Subscriber access provided by UZH Hauptbibliothek / Zentralbibliothek Zuerich
Letter
Adenosine Tetraphosphoadenosine Drives a Continuous ATP-Release Assay for Aminoacyl-tRNA Synthetases and other Adenylate forming Enzymes Adrian J. Lloyd, Nicola J. Potter, Colin W. G. Fishwick, David I. Roper, and Christopher G Dowson ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb400248f • Publication Date (Web): 30 Jul 2013 Downloaded from http://pubs.acs.org on August 1, 2013
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Adenosine Tetraphosphoadenosine Drives a Continuous ATPRelease Assay for Aminoacyl-tRNA Synthetases and other Adenylate forming Enzymes
Adrian J. Lloyd1*#, Nicola J. Potter2#, Colin W.G. Fishwick2, David I. Roper1 and Christopher G. Dowson1* 1
School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, West Midlands,
CV4 7AL, UNITED KINGDOM and 2School of Chemistry, University of Leeds, Leeds, LS2 9JT, UNITED KINGDOM. *To whom correspondence should be addressed: Adrian J. Lloyd email:
[email protected], Tel: +44 (0)2476 522568, Fax: +44(0)2476 523568 and Christopher G. Dowson email:
[email protected], Tel: +44 (0)2476 523534, Fax: +44(0)2476 523568. #
Both authors contributed equally to the manuscript.
1
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 20
Abstract Aminoacyl-tRNA synthetases are essential for the correct linkage of amino acids to cognate tRNAs to maintain the fidelity of protein synthesis.
Tractable, continuous assays are
valuable for characterizing the functions of synthetases and for their exploitation as drug targets. We have exploited the unexplored ability of these enzymes to consume adenosine tetraphosphoadenosine (diadenosine 5’,5’’’ P1 P4 tetraphosphate; Ap4A) and produce ATP to develop such an assay.
We have used this assay to probe the stereo-selectivity of
isoleucyl-tRNAIle and Valyl-tRNAVal synthetases, the impact of tRNA on editing by isoleucyltRNAIle synthetase (IleRS) and to identify analogues of intermediates of these enzymes that might allow targeting of multiple synthetases. We further report the utility of Ap4A based assays for identification of synthetase inhibitors with nM to mM affinities.
Finally, we
demonstrate the broad application of Ap4A utilization with a continuous Ap4A-driven RNA ligase assay. The ability of small molecules to distinguish between eukaryotic and prokaryotic protein synthesis has allowed development of many clinically valuable antibiotics(1) including mupirocin, which targets the active site of Gram positive IleRS(2). However, the lack of systemic efficacy of mupirocin, its inactivity against Gram negative and TB infections(2,3) and emergence of mupirocin resistance in MRSA and MSSA(2) has increased the need for new synthetase inhibitors. Many aminoacyl-tRNA synthetases (AaRSs) cannot bind the correct amino acid accurately enough to ensure the fidelity of protein synthesis(4). Therefore these enzymes remove incorrectly aminoacylated intermediates and tRNAs with pre and post-transfer editing mechanisms. These are partitioned between the active site (pre-transfer) and a posttransfer editing domain(4).
Antimicrobial targeting of the post-transfer editing domain of
leucyl-tRNALeu synthetase (LeuRS) from Gram negative pathogens has been abandoned because of the development of resistance in clinical trials due to mutation(5). This result was 2
ACS Paragon Plus Environment
Page 3 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
heralded by the impact of mutation of the post-transfer editing of Escherichia coli LeuRS which unveiled a pre-transfer-editing capability(6). All AaRSs activate their cognate amino acids with ATP to form an aminoacyladenylate (Figure 1a). AaRSs then catalyse the attack of this intermediate by tRNA to form the correct aminoacyl-tRNA product(4). However, in the absence of tRNA, AaRSs can cleave this aminoacyl-adenylate with pyrophosphate. monitored as [32P]-pyrophosphate Alternatively,
Consequently, AaRS activity can be
exchange into ATP. (Figure 1a reaction 1)(7).
aminoacyl-tRNA formation (Figure 1a, reaction 2) can be followed with
radiolabelled amino acid by trichloroacetic acid precipitation of the radiolabelled aminoacylated tRNA product(7). However, utilization of radiolabels prevents continuous monitoring of AaRS activity and is expensive, which often precludes high throughput and saturating substrate experiments(7). This and the success of mupirocin suggest significant advances in inhibitor screening and AaRS enzymology could be leveraged by continuous non-radioactive assays that monitored AaRS aminoacyl adenylate turnover. We initially identified the assay in(8) as a starting point.
