A Phenotypic Based Target Screening Approach Delivers New

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A phenotypic based target screening approach delivers new antitubercular CTP synthetase inhibitors Marta Esposito, Sára Szadocka, Giulia Degiacomi, Beatrice Silvia Orena, Giorgia Mori, Valentina Piano, Francesca Boldrin, Julia Zemanova, Stanislav Huszár, David Barros, Sean Ekins, Joël Lelièvre, Riccardo Manganelli, Andrea Mattevi, Maria Rosalia Pasca, Giovanna Riccardi, Lluis Ballell, Katarína Mikušová, and Laurent R. Chiarelli ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 6, 2017

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ACS Infectious Diseases

A phenotypic based target screening approach delivers new antitubercular CTP synthetase inhibitors.

Marta Esposito†+, Sára Szadocka‡+, Giulia Degiacomi#, Beatrice S. Orena†, Giorgia Mori†, Valentina Piano†, Francesca Boldrin#, Júlia Zemanová‡, Stanislav Huszár‡, David Barros§, Sean Ekins∆⊥, Joel Lelièvre§, Riccardo Manganelli#, Andrea Mattevi†, Maria Rosalia Pasca†, Giovanna Riccardi†, Lluis Ballell§, Katarína Mikušová‡, Laurent R. Chiarelli†*.

†

Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, via

Ferrata 9, 27100 Pavia, Italy ‡

Department of Biochemistry, Faculty of Natural Sciences, Comenius University in

Bratislava, Mlynská dolina CH1, 84215 Bratislava, Slovakia. #

Department of Molecular Medicine, University of Padova, via Gabelli 63, 35121 Padova,

Italy. §

Diseases of the Developing World, GlaxoSmithKline, Calle Severo Ochoa 2, 28760 Tres

Cantos, Madrid, Spain ∆

Collaborative Drug Discovery Inc., 1633 Bayshore Highway, Suite 342, Burlingame, CA

94010, USA.

E-mail: [email protected]

ACS Paragon Plus Environment


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Despite its great potential the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 µM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labelling experiments, demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

Keywords: drug discovery; phenotypic screening; target-based screening; Mycobacterium tuberculosis; CTP synthetase; pyridine-thiazole.


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Although there has been extensive investment over the last few decades, tuberculosis (TB) still remains an urgent global health issue, with more than 10.4 million estimated new cases and about 1.8 million deaths each year1. Moreover, the increasing spread of Mycobacterium tuberculosis (Mtb) multidrug resistant (MDR) strains (with about 480,000 cases in 2015) of which 10% have been reported as extremely drug resistant (XDR), has led to an increased requirement for new drugs, with new mechanisms of action2. After the first complete sequencing of the Mtb genome, there were great expectations derived from target-based approach for TB drug discovery which proved to be mostly unsuccessful. High-throughput screening (HTS) has been the preferential method adopted for the identification of new hits from large compound libraries3. In the case of Mtb, wholecell screening using HTS can provide compounds able to cross the mycobacterial cell envelope, overcoming one of the main challenges for TB drugs. The large number of phenotypic screening efforts performed until now led to the identification of several active molecules, however, the mechanism of action of many of them is still not known4. The elucidation of the cellular target is an important component of the hit to lead optimization process, enabling the assistance of biochemistry and structural biology to characterize the mechanism of action and to better assess at molecular level potential host cell toxicities. However, for several reasons (e. g. difficulty in obtaining resistant mutants etc.) this task is not trivial4. A great improvement arose from the introduction of next generation sequencing platforms, allowing wide and intensive use of genomics in combination with chemical library screening, for identification of the cellular target of novel compounds4, 5. Furthermore, it is possible to combine target-based and phenotypic screening strategies6. In this context, GlaxoSmithKline (GSK) has identified and released the structures and activities of a large number of anti-mycobacterials (https://www.ebi.ac.uk/chembl/)7 verified to be active



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against M. tuberculosis H37Rv cells and showing low toxicity against human cell-lines8, 9. Different screens of this set allowed the identification of the cellular targets of several hits, not only by the classical genomic-based target assignment10, but also through different strategies11. For instance, a new series of leads targeting DprE1 has been recently identified through the screening of this set against a dprE1 over-expressing strain of Mycobacterium bovis BCG12. Moreover, since new drug candidates should possess a novel mechanism of action to ensure effectiveness against Mtb MDR and XDR strains2,13, the drug discovery exploitation of the latest validated targets seems a reasonable starting point. In this respect, the novel TB drug target CTP synthetase PyrG14 could represent a good platform for target based screening of antitubercular compound libraries. This essential enzyme was demonstrated to be druggable, and, being involved in several biochemical pathways, its inhibition has proven to affect several aspects of mycobacterial physiology14. With this aim, we exploited the GSK antimycobacterial compound set (GSK TB-set) library to explore the possibility of finding new antitubercular CTP synthetase inhibitors.

