High-throughput quantification of ochronotic pigment formation in Escherichia coli to evaluate the potency of human 4-hydroxyphenylpyruvate dioxygenase inhibitors in multi-well format

4-hydroxyphenylpyruvate dioxygenase (HPD) is a key enzyme in the catabolism of tyrosine and therefore of great importance as a drug target to treat tyrosine-related inherited metabolic disorders (TIMD). Inhibition of this enzyme is therapeutically applied to prevent accumulation of toxic metabolites in TIMD patients. Nowadays an ex-herbicide, nitisinone, is used for this purpose and many more inhibitors are being explored and need to be tested. Here, we describe a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of new human HPD inhibitors in a high-throughput and robust fashion. For this high-throughput screening (HTS) system we rely on the capability of recombinant E. coli that express human HPD, to generate a brown ochronotic pigment after the addition of tyrosine, whereafter this brown pigment can be quantified in a very specific and sensitive way by spectrophotometric analysis. Altogether, this robust and simple HTS screening system can be described as non-harmful, non-laborious and cost-effective with the aim to identify and evaluate novel therapeutic human HPD inhibitors for the treatment of TIMD.• This robust high-throughput screening system enables rapid identification and evaluation of potential inhibitors of human 4-hydroxyphenylpyruvate dioxygenase.• Simple and fast colorimetric quantification of the formation of ochronotic pigment.


Method details
The human enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD) plays an important role in the tyrosine catabolic pathway and as such in the management of tyrosine-related inherited metabolic disorders (TIMD), i.e. hereditary tyrosinemia (HT) type I and alkaptonuria (AKU). HPD is an iron-(II)dependent, non-haem, oxygenase and catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) to homogentisate (HGA), which comprises the second step in the catabolism of tyrosine [1][2][3] . Inhibition of this key enzyme prevents the formation of HGA and following toxic intermediates, and as such alleviates the harmful phenotype of these TIMD [1 , 4] ( Fig. 1 ). Such an inhibitor, that is used to treat TIMD, is nitisinone [2-(2-nitro-4-trifluoromethylbenzoyl-1,3-cyclohexadione; NTBC], a member of the β-triketone family of herbicides (i.e. mesotrione, sulcotrione, and tembotrione) but many other inhibitors are being explored that may pave the way to more TIMD treatment options.
In search for new inhibitors of HPD, a screening system that allows to identify and evaluate the potency of new HPD inhibitors for the development of TIMD therapies is necessary. To fill this gap, we have developed a straightforward, colorimetric and non-laborious high-throughput screening (HTS) assay in bacteria which depends on the activity of recombinant expressed human HPD [1] . For this HTS, Escherichia coli is the preferred organism to use for the production of recombinant enzymes because genetic engineering as well as HTS setups in microtiter plates are well-established [5 , 6] . Furthermore, due to the natural presence of transaminases, E. coli is also capable of easily converting tyrosine to HPP, the first step in the metabolism of tyrosine, which is then further metabolized into HGA by the expressed human HPD enzyme. Subsequently, accumulated HGA will auto-oxidize to benzoquinone acetic acid due to the absence of HGD in E. coli and self-polymerize to produce a melanin-like ochronotic pigment. The melanin-like ochronotic pigment is also known as pyomelanin and exhibits a characteristic brown color. Our newly developed colorimetric bioassay is based on the quantification of pyomelanin, derived from tyrosine under aerobic and physiological conditions relevant to humans. In the presence of an HPD-inhibitor, this ochronosis process will be reduced or even prevented when HPD activity is blocked [7][8][9] ( Fig. 2 ).
Other methods, using a coupled enzyme reaction to perform high-throughput screening have been published but these are not the most efficient. Compared to our HTS assay, coupled enzyme HTS systems are more laborious, time-consuming and complex [10][11][12][13] .
Altogether, the main purpose of this bacterial whole-cell HTS assay based on human HPD is to analyze the inhibitory capacity of new potent inhibitors of the human HPD enzyme and thereby to evaluate their therapeutic potential [1] . Tyrosine derives from the diet or hydroxylation of phenylalanine by phenylalanine hydroxylase and is converted to 4hydroxyphenylpyruvate (HPP) by tyrosine aminotransferase, the first step in the tyrosine catabolism. Further, 4-HPD catalyzes the formation of homogentisate (HGA) out of HPP, which is converted to maleylacetoacetate (MAA) by homogentisate dioxygenase. Excess of HGA can accumulate, auto-oxidize and polymerize to an ochronotic pigment, i.e. pyomelanin. The penultimate step comprises the conversion of MAA to fumarylacetoacetate (FAA) by maleylacetoacetate isomerase. Lastly, FAA will be broken down to fumarate and acetoacetate by the final enzyme fumarylacetoacetate hydrolase (FAH). When FAH functionality is deficient, MAA and FAA can also be converted into succinylacetone which inhibits the porphobilinogen synthesis. 5 different TIMD are related to an inefficient or deficient enzyme in this pathway.  -Pipette (10 μL -100 μL -10 0 0 μL) (Eppendorf).