Here, the pyrophosphate
attacking the aminoacyl adenylate intermediate exchanged for the imidodiphosphate moiety of 5'-adenylyl-β,γ-imidodiphosphate (ADPNP) in the presence of the amino acid to generate ATP that could be assayed spectrophotometrically (Supporting Information Figure 1 (Figure S1a)). We therefore obtained an assay for E. coli and Streptococcus pneumoniae alanyltRNAAla and S. pneumoniae seryl-tRNASer synthetases (AlaRS and SerRS) that we characterised (Table S1 and Figure S2a-i). However the E. coli class I IleRS and valyl-tRNAVal synthetases (ValRS) could not utilise ADPNP (Figure S1b), possibly because of differential modes of ATP binding to class I and II synthetases(9). Furthermore, the low micromolar ADPNP KmApp of E. coli AlaRS (Table S1) reduced the sensitivity of the assay to competitive inhibitors. To enable access to the 3
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 20
enzymology of both class I and II AaRSs, that was highly sensitive to hit detection, we sought another adenylate to support pyrophosphate exchange activity. The synthetases discussed above synthesise Ap4A where the aminoacyl adenylate formed with the concomitant generation of pyrophosphate, is cleaved by a second ATP(12) to form Ap4A (Figure 1a, reaction 3)(10).
Reversal of this reaction would generate two
molecules of ATP which could be continuously detected by spectrophotometry, fluorometry or bioluminescence (Figure 1b). Assays following conversion of Ap4A to ATP require detection of ATP in the presence of Ap4A.
We found that glucose phosphorylation with 0.1 mM ATP by hexokinase when
coupled to glucose 6-phosphate dehydrogenase to generate NADPH (Figure 1b) caused an instantaneous increase in absorbance at 340 nm in the presence of 6.33 mM Ap4A which itself, caused no significant absorbance change. The absorbance increase was consistent with the ATP concentration added to the assay.
This suggested the coupling system
discriminated between ATP and Ap4A and was not significantly inhibited by the dinucleotide. We then tested the ability of E. coli AlaRS, E. coli ValRS and E. coli IleRS to generate ATP from Ap4A. All three enzymes showed linear Ap4A, pyrophosphate, synthetase and amino acid dependent ATP production as reported at 340nm (Figure 2a-c). The linear initial rates in Figure 2a-c indicated the products of the coupling enzymes (Figure 1b) did not significantly impact on AaRS activity. To prove that the assay followed the pyrophosphorolytic cleavage of Ap4A we followed
Ap4A consumption by S.pneumoniae AlaRS in the presence of L-alanine and
excess pyrophosphate. At the end of the reaction, two ATP were produced per Ap4A (Figure 2d), consistent with the stoichiometry of this reaction. Amino acid-dependent AaRS catalysed Ap4A pyrophosphorolysis
would not be
limited by the amino acid which is regenerated per catalytic cycle (Figure 1b). Therefore, we 4
ACS Paragon Plus Environment
Page 5 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
assayed IleRS with limiting (10 µM) L-isoleucine. The absorbance of the assay extended beyond that where L-isoleucine was consumed (0.062 AU), to that consistent with consumption of the next limiting substrate in the assay (pyrophosphate; Figure 2c) and regeneration of L-isoleucine. Having characterised AaRS pyrophosphorolysis of Ap4A, we examined the kinetics of this process for IleRS, ValRS and AlaRS. Dependences of activity on Ap4A, cognate amino acids and pyrophosphate were hyperbolic (Figures 2e and S3a-i). In many of these assays, the AaRS concentration approached that of the substrate (especially pyrophosphate). Therefore, we fitted the data to a rate equation that took this into account (Table 1 legend). This analysis revealed that for E. coli AlaRS, the KmApp for Ap4A was 204-fold greater than that for ADPNP (Tables 1 and S1, Figures S2h and S3h) . Comparison of Table 1 and Figure 3 with published data suggested that the KmApp of E. coli AlaRS, IleRS and ValRS for Ap4A was 16.8-(11), 45.2-(12) and 21.6-fold(13) greater than for ATP.
Thus, Ap4A utilization
afforded a continuous assay, with kinetic constants that would be more sensitive to inhibitor detection than assays that utilised ATP. The KmApp values in the Ap4A assay for the cognate amino acids for ValRS, AlaRS and IleRS (Table 1, Figures 2e, S3f, i and c) were elevated by 101-fold, 70.2-fold and 9.03fold respectively relative to published data(11,14,15). This may have resulted from a reduced affinity of the amino acid for synthetases bound to Ap4A, relative to that in the presence of ATP. As also deduced from the Ap4A kinetics, the sensitivity of assays to amino acid or aminoacyl-adenylate synthetase-targeted inhibitors would be expected to be vastly enhanced by the relatively high KmApp values of these enzymes for their cognate amino acids. ValRS(16), IleRS(16) and AlaRS(17) form adenylates and aminoacyl-tRNA species with non-cognate amino acids. We therefore tested the ability of these enzymes to utilise Ap4A with L-threonine, L-valine and L-serine, the respective non-cognate editing substrates of these enzymes(16,17). Activation of L-valine by IleRS and L-serine by S. pneumoniae AlaRS 5
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 20
was easily detectable (Figure 2f and Figure S4). The efficiencies ( kcatApp/KmApp values) of Lvaline and L-isoleucine activation by E. coli IleRS (Table 1) were 1:207.5, in excellent agreement with previous data (1:200)(18) and consistent with the requirement for editing by IleRS.