Results and Discussion Screening of the GSK TB-set against PyrG affords new inhibitors A previous whole cell phenotypic screening of GSK compounds identified a set of 177 molecules (GSK TB-set) active against M. tuberculosis cell growth, whose targets are largely unknown8. For this reason, to identify compounds targeting PyrG, the GSK TB-set was assayed against the recombinant M. tuberculosis enzyme. The assays, performed at a final concentration of 100 µM for each compound, and at subsaturating concentrations of ATP, identified 16 compounds (9% hit rate) inhibiting more than 50% of enzymatic activity (Table 1). ACS Paragon Plus Environment

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Table 1: compounds of the GSK TB-set showing activity against M. tuberculosis PyrG PyrG

PyrG GSK ID

structure

inhibition GSK ID

structure

a

inhibition (%)a

(%) 1570606A

90.8

735816A

62.6

735826A

79.2

829969A

60.5

920684A

74.3

275984A

59.5

1611550A

66.9

345724A

53.8

1729177A

66.8

547543A

55.1

1668869A

66.7

921190A

52.2

445886A

68.1

636544A

51.1

920703A

60.0

2200160A

51.1

a

Enzyme assays were performed in the presence of 100 µM of each compound

About half of these active compounds, in particular the most active, were characterized by a 4-(pyridin-2-yl)thiazole group, suggesting a particular affinity of PyrG for this moiety. Among them, GSK1570606A (1), GSK920684A (2), and GSK735826A (3) (Table 1), (Mtb Minimal Inhibitory Concentration, MIC: 16 µM, 7.6 µM and 1.4 µM, respectively) showed ACS Paragon Plus Environment


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higher inhibitory effects against PyrG activity (IC50 values of 2.9 ± 0.6 µM, 23.3 ± 2.2 µM and 19.7 ± 2.2 µM, for compounds (1) (2) and (3), respectively), and were selected for further investigation. Firstly, in an attempt to gain insight into the binding between PyrG and the three inhibitors, a computational analysis of the possible poses of the compounds was performed by docking in the PyrG ATP binding site14 (Fig. 1) using Discovery Studio 4.1 (Biovia, San Diego, CA). The superimposition with the UTP molecule shows a partial overlap. The 4(pyridin-2-yl)thiazole moiety is in a comparable position for (2) and (3), while (1) has a flipped orientation in the protein. However, all 3 molecules have phenyl rings that are suggested to pi-stack with Arg223. This superimposition is very similar to that of the 11426026 compound, a previously described PyrG inhibitor, with a IC50 value (35 µM) similar to that of the three GSK compounds, which has been demonstrated to be competitive towards the ATP binding site14.


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Figure 1. Docking GSK compounds in CTP synthetase. (A) compound (1) libdock score 88.16; (B) compound (2) libdock score 92.96; (C) compound (3) libdock score 87.23. All molecules (grey) are compared to UTP (Yellow); (D) compound 11426026, previously reported as a PyrG ATP binding site competitive inhibitor.



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To confirm the hypothesis that the compounds bind to the ATP site, PyrG steady state kinetic analysis was performed in the presence of different concentrations of the compounds. The analysis revealed that all the compounds behave as competitive inhibitors towards the ATP binding site, showing apparent Ki values of 3.5±0.4 µM (1); 22.0±0.6 µM (2); and 16.3±0.5 µM (3) (Fig. 2). By contrast, all of them were uncompetitive toward the pyrimidine nucleotide binding site (Fig. S1).

Figure 2. Double reciprocal plots of PyrG kinetic analysis towards ATP, in the presence of different concentrations of compound (1), (2) and (3) (panel A, B and C, respectively).

Finally, as the CTP synthetases physiologically use glutamine as an ammonia donor to fulfill the catalysis, the effects of the three compounds against the glutaminase activity of PyrG were evaluated. As depicted in Fig. S2, the presence of the three compounds leads to a decrease in the Vmax values, but leaving unchanged the Km, thus excluding a direct interaction of the compounds with the glutamine binding site.

Effects of GSK PyrG inhibitors against human CTP synthetase Due to the relatively large similarity between Mtb and human CTP synthetases (about 44% sequence identity), the type 1 human CTP synthetase (Hu-CTPS1) was obtained, to ACS Paragon Plus Environment

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assess the compounds’ specificity. The recombinant Hu-CTPS1 was produced using Pichia pastoris expression system and purified as described in Methods (Fig. S3), affording about 3 mg of protein from 1 liter of yeast culture, with a specific activity of 6.5 s-1, and Km values of 0.2 mM towards either UTP or ATP. The enzymatic activity assay of Hu-CTPS1 in the presence of the compounds, performed at sub-saturating substrate concentrations, revealed that all the three 4-(pyridin-2-yl)thiazole derivatives inhibited the human CTP synthetase (Fig. 3). By contrast, the 11426026 compound, a Mtb PyrG inhibitor previously identified14, displayed a significantly higher IC50 against Hu-CTPS1 (>1 mM for Hu-CTPS1 vs 13.5 ± 1.3 µM for the Mtb PyrG). However, although compounds (2) and (3) showed IC50 values against Hu-CTPS1 (38.0 ± 3.1 µM and 24.2 ± 3.7 µM, for (2) and (3), respectively), similar to that against PyrG, compound (1) was a weaker inhibitor of the human enzyme, with an IC50 10-fold higher (27.8 ± 2.3 µM).



