Abstract
Background: Human 4-hydroxyphenylpyruvate dioxygenase (HPD) is the therapeutical target for the treatment of tyrosinemia type 1 (HT1). HT1 patients are unable to break down tyrosine due to a defect in fumarylacetoacetase (FAH), the final enzyme of the tyrosine catabolism. As a consequence toxic derivatives will accumulate and cause mainly liver damage. The life-saving drug, nitisinone, inhibits the upstream HPD enzyme, as such production of toxic metabolites is prevented. Due to possible side-effects of nitisinone, new HPD inhibitors are being developed. We aim to develop a bacterial whole-cell high-throughput screening assay that allows to analyse the inhibitory capacity of new HPD inhibitors.
Method: A colorimetric assay was developed using recombinant Escherichia coli expressing HPD. Human HPD was subcloned into pET42b(+) and subsequently several E. coli strains were transformed with the construct to identify the most optimal strain for reliable expression of HPD. Next, growth and expression conditions were optimised. Finally, as a proof-of-principle, dose-response inhibition curves were generated for nitisinone and the herbicides mesotrione, sulcotrione and tembotrione.
Results: E. coli C43(DE3) was the most optimal strain to reliably express human HPD. Optimisation of the growth and expression conditions showed that expression should be induced in glucose-free LB medium with 1 mM isopropyl-β-D-thiogalactopyranoside and incubated at 37°C for 24h. But human HPD has a short half-life under applied conditions and therefore the substrate sodium-L-tyrosine is added at the start of the induction. The assay was developed and challenged by a concentration range of nitisinone, mesotrione, sulcotrione and tembotrione. Colorimetric analyses were performed at OD405 as HPD causes the medium to turn brown due to the formation of a melanin-like pigment. Reliable dose-response inhibition curves were obtained.
Conclusion: This study resulted in a robust high-throughput 96-well MTP screening assay allowing the identification and evaluation of novel therapeutic inhibitors of the human HPD enzyme.
Method: A colorimetric assay was developed using recombinant Escherichia coli expressing HPD. Human HPD was subcloned into pET42b(+) and subsequently several E. coli strains were transformed with the construct to identify the most optimal strain for reliable expression of HPD. Next, growth and expression conditions were optimised. Finally, as a proof-of-principle, dose-response inhibition curves were generated for nitisinone and the herbicides mesotrione, sulcotrione and tembotrione.
Results: E. coli C43(DE3) was the most optimal strain to reliably express human HPD. Optimisation of the growth and expression conditions showed that expression should be induced in glucose-free LB medium with 1 mM isopropyl-β-D-thiogalactopyranoside and incubated at 37°C for 24h. But human HPD has a short half-life under applied conditions and therefore the substrate sodium-L-tyrosine is added at the start of the induction. The assay was developed and challenged by a concentration range of nitisinone, mesotrione, sulcotrione and tembotrione. Colorimetric analyses were performed at OD405 as HPD causes the medium to turn brown due to the formation of a melanin-like pigment. Reliable dose-response inhibition curves were obtained.
Conclusion: This study resulted in a robust high-throughput 96-well MTP screening assay allowing the identification and evaluation of novel therapeutic inhibitors of the human HPD enzyme.
| Translated title of the contribution | Ontwikkeling van een high-throughput bacteriële inhibitie analyse voor humaan 4-hydroxyfenylpyruvaat dioxygenase in de context van tyrosinemie type 1 |
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| Original language | English |
| Publication status | Unpublished - 2018 |
| Event | PhD day - VUB Campus Jette, Jette, Belgium Duration: 20 Mar 2018 → 20 Mar 2018 |
Conference
| Conference | PhD day |
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| Country/Territory | Belgium |
| City | Jette |
| Period | 20/03/18 → 20/03/18 |