However, Ap4A consumption by ValRS was barely detectable with 15 mM L-
threonine. It is probable that the reduced affinity for both non-cognate amino acid and the substitution of ATP by Ap4A severely reduced ValRS activity. tRNAIle stimulates IleRS editing(16) (Figure S5a). Therefore, we assessed the ability of Ap4A to support this process. tRNA stimulated Ap4A utilization by IleRS with L-valine but not L-isoleucine (Figure S5b). (Supporting Information section 4.1 (SI 4.1)) This showed that Ap4A assays could provide a valuable non-radioactive alternative to the current radiolabel and TLC-based procedures used to study this process(16). We extended our IleRS and ValRS studies to the utilization of D-isoleucine and Dvaline in the Ap4A assay.
D-valine did not support ValRS or IleRS activity, as previously
observed(19,20). In contrast, D-isoleucine was activated by IleRS (Figure 2(g)) where IleRS was only 189.2-fold more efficient with the L-stereoisomer (Table 1).
This level of
discrimination is similar to that between L-isoleucine and L-valine which has required the evolution of editing mechanisms to eliminate mis-valylation of tRNAIle
(4,16,18)
.
How mis-
activation of D-isoleucine by IleRS is dealt with is relevant because D-isoleucine is found in the peptidoglycan of stationary phase bacterial cultures(21). This raises the possibility that Disoleucyl-tRNAIle may challenge the stereochemical fidelity of protein synthesis, a possibility enhanced by our inability to demonstrate any editing activity with respect to D-isoleucine by E. coli IleRS (Lloyd unpublished data). To validate the Ap4A assay for inhibitor detection, we tested the response of AlaRS, ValRS, SerRS and ThrRS to a series of aminoacyl-adenylate analogues. Data (Figure S6) were analysed as in(22) to determine Ki values for inhibition (Equation 2 and 3, Figure S7 Legend), as AaRS and inhibitor concentrations were comparable in some of the assays. 6
ACS Paragon Plus Environment
Page 7 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Mupirocin is a tight binding analogue of isoleucyl-adenylate(2). In the Ap4A assay, mupirocin was a potent IleRS inhibitor (Figure 2c) with an IC50 of 0.35 ± 0.06 µM (Figure S6a,e) approximating to half the concentration of IleRS (1.13 µM), where the Ki for this interaction was 0.52 ± 0.12 nM (Figure S7a). Conversely, mupirocin was a weak ValRS inhibitor (37% at 0.5 mM), consistent with the specificity of the drug towards prokaryotic IleRS. Aminoacyl-5’-sulphamoyl adenosines are potent AaRS inhibitors(23) and E. coli AlaRS was inhibited by alanyl-5’-sulphamoyl adenosine (Figure 2a) with an IC50 of 0.403 ± 0.005 µM (Figure S6b,e, Figure S7b).
The impact of cognate adenylate analogues on their
cognate AaRSs as shown here with AlaRS and IleRS confirmed the utility of the Ap4A assay. We then probed the ability of aminoacyl-sulphamoyl adenosines to inhibit both cognate and non-cognate AaRSs. Our data strongly suggest that multiple targeting of AaRSs by single potent adenylate based inhibitors is achievable (SI 4.2). The Ap4A assay was originally developed to assess inhibition by molecules selected or designed by in silico drug design. We used the de novo molecular design programme SPROUT(24) to identify small molecules pockets of E. coli IleRS and ValRS.