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Figure 3. Effects of PyrG inhibitors on human CTP synthetase 1 activity. IC50 determination of 11426026 compound (panel A) and GSK compounds (1) (panel B), (2) (panel C) and (3) (panel D) on the M. tuberculosis PyrG (filled symbols) and Hu-CTPS1 (open symbols).

The possibility to screen compound libraries against both Mtb and human CTPsynthetases could be a valuable “double tool” for identification of inhibitors affecting almost exclusively mycobacterial PyrG, such as 11426026. It is worth noting that, despite their activity against the human enzyme, the GSK compounds showed low toxicity against a human cell-line8. Moreover, the fact that compound (1) is tenfold less active toward the human enzyme, highlights the possibility to develop analogs that are more specific for the mycobacterial CTP synthetase. ACS Paragon Plus Environment

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Validation of PyrG as a target of the three GSK inhibitors As the GSK compounds have been shown to be ATP binding site inhibitors, their specificity towards the CTP synthetases was assessed. In order to do this, the compounds were assayed against a small panel of diverse kinases or ATP binding enzymes, from different sources (Escherichia coli, rabbit muscle, and human pyruvate kinases, human adenylate kinase, M. tuberculosis phosphoribosyl pyrophosphate synthetase, human phosphoglycerate kinase, yeast hexokinase). As shown in table S1, none of these enzymes is inhibited by the three compounds, thus excluding a broad specificity of these compounds. To confirm the role of PyrG as a target of the compounds, the isolation of Mtb resistant mutants was attempted, to check for the presence of possible mutations in the pyrG gene. Concentrations of compounds ranging from 5-fold to 20-fold the MIC were used, but despite several attempts no resistant colonies could be isolated. The inability to obtain spontaneous resistant mutants could be related to the strict essentiality of the CTP synthetase enzyme14,15, and clearly reflects a very low mutation frequency of the pyrG gene, reinforcing its significance as a drug target. However, as the three compounds were demonstrated to compete with the ATP binding site of PyrG, mutations that could hamper their binding, but without detrimental effects on the enzyme activity, could confer drug resistance. For this reason, the GSK compounds were also tested on a previously characterized M. tuberculosis mutant, harboring the V186G mutation in PyrG, and resistant to the already described 11426026 compound, an ATP binding site PyrG competitive inhibitor14. The Mtb mutant strain did not show cross resistance with compound (3) and was weakly resistant to the other two compounds (MIC values 2-fold higher than that of the wild type) (Table S2). The three compounds assayed against the recombinant PyrG V186G, were found to maintain activity against the mutant enzyme,



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showing IC50 values slightly increased with respect to the wild type, thus explaining the sensitivity of the Mtb mutant strain (Fig. S4). To confirm that also the intracellular mechanism of action of the compounds effectively involves the CTP synthetase, the compounds were assayed against an Mtb conditional knock-down (cKD) mutant strain (TB456), constructed using the Pip-ON inducible system. In this system pyrG gene was under the control of the inducible promoter Pptr and its expression was allowed only in the presence of the inducer (Pristinamycin I). The conditional mutant TB456 was able to grow only on plates containing Pristinamycin I (100 ng/ml), confirming the essentiality of the gene14 (Fig. S5). Moreover, TB456 growth was shown to be induced in a dose-dependent manner in the presence of different amounts of Pristinamycin I in liquid media. Standing cultures of TB456 were grown using different concentrations of inducer (0 to 100 ng/ml) for 72 hours; then bacteria were diluted to OD540 0.05 in the same media conditions. After 72 hours it was clearly visible that the mutant growth depended on Pristinamycin concentration. After the second re-fresh the growth of bacteria exposed to the lowest concentrations of Pristinamycin I was arrested (Fig. S6) Then, TB456 cultures grown in different Pristinamycin I concentrations were exposed to the compounds object of the study, and to 11426026, which is known to target PyrG, as control (Table 2). Actually, the MIC for compound (2) did not show any variation in function of Pi amount in which the bacteria were grown. On the contrary the MICs for compounds (1), as well as to 11426026, were halved at lower concentration of the inducer, increasing only at the highest Pi concentration. Furthermore, in the presence of compound (3) the TB456 strain did not grow at minimal Pi concentrations, showing a four- fold lower MIC value even at the highest inducer. The dependence of the effects of compound (1) and particularly of compound (3) on the PyrG intracellular level demonstrate its role in the mechanism of their action, thus supporting the hypothesis that PyrG is an intracellular target of these two compounds. ACS Paragon Plus Environment

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Table 2: MIC values of Mtb wild-type and TB456 cKD strains.

