and loses its enzymatic action. Utilizing httQ72-Luc with Q80(CFP/YFP) as seeds, we screened the Johns Hopkins Medical Compound Library and identified leflunomide, a dihydroorotate dehydrogenase inhibitor with immunosuppressive and anti-psoriatic activities, like a novel drug that stops polyQ aggregation.

Leflunomide and its energetic metabolite teriflunomide inhibited protein aggregation independently of their known function in pyrimidine biosynthesis, considering that neither uridine remedy nor other pyrimidine biosynthesis inhibitors affected polyQ aggregation. Inducible cell line and cycloheximide-chase experiments indicate that these medication prevent incorporation of expanded polyQ into an aggregate.

This research demonstrates the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. As current trials are under-way for teriflunomide in the therapy of several sclerosis, we propose this drug be deemed a doable therapeutic agent for polyQ diseases.