Stock solutions and reagents
-Lysogeny Broth (LB) medium : 5 g yeast extract (Sigma-Aldrich), 10 g peptone from casein, acid digest (Sigma-Aldrich) and 10 g sodium chloride (Sigma-Aldrich) are dissolved in 900 mL ultrapure water. Adjust the pH of the solution to 6.7 General safety note E. coli C43 (DE3) is classified as Biosafety Level 1 (BSL1), which means they pose a minimal potential threat to laboratory workers and do not cause infection or disease in healthy adults.

Protocol
Before commencing any work, sterilize the workspace using 70% ethanol. Always use personal protective equipment!

Inoculation and incubation of bacterial cells containing 4-hydroxyphenylpyruvate dioxygenase
The importance of a pre-culture is that cell growth should be monitored before induction and this can be accomplished by using LB medium, a nutrient-rich media commonly used to culture bacteria. Leaky expression of the toxic mRNA/protein, resulting in slower growth rate of the bacterial cells, can be avoided by the addition of 1% w/v glucose, i.e. a carbon source [5] .
Inoculation and growth of the preculture -Add 6 mL LBG to a test tube and add kanamycin to a final concentration of 50 μg/mL. -From the frozen glycerol stock (25%) of E. coli C43 (DE3) -EV and C43 (DE3) -HPD, a pinch is taken by means of a sterile pipette tip and dropped into the liquid LBG + antibiotic. Swirl the tube and close with a non-airtight cap. -The preculture is incubated and grown overnight (18 ± 2 h) at 250 rotations per minute (rpm) and 37 °C in a shaking incubator.

Colorimetric bioassay high-throughput screening
After overnight inoculation (18 ± 2 h), the preculture is diluted in fresh medium as it allows the cells to return to their logarithmic growth phase whereafter they reach an optimal cell density for induction of protein expression [14] . As soon as HPD expression is induced, HPP will be converted to HGA which will result in the formation of ochronotic pigment and the medium will turn brown. In case you have a potent inhibitor of HPD, the medium remains yellowish at a certain concentration. NOTE: Ensure that the overnight culture is well mixed and that the cells are not settled before taking an aliquot. Use clean cuvettes and insert it in the proper orientation in the Biophotometer.
-Use the overnight culture to inoculate a volume of LB ( + add kanamycin to a final concentration of 50 μg/mL) in an Erlenmeyer culture flask to an OD 600 = 0.1 by using following formula: NOTE: At the mid-log phase the bacteria are in the exponential growth phase which means they express chaperones that help to remove misfolded proteins and will reduce formation of inclusion bodies. If you induce too early, the protein yield will be reduced and/or bacterial growth will be affected.
-Add sodium-L-tyrosine to a final concentration of 0.75 mg/mL in the expression culture in the Erlenmeyer flask.