Compound

H37Rv

M. tuberculosis MIC (µ µM) TB456 [pristinamycin] (ng/ml)

0 5 (1) 20 n.d. 10 (2) 38 n.d. 38 (3) 4.3 n.d. n.d. 11426026 42.5 n.d. 21.2 n.d.: not determined because of lack of cells growth

15 10 38 1.1 21.2

25 10 38 1.1 21.2

50 20 38 1.1 42.5

GSK PyrG inhibitors cause several metabolism alterations in M. tuberculosis The role of the CTP synthetase in the mechanism of action of these compounds was also examined by metabolic labeling of the nonpathogenic model strain M. tuberculosis H37Ra (MIC of 20.8 µM, 15.2 µM and 0.28 µM, for compound (1), (2) and (3), respectively) with [14C]-uracil. Within mycobacterial cells [14C]-uracil is incorporated into [14C]UMP by uracil phosphoribosyltransferase(Upp) from the pyrimidine salvage pathway16, being then further metabolized to the whole range of nucleotides. M. tuberculosis H37Ra cells were grown in the absence or presence of the compounds (8 x MIC) for one hour, then [14C]-uracil was added and radiolabeling continued for three hours. The radiolabeled nucleotides were then extracted from cell pellets with 9% formic acid17, and separated by thin layer chromatography. As depicted in Figure 4A, addition of the compounds to the media during cultivation of mycobacteria led to a significant increase in the ratios of radioactivity incorporated to [14C]UTP and [14C]CTP compared to the control, pointing to inhibition of PyrG. A similar trend was found in the positive control 11426026, while for benzothiazinone BTZ 043, the drug with an unrelated mode of action targeting cell wall enzyme DprE118, the [14C]UTP/[14C]CTP ratio was comparable to the untreated control cells (Table S3).



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Clearly, inhibition of CTP synthetase activity would affect numerous catabolic and anabolic reactions in mycobacteria, due to the requirement of CTP by bifunctional CoaBC enzyme involved in biosynthesis of coenzyme A. This enzyme was recently shown as a validated bactericidal target in M. tuberculosis19. CTP is critical for lipid metabolism20 also for producing of activated CDP-derivatives required for biosynthesis of phospholipids. We thus investigated the effects of the compounds on lipid biosynthesis by [14C]-acetate metabolic labeling in the same conditions as described for [14C]-uracil. Analysis of lipids extracted from the radiolabeled cells revealed that incorporation of [14C]-acetate was decreased, in the cells treated with compounds (1), (2), (3) and 11426026. On the contrary, the presence of BTZ043 resulted in an increase of the radiolabel incorporation due to accumulation of TDM, as expected (Fig. 4 B and C). These experiments confirm that the effects of the compounds are pleiotropic, as expected, but it is not possible to conclude, if they are the result of only PyrG inhibition, or due to targeting additional enzyme(s).


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Figure 4. Examination of effects of (1), (2) and (3) on M. tuberculosis H37Ra cells by metabolic labeling. Bacterial cells were pre-treated with 8-fold MIC of the drugs for 1 h, followed by 3 h of radiolabeling with [14C]uracil (A) or [14C]-acetate (B, C). (A) Ratios of radioactivity incorporated to [14C]UTP and [14C]CTP extracted from [14C]uracil-labeled cells. Nucleotide extracts were separated by TLC on PEI-cellulose plate. Columns and error bars represent mean ± SD of triplicate measurement of radioactivity in the [14C]UTP and [14C]CTP spots cut from TLC plates corresponding to two separate experiments; from each experiment two plates were analyzed. The autoradiograph is representative image from three separate experiments. (B) TLC analysis of the lipids from [14C]-acetate-labeled cells. Lipids were extracted as in “Methods”, aliquots corresponding to the same amounts of extracted material (as shown in 5C) were loaded on a silica gel TLC plate and ACS Paragon Plus Environment


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separated in Solvent I: CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4); Solvent II CHCl3/CH3OH/H2O (20:4:0.5); and Solvent III: petroleum ether/ethyl acetate (98:2; 3 times).The plates were exposed to an autoradiography film at −80 °C for 6 days. TDM, trehalosedimycolates; TMM, trehalosemonomycolates; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; CL, cardiolipin; TAG, triacylglycerol. Autoradiographs are representative images from three separate experiments. (C) Visualization of the cold lipids by cupric sulfate staining.