that bound to the aminoacyl-adenylate binding Focussed libraries of molecules were synthesised
based around these scaffolds (SI 1.1,1.2,2.1,2.2). This and additional virtual high throughput screening (vHTS) generated a library of 63 compounds (SI 1.3, Tables S2-S5, Figure S9). Screening assays sensitive to ValRS and IleRS inhibitors with as low as millimolar affinities were designed (SI 4.3) and implemented to detect inhibitors of these enzymes within this library. Although we were unable to identify dual targeted inhibitors by this route, we identified
two
IleRS
inhibitors
we
synthesised
(SI
2.2):
N-tert-butyl-2-(3-{[3-(tert-
butylcarbamoyl-methyl)benzyl-amino]-methyl}-phenyl)-acetamide (1) and N-isopropyl-2-(3{[3-(isopropyl-carbamoyl-methyl)-benzylamino]-methyl}-phenyl)-acetamide (2) (Table S6) with Z’-factors(25) of 0.73 and 0.55 and IC50 values of 0.22 ± 0.035 mM and 0.895 ±0.057 7
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 20
mM respectively (Figure 3a and b). These molecules exerted no effect on the hexokinase/6phospho-gluconate dehydrogenase coupling system. Both 1 and 2 were competitive inhibitors against L-isoleucine and Ap4A (Figures 3c-f), suggesting that they bound to IleRS at the site they were designed to target. The Ki values of 1 and 2 (Figures 3c-f) suggested they bound weakly to IleRS, but are consistent with the starting point for fragment based drug design(26). Screening of this library with ValRS (Table S7) identified 3 (4-Amino-2-[2-oxo-2-(3phenyl-isoxazol-5-yl)ethylsulphanyl]-pyrimidine-5-carboxylic acid)) as a ValRS inhibitor (Z’ factor = 0.74) that was not an IleRS inhibitor. 3 did not inhibit the coupling enzymes at 1 mM and on re-screening displayed a sigmoid dependence of inhibition on concentration (IC50 = 1.51 ± 0.32 mM, Hill coefficient of 3.65 ± 0.68; Figure S10d)). To gain additional insight into molecules that might inhibit ValRS, we identified three triazinyl dyes: Procion Red HE-3B, Procion Green HE4BD (Table S7) and Trypan Blue that inhibited the enzyme. The effect was specific (the coupling enzymes were unaffected by up to 0.1 mM dye as judged by the response of an inhibited ValRS assay to 0.1 mM ATP). Furthermore, these dyes did not inhibit IleRS. IC50//Ki values(22) for Procion Red HE-3B, Procion Green HE4BD and Trypan Blue were 2.14 ± 0.29 µM/0.49 ± 0.06 µM, 6.98 ± 1.23 µM/2.21 ± 0.52 µM and 19.2 ± 8.6 µM/13.2 ± 1.47 µM respectively (Figures S10a-c). Procion Green HE4BD inhibits methionyl-tRNAMet synthetase(27) suggesting that this dye could facilitate development of multi-targeted AaRS inhibitors.
Trypan blue has anti-
trypanosomal activity(28). Our data suggest that this molecule could target ValRS, which itself may be a viable strategy in treatment of trypanosomiasis. The utility of the Ap4A assay led us to investigate its use with other adenylate utilizing enzymes.
T4 RNA ligase type 1 self adenylates a lysine residue (K99) prior to RNA
ligation(29) and also synthesises Ap4A(30). Therefore we tested the ability of RNA ligase to 8
ACS Paragon Plus Environment
Page 9 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
generate ATP from Ap4A as we would anticipate the enzyme to self-adenylate using Ap4A to generate ATP.
Subsequently, pyrophosphate could cleave the enzyme lysyl adenylate
generating a second ATP (Figure S11a). As anticipated, T4 RNA ligase catalysed the pyrophosphorolysis of Ap4A in a manner dependent on enzyme, pyrophosphate and Ap4A ( Figure S11b), which showed a linear rate that is stable for at least 30 minutes. This demonstrates the potentially great utility of Ap4A as an assay with respect to other adenylate forming enzymes and may be an improvement on current ligase adenylation assays (SI 4.4 and SI. 4.6). Other continuous AaRS assays have been developed, including the method(8) which initiated this work. These assays are compared to the Ap4A assay in the SI 4.5 and SI. 4.7. Finally, many drug targets turnover Ap4A(31). Indeed in TB there are over sixty enzymes that utilise adenylate intermediates(32) Furthermore, with respect to Ap4A assay implementation, NADPH utilization as a reporter of activity suggests an additional fluorescence format for these assays, and generation of ATP from Ap4A indicates a potential
luciferase based
luminescence readout, yielding three separate techniques for Ap4A utilization assays. Methods Methods are incorporated into Supporting Information (SI 3) Associated Content Methods, additional figures and tables as described in the text are available free of charge in Supporting Information via the Internet at http://pubs.acs.org. Acknowledgments We thank D. Söll for critical reading of the manuscript and the MRC for financial support. A. Lloyd thanks the Birmingham-Warwick Science City Translational Medicine Initiative for support. 9
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
References 1. Sutcliffe, J. A. (2011) Antibiotics in development targeting protein synthesis. Ann. N. Y. Acad. Sci. 1241, 122-152. 2. Hurdle, J. G., O'Neill, A. J., and Chopra, I. (2005) Prospects for aminoacyl-tRNA synthetase inhibitors as new antimicrobial agents. Antimicrob. Agents Chemother. 49, 4821-4833. 3. Sassanfar, M., Kranz, J. E., Gallant, P., Schimmel, P., and Shiba, K. (1996) A eubacterial Mycobacterium tuberculosis tRNA synthetase is eukaryote-like and resistant to a eubacterial-specific antisynthetase drug. Biochemistry 35, 9995-10003. 4. Yadavalli, S. S., and Ibba, M. (2012) Quality control in aminoacyl-tRNA synthesis its role
in translational fidelity. Adv. Protein Chem. Struct. Biol. 86, 1-43. 5. Hernandez, V., Crépin, T., Palencia, A., Cusack, S., Akama, T., Baker, S. J., Bu, W., Feng, L., Freund, Y. R., Liu, L., Meewan, M., Mohan, M., Mao, W., Rock, F. L., Sexton, H., Sheoran, A., Zhang, Y., Zhang, Y. K., Zhou, Y., Nieman, J. A., Anugula, M. R., Keramane, E. M., Savariraj, K., Reddy, D. S., Sharma, R., Subedi, R., Singh, R., O'Leary, A., Simon, N. L., De Marsh, P. L., Mushtaq, S., Warner, M., Livermore, D. M., Alley, M. R., and Plattner, J. J. (2013) Discovery of a Novel Class of Boron-based Antibacterials with Activity against Gram-negative Bacteria. Antimicrob. Agents Chemother. 57, 1394-1403. 6. Williams, A. M., and Martinis, S. A. (2006) Mutational unmasking of a tRNA-dependent pathway for preventing genetic code ambiguity. Proc. Natl. Acad. Sci. U S A. 103, 3586-3591. 7. Francklyn, C. S., First, E. A., Perona, J. J., and Hou, Y. M. (2008) Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases. Methods 44, 100-118. 8.