Spectroscopic quantification of ochronotic pigment
Depending on the potency of the inhibitor and its concentration, the formation of the ochronotic pigment can be visually seen after 24 h as a brown colored medium. (4) Supplementary material and/or Additional information : Human HPD is an important drug target that is involved in HT1, however, a simple and robust HTS assay is lacking in drug discovery. As such, we have developed an HTS colorimetric whole-cell bioassay that can be used as a first and fast screening and especially for labs with limited funding. Many advantages are related to this assay. It is a simple, cheap and fast assay as the induction time of the recombinant HPD enzyme is only 24 h where other assays have induction times of 48 or even 72 h [7] . To allow high-throughput screening, the bioassay was adapted to the 96-well microplate format. Previously reported 96-well plate format assays are based on the coupled enzyme reaction of the isolated HPD enzyme with the crude extract of HGD and the formation of maleylacetoacetate, measured at 318 nm. This is also a reliable assay, however, it is laborious, time-consuming and there is a need for addition of exogenous co-factors what makes it more expensive [11-13 , 15-18] . Another advantage of this whole-cell bioassay is that you can use L-tyrosine as a substrate instead of the much more expensive HPP due to the natural presence of tyrosine aminotransferases in E. coli [19][20][21][22] . Furthermore, we can conclude that we have developed a very robust HTS assay that was rigorously validated both for pharmacological relevance, as well as for robustness of assay performance according to the NCGC guidelines [1] . This validation consists of a 3-day Plate Uniformity study and a Replicate-Experiment study, and, in addition, signal variability and spatial uniformity were assessed. Moreover, this HTS assay has been executed in two different labs, independent from each other, without undergoing any substantive changes. The plate uniformity and signal variability results, in 96-well microplate format, are represented in Table 1 and were used to calculate the CVs and Z factor values, taking into account 1 replicate per inhibitor or inhibitor concentration, in order to represent a real-life HTS assay. Maximum signal plate CVs were 1.82-4.06%, midpoint signal CVs were 2.74-7.56%, and minimum signal CVs were 2.28-5.86%. These CVs all pass the < 20% criterion. Plate Z values were 0.87 ± 0.03 whilst the recommended acceptance criterion is Z factor ≥0.40 and related to the screening it can be seen as an excellent and robust assay. The inter-plate tests, all within-day fold shifts < 2 and all average (between-) day fold shifts < 2 also meet the criteria [1 , 23] .
Due to evaporation during the incubation periods or plate stacking, plate edge or side effects can occur in HTS assays and as such spatial uniformity needs to be examined. Plates that do not exhibit material edge or drift effects are the overall requirement. Row and column drift were assessed using the high and medium signals from left-to-right and top-to-bottom. No drift effects that exceed 20% were observed, i.e. maximum drift observed for the high signal and medium signal was 6.23% and 13.79% resp., so material drift effects can be excluded [1] . We foresee that the throughput and robustness can even be further improved by miniaturizing the assay to 384 and 1536 well-format using adjusted equipment including automatic pipetting systems and liquid-handling robotics. Table 1 Results of the (intra) plate uniformity study. H, high signal; M; medium signal; L, low signal: CV, coefficient of variation; Mid. %, normalised mid signal; SW, signal window; Z , Z factor. All max signal and all mid signal (unnormalised) CVs are < 20%. All normalised mid signal (mid. %) SD's < 20, SW's type = "Other" > 2 and Z factors > 0.4. All min (low) SD's < Min (max (High) SD, mid SD). Z and CV values were calculated taking into account 1 replicate per compound or concentration, thereby mimicking a real-life HTS assay. There is one major concern involving variable absorption effects of different drugs in E. coli , causing incomparable inhibitory effects as knowledge of the influx and efflux transporters for xenobiotic, present in bacteria, is remarkably limited. However, a recent study revealed that, besides the diffusion of xenobiotics across whatever phospholipid bilayer, many transporters interact with xenobiotics [24] . If E. coli cells are capable of taking up large antibiotic molecules, we assume that the absorption (and absorption rate) of small chemical molecules, i.e. triketone inhibitors and derivatives, is not affected. Furthermore, the aim of our HTS system is not to establish therapeutical concentrations of each inhibitory compound. This assay is used as a first and fast HTS system to screen a large library of different inhibitors of human HPD in the search of possible hits.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.