Conclusion The use of whole cell phenotypic screening in combination with target based assay has been recently demonstrated a useful strategy for the identification of new leads12. With this aim an antitubercular compound library was screened against CTP synthetase PyrG, identifying a new series of 4-(pyridin-2-yl)thiazole compounds, able to inhibit the enzyme activity. These compounds, which have been shown to be quite specific for the CTP synthetases, confirm the high druggability of PyrG. Indeed, several lines of evidence, which emerged from the different approaches used, point to PyrG as a main target of this series, although further additional targets could not be completely excluded, demonstrating the effectiveness of such a screen against newly validated targets, to identify novel scaffolds for drug development. Moreover, the parallel screen against the corresponding human enzyme CTPS-1, could help in defining the selectivity of the compounds. Indeed, among the three hit molecules identified, compound (1) was more selective for the mycobacterial enzyme and represents a useful scaffold for further target-assisted structure activity relationship studies in order to improve its efficacy.


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METHODS Compound library screening against PyrG enzyme activity. Wild-type and mutant M. tuberculosis PyrG recombinant proteins were obtained in E. coli, as previously reported 14. The publically available GlaxoSmithKline antimycobacterial compound set (GSK TB-set), consisting of 177 compounds active against M. tuberculosis growth8, was screened against PyrG activity, at a final concentration of 100 µM for each compound. PyrG enzyme activity was determined in a final volume of 100 µl at 37°C by continuous spectrophotometric assay following the rate of increase in absorbance at 291 nm due to the conversion of UTP to CTP (ε= 1.34 mM-1 cm-1)21. The reaction mixture contained: 50 mM HEPES pH 8.0, 10 mM MgCl2, 1 mM UTP, 0.2 mM ATP, 0.5 µM PyrG, 1 µl of compound (10 mM in dimethylsulfoxide, DMSO), and the reaction was started by the addition of 100 mM NH4Cl. Blank reactions were performed by adding 1 µl of DMSO. All assays were performed in triplicate. The compounds inhibiting more than 75% of PyrG activity in these conditions, and selected

for

further

investigation,

were

re-purchased

from

MolPort

(Riga,

Latvia):GSK1570606A,(2-(4-fluorophenyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)acetamide, #MolPort-003-158-205;

GSK735826A,

N-(4-(pyridin-2-yl)thiazol-2-yl)-

[1,3]dioxolo[4',5':4,5]benzo[1,2-d]thiazol-6-amine #MolPort-003-038-940; GSK920684A, 2(3-fluorophenoxy)-N-(4-(pyridin-2-yl)thiazol-2-yl)acetamide, #MolPort-004-106-239. PyrG inhibition was reconfirmed, then IC50 and Ki values were determined. For IC50 determinations, the enzyme activities were measured in the presence of compound and values were estimated according to the Equation 1, where A[I] is the enzyme activity at inhibitor concentration [I] and A[0] is the enzyme activity without inhibitor. A[I] =A[0] × ቀ1- ሾIሿ+IC ቁ [I]

equation 1

50



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The inhibition constant (Ki) values were determined by assaying the PyrG enzymatic activity at different substrates and compound concentrations. The assays were performed at a UTP final concentration of 2 mM when ATP was the variable substrate, and at 2 mM ATP when UTP was the variable substrate. Ki values were calculated using an adapted equation for competitive inhibition (Equation 2)22 with Origin 8 software. v=

Vmax [S]

ሾIሿ Ki

ሾSሿ+ Km ൬1+ ൰

equation 2

PyrG glutaminase activity was assayed by using a glutamate dehydrogenase (GDH, Sigma-Aldrich) coupled assay, which converts the produced glutamic acid into αketoglutaric acid through the reduction of NAD+. Thus the activity was determined measuring the increasing in absorbance at 340 nm of the produced NADH (ε = 6220 M-1 cm-1). Assays were performed at 37°C in 50 mM Hepes pH 8.0, 10 mM MgCl2, 1 mM NAD+, 100 µM GTP, 0.2 mM ATP, 2 mM UTP, 1 U GDH, PyrG 1 µM, and reaction started by adding the glutamine solution. Steady state kinetics toward Gln were performed at 3 and 6 µM for cpd (1) and 20 and 40 µM for cpds (2) and (3).

Screening of the compounds against different kinases. E. coli pyruvate kinase, human R type pyruvate kinase, human adenylate kinase, M. tuberculosis phosphoribosyl pyrophosphate synthetase and human phosphoglycerate kinase were prepared, and their activity assayed, as previously reported23-27. Rabbit muscle pyruvate kinase and yeast hexokinase were purchased from Sigma-Aldrich, and their activity was assayed according to the manufacturer. Enzyme activities were determined at their subsaturating ATP concentrations, in the presence and in the absence of 100 µM of compound (1), (2) or (3).