Roy, S. (1983) A continuous spectrophotometric assay for Escherichia coli alanyltransfer RNA synthetase. Anal. Biochem. 133, 292-295.
10
ACS Paragon Plus Environment
Page 10 of 20
Page 11 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
9. First, E. A. (2005) catalysis of the tRNA aminoacylation reaction. In The aminoacyl-tRNA synthetases (Ibba, M., Francklyn, C., and Cusack, S. Eds.) Chp. 30, pp 328-352, Landes Bioscience, Texas, USA. 10. Zamecnik, P. (1983) Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A): its role in cellular metabolism. Anal. Biochem. 134, 1-10. 11. Davis, M. W., Buechter, D. D., and Schimmel, P. (1994) Functional dissection of a predicted class-defining motif in a class II tRNA synthetase of unknown structure. Biochemistry 33, 9904-9911. 12. Yanagisawa, T., Lee, J. T., Wu, H. C., and Kawakami, M. (1994) Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase. J. Biol. Chem. 269, 24304-24309. 13. Myers, G., Blank, H. U., and Söll, D. (1971) A comparative study of the interactions of Escherichia coli leucyl-, seryl-, and valyl-transfer ribonucleic acid synthetases with their cognate transfer ribonucleic acids. J. Biol. Chem. 246, 4955-4964. 14. Owens, S. L., and Bell, F. E. (1970) Specificity of the valyl ribonucleic acid synthetase from Escherichia coli in the binding of valine analogues. J. Biol. Chem. 245, 55155523. 15. Fersht, A. R. (1977) Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase. Biochemistry 16, 1025-1030. 16. Dulic, M., Cvetesic, N., Perona, J. J., and Gruic-Sovulj, I. (2010) Partitioning of tRNAdependent editing between pre- and post-transfer pathways in class I aminoacyltRNA synthetases. J. Biol. Chem. 285, 23799-23809. 17. Beebe, K., Ribas De Pouplana L., and Schimmel, P. (2003) Elucidation of tRNAdependent editing by a class II tRNA synthetase and significance for cell viability. EMBO J. 22, 668-675.
11
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
18. Schmidt, E., and Schimmel, P. (1994) Mutational isolation of a sieve for editing in a transfer RNA synthetase. Science 264, 265-267. 19. Owens, S. L., and Bell, F. E. (1968) Stereospecificity of the Escherichia coli valyl-RNA synthetase in the ATP- 32PPi exchange reaction. J. Mol. Biol. 38, 145-146. 20. Bergmann, F. H., Berg, P., and Dieckmann, M. (1961) The enzymic synthesis of amino acyl derivatives of ribonucleic acid: II. The preparation of leucyl-, valyl-, isoleucyland methionyl ribonucleic acid synthetases from Escherichia coli. J. Biol. Chem. 236, 1735–1740. 21. Cava, F., de Pedro, M. A., Lam, H., Davis, B. M., and Waldor, M. K. (2011) Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J. 30, 3442-3453. 22. Morrison, J.F. (1969) Kinetics of the reversible inhibition of enzyme catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta 185, 269-286. 23. Vondenhoff, G. H., and Van Aerschot, A. (2011) Aminoacyl-tRNA synthetase inhibitors as potential antibiotics. Eur. J. Med. Chem. 46, 5227-5236. 24. Gillet, V. J., Newell, W., Mata, P., Myatt, G., Sike, S., Zsoldos, Z., and Johnson, A. P. (1994) SPROUT: recent developments in the de novo design of molecules. J. Chem. Inf. Comput. Sci. 34, 207-217. 25. Iversen, P. W., Eastwood, B. J., Sittampalam, G. S., and Cox, K. L. (2006) A comparison of assay performance measures in screening assays: signal window, Z' factor, and assay variability ratio. J. Biomol. Screen. 11, 247-252. 26. Leach, A. R., and Hann, M. M. (2011) Molecular complexity and fragment-based drug
discovery: ten years on. Curr. Opin. Chem. Biol. 15, 489-496. 27. McArdell, J. E., Duffield, M., and Atkinson, T. (1989) Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD. Biochem. J. 258, 715-721.