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Minimal inhibitory concentration determinations and isolation of M tuberculosis spontaneous resistant mutants. The MIC of the compounds was determined in solid medium. A single colony of each M. tuberculosis strain was inoculated in complete Middlebrook 7H9 supplemented with 10% OADC Middlebrook Enrichment, and grown at 37°C until exponential growth phase (~108 CFU/ml). Dilutions to the final concentration of ~106CFU/ml were performed and about 1 µl of cell culture was streaked onto plates containing two-fold serial dilutions of appropriate compound. MIC values were assigned as the lowest drug concentrations inhibiting bacterial cell growth. All experiments were performed in triplicate. The isolation of M. tuberculosis mutants was attempted by plating ~1010 cells from an exponential growth phase wild-type culture onto 7H11 medium containing different concentrations of compounds, ranging from 5 to 20-fold the MIC. MICs of the compounds for M. tuberculosis H37Ra were established by resazurine microplate assay (REMA)28.

Construction and characterization of a pyrG conditional knock down mutant. A Mtb pyrG conditional knockdown mutant was constructed using the PIP-ON system29. At this purpose the first 694bp nucleotides of the pyrG coding sequence were amplified with RP1609

(5’-ttttatgcatcgaaagcacccgcaaacc-3’)

and

RP1610

(5’-

ttttactagtcatcaacgcaatcttgtttttca-3’) primers and cloned in a suicide plasmid in frame with the pristinamycin-inducible promoter Pptr. The obtained plasmid pGi11 was than electroporated in an H37Rv-derivative containing the pip gene integrated at the L5 attB site obtaining TB456. To characterize the growth of the pyrG cKD mutant, TB456 was grown to mid-logarithmic phase and then diluted to a theoretical OD540of 0.05 in medium with different amounts of Pristinamycin I (0,5,7, 15, 25, 50, 75, 100 ng/ml). Serial dilutions



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of standing culture of TB456 grown in Middlebrook 7H9 without Pristinamycin I for 24-48 hrs, were plated onto solid media with/ without Pristinamycin I (100 ng/ml).

MIC determinations in pyrG conditional knock down mutant. Compounds sensitivity of the Mtb pyrG knockdown mutant was determined in presence of different pristinamycin concentrations using the resazurine microplate assay (REMA) as previously described28.

Docking. The PyrG protein was prepared as described previously14 for docking using the default settings of the ‘prepare protein’ protocol in Discovery Studio 4.1 (Biovia, San Diego, CA). The PyrG protein (PDB ID: 4ZDJ)14 was used for docking using LibDock30. The protocol included 100 hotspots and docking tolerance (0.25). The FAST conformation method was also used along with steepest descent minimization with CHARMm.

Metabolic labeling of M. tuberculosis H37Ra. The effects of the PyrG inhibitors on the mycobacterial metabolism were assessed through metabolic labelling of M. tuberculosis H37Ra with [14C]-uracil, or [14C]-acetate. Briefly, M. tuberculosis H37Ra was grown statically in 7H9 medium supplemented with 10% ADC and 0.05% Tween 80 until OD600~0.25-0.35. The culture was then divided to 5 ml aliquots and the compounds (1), (2) and (3) dissolved in DMSO were added at 8x MIC; control drugs 11426026 and BTZ043 were added at 16 µg/ml or 0.4 µg/ml, respectively. Final concentration of DMSO in all cultures was 1%. After 1 hr drug treatment [14C]-uracil (American Radiolabeled Chemicals, specific activity 53 mCi/mmol) was added to a final concentration 0.5 or 1 µCi/ml, or [14C]acetate (American Radiolabeled Chemicals, specific activity 106 mCi/mmol) was added to a final concentration 0.5 µCi/ml, and the incubation continued for next three hours.


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Inhibition of mycobacterial growth by all tested compounds after 4 hr treatment was between 2 to 10 % based on OD600 measurement. Extraction of [14C]-uracil-labeled nucleotides with ice-cold 9% formic acid was carried out as previously reported14. For TLC analysis the extracts were evaporated under vacuum, mixed with 10 nmol each of UTP and CTP and loaded on PEI Cellulose F plate (Millipore). After 15 min soaking in CH3OH, the air-dried TLC plate was developed in 0.75 M KH2PO4, pH adjusted to 3.5 with 0.75M H3PO417 for 3 hrs. Spots corresponding to UTP and CTP were visualized under UV (λ=254 nm), then they were cut from the plate and placed to scintillation vials with EcoLite scintillation liquid for determination of radioactivity. Bacteria labeled with [14C]-acetate were subjected to extraction with 1.5 ml CHCl3/CH3OH (1:2), followed by extraction with 1.5 ml CHCl3/CH3OH (2:1). Each extraction was carried out for 2 hrs at 56°C; the extracts were combined, dried and subjected to fractionation in CHCl3/CH3OH/H2O (4:2:1). Upper (water) phase was discarded and bottom (organic) phase was dried and dissolved in Solvent I (see below) in the ratio 75 µl/OD600 0.5/2 ml of the culture.5 µl aliquots of the lipid extracts were analyzed by TLC on Silica gel plates (Merck) in Solvent I: CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4); Solvent II CHCl3/CH3OH/H2O (20:4:0.5); and Solvent III: petroleum ether/ethyl acetate (98:2; 3 times). After chromatography the plates were exposed to autoradiography film (BioMax MR) at -80°C. For visualization of the cold lipids the plates were sprayed with cupric sulfate (10% in 8% phosphoric acid).

Cloning, expression and purification of the human CTPS1. Recombinant human CTPsynthetase 1 (HuCTPS-1) was expressed in P. pastoris MutS strain KM71H (Invitrogen)31. The full length HuCTPS-1 cDNA was purchased from Dharmacon. The cDNA was inserted into BamHI/NotI restriction sites of pPICZ-B-eGFP-6His vector, affording the pPICZ-BHuCTPS-1-eGFP-6His, to produce the recombinant protein fused with C-terminal



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enhanced Green Fluorescent Protein (eGFP) and 6Xhistidines tags. The pPICZ-B-eGFP plasmid is a modified pPICZ-B vector (Invitrogen), in which the PreScission protease recognition sequence and the eGFP-6His tag sequence were inserted at the 3’ of the multi-cloning site. P. pastoris KM71H transformation was performed according to Lin-Cereghino et al.32, using the pPICZB-HuCTPS-1-eGFP-6His plasmid linearized with SacI (Promega). Transformed cells were plated on YPD (2% Bacto-Yeast Extract, 2% dextrose,1% peptone) agar supplemented with 100 µg/ml zeocin, and incubated at 30 °C for 2-3 days, until colonies appeared. In order to identify the clone showing the best protein expression, each colony was inoculated in a 24-deep wells plate containing 2 ml of BMGY medium (100 mM HK2PO4 pH 6.0, 6.34 % Yeast Nitrogen Base with Ammonium Sulfate without amino acids, 1 % glycerol, 4× 10–5 % biotin), incubated al 30 °C shaking at 280 rpm for 60 hours. Successively, for protein expression induction, the medium was exchanged with BMM (100 mM HK2PO4pH 6, 1.34 %Yeast Nitrogen Base with Ammonium Sulfate without amino acids, 4 x 10-50 biotin, 0.5% methanol), and eGFP fluorescence was measured after 24, 48 and 72 hours, using the Clariostar plate reader (BMG Labtech; excitation 489 nm, emission 509 nm), thus identifying the clone having the highest fluorescence signal. For large scale protein production, a pre-inoculum of the selected colony was grown overnight at 30°C and diluted 80-fold in a 5 liter flask containing 1 liter of BMGY, and incubated at 30 °C in 200 rpm shaking incubator. After 72 hours, cells were harvested by centrifugation and resuspended in half-volume of BMM medium, and 0.5% methanol was added every 24 hours. After 48 hours, cells were collected and resuspended in Buffer A (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 10% glycerol, 1 mM phenyl-methyl sulfonyl fluoride (PMSF; Sigma-Aldrich), protease inhibitors Complete EDTA-Free (Roche), 1 mg/ml DNAse). An equal volume of zirconia beads (0.5 µm, BioSpech) was ACS Paragon Plus Environment

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added to the suspension, and yeast cells were mechanically disrupted with a BioSpec Mini Bead-Beater. The mixture was passed through a cloth mesh straines to remove zirconia beads, and centrifuged at 70000 x g for 30 minutes at 4 °C. The supernatant was applied on a HisTrap column (1 ml, GE Healthcare) previously equilibrated in Buffer A, the column was washed with 20 mM of imidazole, and then CTPS-1 was eluted with 500 mM imidazole in the same buffer. The purified enzyme was dialyzed against Buffer B (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mM DTT),digested with PreScission protease (GE Healthcare, 400 mU/ml), and further purified by a second affinity chromatography, followed by size exclusion chromatography on a HiLoad Superdex 200 column (GE Healthcare). Samples purity was checked by SDS-PAGE and protein concentration evaluated by Bradford reagent33.

ASSOCIATED CONTENTS Supporting Information Effects of GSK compounds against different ATP binding enzymes; MIC values of M. tuberculosis wild-type and pyrG mutant strains to GSK compounds; ratios of radioactivity incorporated to [14C]UTP and [14C]CTP of the control experiments; PyrG kinetic analysis towards UTP in the presence compounds; effects of the GSK compounds against PyrG glutaminase activity; SDS-PAGE of the purification steps of HuCTPS-1; IC50 values of compounds to the wild-type and the V186G mutant PyrG enzyme; characterization of the pyrG conditional mutant TB456. This information is available via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Authors *(L.R.C.) E-mail: [email protected] ACS Paragon Plus Environment


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Current address ⊥

S.E.: Collaborations Pharmaceuticals Inc., Fuquay Varina, NC 27526, USA

Author Contributions +

M.E. and S.S. contributed equally and are considered as co-first authors to this work.