12
ACS Paragon Plus Environment
Page 12 of 20
Page 13 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
28. Morgan, H. P., McNae, I. W., Nowicki, M. W., Zhong, W., Michels, P. A., Auld, D. S., Fothergill-Gilmore, L. A., and Walkinshaw, M. D. (2011) The trypanocidal drug suramin and other trypan blue mimetics are inhibitors of pyruvate kinases and bind to the adenosine site. J. Biol. Chem. 286, 31232-31240. 29. El Omari, K., Ren, J., Bird, L. E., Bona, M. K., Klarmann, G., LeGrice, S. F. J. and Stammers, D. K. (2006)
Molecular architecture and ligand recognition
determinants for T4 RNA ligase J. Biol. Chem. 281, 1573-1579. 30. Atencia, E. A., Madrid, O., Günther Sillero, M. A., and Sillero, A. (1999). T4 RNA ligase catalyses the synthesis of dinucleoside polyphosphates Eur. J. Biochem. 261, 802-811. 31.
Fraga, H., and Fontes, R. (2011) Enzymatic synthesis of mono and dinucleoside polyphosphates. Biochim. Biophys. Acta. 1810, 1195-1204.
32. Duckworth, B. P., Nelson, K. M., and Aldrich C. C. (2012) Adenylating enzymes in Mycobacterium tuberculosis as drug targets Curr. Top. Med. Chem. 12, 766–796. 33. Segel, I. H. (1975) Enzyme Kinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems. pp. 18–99, Wiley-Interscience, New York.
Table Legends Table 1: Characterization of the substrate dependence of E. coli AlaRS, IleRS and ValRS on the rate of pyrophosphorolysis of Ap4A
Assays were performed as
noted in the table. Due to the high concentrations of enzyme used relative to the lower concentrations of some substrates (particularly pyrophosphate) all data for consistency were fitted to an equation which takes the concentration of enzyme in the assay into account(33): [E] and [S] are enzyme and substrate concentrations: Vo =
kcatApp 2
([E] + [S] + KmApp)2 – (4.[E].[S])
13
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 14 of 20
vo vs [S] plots (Figures 2(e)-(g), S2 and S3) were assessed for goodness of fit using the correlation coefficient (r2) which in all cases was >0.92. Figure Legends Figure 1: Mechanism of Ap4A production and exploitation of its consumption by AaRSs.
Figure 1a: Catalytic activities of AaRSs. Formation of the central
aminoacyl-adenylate intermediate and inorganic pyrophosphate from ATP and an amino acid(4), and reaction of this intermediate with the C-2 or C-3 hydroxyl of the 3’ ribose of tRNA (reaction 2) to form an aminoacyl-tRNA. The aminoacyl-adenylate can also be cleaved by pyrophosphate (reaction 1), displacing the amino acid to regenerate ATP, or by the oxygen atom appended to the γ-phosphorus of a second molecule of ATP which results in the amino acid-dependent formation of adenosine tetraphospho-adenosine (Ap4A; reaction 3).
Figure 1b:
Reversal of Ap4A
synthesis by AaRS. Cleavage of Ap4A by an amino acid substrate and pyrophosphate yielding two molecules of ATP which can be continuously monitored by coupling to NADP+ reduction to NADPH. Figure 2:
Kinetics, stoichiometry and amino acid specificity of AaRS-catalysed
pyrophosphorolysis of Ap4A to ATP.