Author Contributions M.E. and L.R.C. performed the screening of the TB-set library, provided by D.B., J.L. and L.B.; M.E. and L.R.C. performed proteins expression, purification and characterization; S.E. performed docking experiments; B.S.O. and G.M. performed MIC determinations and isolation of resistant mutants; G.D. and F.B. constructed pyrG conditional knock down mutant and performed MIC determinations; S.S., S.H. and J.Z. performed metabolic labeling experiments; M.E. and V.P. cloned the human CTPS1; R.M., D.B., J.L., M.R.P., G.R., L.B., K.M. and L.R.C. analysed data; R.M., A.M., M.R.P., G.R., K.M., L.R.C. supervised and directed the work; S.E., R.M., K.M., L.R.C. wrote the paper. All authors discussed the results and commented on the manuscript.

Competing Financial Interests SE was a consultant for Collaborative Drug Discovery, Inc. All other authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by European Community’s Seventh Framework Program (Grant 260872); by University of Pavia, Italy (“Universitiamo–Tubercolosi: un killer riemergente” to G.R.); by the Slovak Research and Development Agency (Contract No. DO7RP-0015-11 to K.M.). ACS Paragon Plus Environment

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ABBREVIATIONS HTS, high-throughput screening; Hu-CTPS1, human CTP synthetase 1; MDR multi drug resistant; MIC, Minimal Inhibitory Concentration; Mtb, M. tuberculosis; PyrG, Mtb CTP synthetase; TB, tuberculosis; XDR, extremely drug resistant.



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For Table of Contents Use Only

A phenotypic based target screening approach delivers new antitubercular CTP synthetase inhibitors.

Marta Esposito, Sára Szadocka, Giulia Degiacomi, Beatrice S. Orena, Giorgia Mori, Valentina Piano, Francesca Boldrin, Júlia Zemanová, Stanislav Huszár, David Barros, Sean Ekins, Joel Lelièvre, Riccardo Manganelli, Andrea Mattevi, Maria Rosalia Pasca, Giovanna Riccardi, Lluis Ballell, Katarína Mikušová, Laurent R. Chiarelli.

The combination of whole cell phenotypic screening and biochemical target based assay can be fruitfully exploited for the identification and development of novel antitubercular leads, as demonstrated by our small screening against the recently validated target CTP synthetase.


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Figure 1. Docking GSK compounds in CTP synthetase. (A) compound (1) libdock score 88.16; (B) compound (2) libdock score 92.96; (C) compound (3) libdock score 87.23. All molecules (grey) are compared to UTP (Yellow); (D) compound 11426026, previously reported as a PyrG ATP binding site competitive inhibitor. 156x202mm (300 x 300 DPI)



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Figure 2. Double reciprocal plots of PyrG kinetic analysis towards ATP, in the presence of different concentrations of compound (1), (2) and (3) (panel A, B and C, respectively). 69x27mm (300 x 300 DPI)


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Figure 3. Effects of PyrG inhibitors on human CTP synthetase 1 activity. IC50 determination of 11426026 compound (panel A) and GSK compounds (1) (panel B), (2) (panel C) and (3) (panel D) on the M. tuberculosis PyrG (filled symbols) and Hu-CTPS1 (open symbols). 137x135mm (300 x 300 DPI)



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Figure 4. Examination of effects of (1), (2) and (3) on M. tuberculosis H37Ra cells by metabolic labeling. Bacterial cells were pre-treated with 8-fold MIC of the drugs for 1 h, followed by 3 h of radiolabeling with [14C]uracil (A) or [14C]-acetate (B, C). (A) Ratios of radioactivity incorporated to [14C]UTP and [14C]CTP extracted from [14C]uracil-labeled cells. Nucleotide extracts were separated by TLC on PEI-cellulose plate. Columns and error bars represent mean ± SD of triplicate measurement of radioactivity in the [14C]UTP and [14C]CTP spots cut from TLC plates corresponding to two separate experiments; from each experiment two plates were analyzed. The autoradiograph is representative image from three separate experiments. (B) TLC analysis of the lipids from [14C]-acetate-labeled cells. Lipids were extracted as in “Methods”, aliquots corresponding to the same amounts of extracted material (as shown in 5C) were loaded on a silica gel TLC plate and separated in Solvent I: CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4); Solvent II CHCl3/CH3OH/H2O (20:4:0.5); and Solvent III: petroleum ether/ethyl acetate (98:2; 3 times).The plates were exposed to an autoradiography film at −80 °C for 6 days. TDM, trehalosedimycolates; TMM, trehalosemonomycolates; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; CL, cardiolipin;


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TAG, triacylglycerol. Autoradiographs are representative images from three separate experiments. (C) Visualization of the cold lipids by cupric sulfate staining. 154x190mm (300 x 300 DPI)


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