Panel (a) – (d); Time courses of AaRS
pyrophosphorolysis PPi denotes pyrophosphate. Background rates were followed before addition of the missing component of the assay at arrow. Panel (a): E. coli AlaRS in the presence of 0.41 mM Ap4A, 2 mM L-alanine, 20 µM pyrophosphate and 0.92 µM AlaRS; Panel (b) E. coli ValRS in the presence of 0.388 mM Ap4A, 4 mM L-valine, 20 µM pyrophosphate and 2.41 µM ValRS; Panel (c) E. coli IleRS in the presence of 0.7 mM Ap4A, 10 µM L-isoleucine, 20 µM pyrophosphate and 2.4 µM ValRS. For panels (a) - (c) assays were run without (maroon trace, DMSO control) or with 5 µM alanyl-5’-sulphamoyl adenosine (ASA), 50 µM threonyl-5’-sulphamoyl adenosine (TSA) or 5 µM mupirocin (blue trace). Panel (d): Stoichiometry of Ap4A 14
ACS Paragon Plus Environment
Page 15 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
conversion to ATP in the presence of L-alanine and S. pneumoniae AlaRS. Reaction was allowed to approach equilibrium in the presence of 1.8 µM AlaRS, 0.471 mM pyrophosphate and 27.2 µM Ap4A. Final [ATP] inferred from the ∆A340 on completion of the reaction was 59.2 µM, implying conversion of Ap4A to 2.16 equivalents of ATP. Panel (e) – (g): dependence of initial velocity of IleRS on [L-Isoleucine], [L-valine] and [D-Isoleucine] respectively. For panels (e) to (g) Assays were performed at [cosubstrate] and [enzyme] in Table 1. Data were fitted to the equation in the figure legend to Table 1(22) Figure 3: Evaluation of 1 and 2, IleRS inhibitors identified with the Ap4A assay. [IleRS] = 3.14 µM IleRS, [pyrophosphate] = 20 µM. Panels (a) and (b) dependence of inhibition on 1 or 2 respectively. [Ap4A] and [L-isoleucine] were 0.7 mM and 10 µM respectively. Panels (c) and (d): (IleRS activity)-1 vs. [L-isoleucine]-1 in the presence of 0-3 mM 1 (c) or 0-3.6 mM 2 (d). Panels (e) and (f) ): (IleRS activity)-1 vs. [Ap4A]-1 in the presence of the same range of 1 (e) or 2 (f) concentrations. r2 values for data collected at each [inhibitor] are in purple. Inserts in panels (c) to (f) are secondary plots of slopes of reciprocal plots vs. [inhibitor]. Ki values were determined from the abscissa intercept of secondary plots.
15
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Page 16 of 20
Table 1
AaRS
AlaRS
IleRS
ValRS
[AaRS]Assay
Nonvaried substrate
[Nonvaried substrate] (mM)
Varied Substrate
KmApp (µ µM)
kcatApp (min-1)
kcatApp/KmApp (min-1.µ µM-1)
Goodness of fit (r2)
0.92 µM
L-Alanine
10.0
L-Alanine
14200 ± 1540
39.3 ± 1.93
0.00280
0.984
0.92 µM
Ap4A
3.99
Ap4A
870 ± 171
21.8 ± 1.41
0.0251
0.961
0.92 µM
PPi
0.471
PPi
12.2 ± 1.62
19.5 ± 0.800
1.600
0.979
1.13 µM
L-Isoleucine
14.4
L-Isoleucine
36.1 ± 3.12
6.960 ± 0.180
0.193
0.994
1.13 µM
Ap4A
5.00
Ap4A
6330 ± 929
16.50 ± 1.29
0.00261
0.990
1.13 µM
PPi
0.470
PPi
2.46 ± 0.470
7.53 ± 0.360
3.060
0.923
0.74 µM
L-Valine
3950 ± 694
3.62 ± 0.210
0.000930
0.980
0.74 µM
D-Isoleucine
4090 ± 411
4.17 ± 0.174
0.00102
0.993
1.10 µM
L-Valine
14.4
L-Valine
12700 ± 1700
10.70 ± 0.790
0.000840
0.991
1.10 µM
Ap4A
1.53
Ap4A
1740 ± 161
9.92 ± 0.377
0.00570
0.978
1.10 µM
PPi
0.471
PPi
1.80 ± 0.190
4.08 ± 0.0870
2.267
0.988
16
ACS Paragon Plus Environment
Page 17 of 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Figure 1a O NH2
NH2
Adenosine tetraphospho Adenosine (Ap (AP44A) N
N
O OH OH
O O
O O O P P P O OO OO O
OH OH
N
N
O O O O O O P P P P O OO O O O O
Amino Acid
R
N
N NH2
N
N
N
N
N
NH2
O
Pyrophosphate
O O P OO O P O
ATP
N
O OH OH
H2N
R
O
NH2
O O
3
NH2
O
N
O O
N
N
O P OO O
O O
O
O
P P P O OO OO O
O OO O P P O O
O
Central Aminoacyl Adenylate Intermediate
OH OH
2 tRNA
R
NH2
Aminoacyl-tRNA
H2N O
N
N
N
N
O O
5’-AMP
O O O P P P O OO OO O
OH OH
O
O
O
NH2
OH OH N
N
ATP*
N
N
R
N
N
N
1
N
N
R
NH2
O
NH2
O P OO
OH OH
Figure 1b Pyrophosphate -O
Amino acid O NH2
-O
N
N
NH3+
N O OH OH
N
N
R
N
OH OH N
O OP P -O O O O-
NH2
O PO O -O O O OP P P O -O O O O -O
Ap AP4A 4A:
O
-O R
N NH2
R
OH OH O -O O O OP -O P O P O O -O
NH2
NH3+
N N
N
N
ATP
N
2 Glucose
2 6-Phosphogluconate
Hexokinase
2 Glucose-6-Phosphate NH2
ADP
2 NADPH + 2 H
+
N
+
2 NADP
HO
O
N
O
P P -O O O O-
∆A340
N
O OH OH
17
ACS Paragon Plus Environment
N N
2
O O O -O P P P -O O O O- O
O O
OH OH
NH2
Glucose 6-phosphate Dehydrogenase
NH3 +
N
N
AaRS
O
ATP
O
O
OH OH
N
N
Amino acid
O
P -O O
O
O N
N
ACS Chemical Biology
Figure 2
0.24 0.20
0.24
(a) E. coli AlaRS
0.08
20 µM PPi
20 µM PPi
0.12 0.08
0.04 4
8
12
16
+ 50 µM TSA
0.04
+ 5 µM ASA
0.00 0
0.00 0
20
2
4
Time (min) 0.20
(c) E. coli IleRS
DMSO
20 µM PPi
0.08
Net: AP4A + PPi
30
0 8
12
14
12
16
20
0
Time (min)
20
40
(e)
59.2 µM ATP
2 ATP
60
80
100
Time (min)
25 mM L-Alanine
(f)
6
Alanine
Initial [Ap4A] = 27.2 µM Final [ATP] = 59.2 µM Expected [ATP]/[AP4A] = 2.00 Observed [ATP]/[AP4A] = 2.18
15
+ 5 µM mupirocin
4
10
45
0.04 0.00 0
8
(d) S. pneumoniae AlaRS
60
µ M ATP
0.12
6
Time (min)
0.16
∆ A340
DMSO
0.16
∆ A340
∆ A340
0.16 0.12
(b) E. coli ValRS
0.20
DMSO
(g)
3.2
3.2
2.4
2.4
4 3 2
v0 (min-1)
vo (min-1)
5
Vo (min-1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 18 of 20
1.6 0.8
1.6 0.8
1 0 0.00
0.04
0.08
0.12
0.16
[L-Isoleucine] (mM)
0.20
0.0 0
3
6
9
12
15
[L-Valine] (mM)
18
ACS Paragon Plus Environment
18
0.0 0
3
6
9
[D-Isoleucine] (mM)
12
Page 19 of 20
Figure 3
(a)
(b) 75
100 % Inhibition
% Inhibition
60 2
75
(r = 0.971) O
50
(IC50 = 0.220 ± 0.035 mM)
N H
O
H N
25
(r2 = 0.923)
45
0 0.0
0.4
0.8 1.2 [1] (mM)
1.6
Slope (Km app/Vmaxapp) µ M.min) (µ
250
Ki = 0.184 mM
200
r2 = 0.985
(d) [1] (mM)
50 0 0.0 0.6 1.2 1.8 2.4 3.0 [1] (mM)
1.0 1.5 [2] (mM)
3.5 2.0
2.5
Ki = 0.351 mM
[2] (mM)
r2
5 4
v-1 (min)
2.25 1.80 1.50 1.00 0.75
2.0
3.6 0.997
r2
3.00 0.997
5.0
v (min)
0.5
r2 = 0.971 0.934
100
-1
N H
Inhibition with respect to isoleucine
150
6.5
O
H N
0 0.0
2.0
(IC50 = 0.895 ± 0.057 mM)
N H
15
Inhibition with respect to isoleucine
(c)
O
30
N H
0.995 0.999 0.987 0.995 0.997
3.0 0.993
[2] (mM)
2.4 0.986
3
1.8 0.997
2
1.0 0.975 0
1
0.966
0.00 0.866
0.5 -0.01
-1.0
0.01
0.000 0.025 0.050 0.075 0.100 0.125
0.02
-1 [L-Isoleucine] µM-1) [Isoleucine] (µ µM-1) (µ
-1 (µ µM-1) -1
[L-Isoleucine] [Isoleucine] (µ µM )
12
9.0
9
Inhibition with respect to Ap4A
(f)
Ki = 0.140 mM r2 = 0.959 [1] (mM)
3 0 0.0 0.6 1.2 1.8 2.4 3.0 [1] (mM)
r2
9.0 7.5
3.00 0.984
7.5
[2] (mM)
2.25 0.994
6.0
-0.2
1.80 0.989 1.50 0.982
4.5 3.0
0.75 0.986 0.30 0.999 0.00 0.902
1.5 -1.5 -3.0
0.2
Ki = 0.730 mM r2 = 0.930 0.934
6
v-1 (min)
(e)
Slope (Km app/Vmaxapp) (mM.min)
Inhibition with respect to Ap4A
v-1 (min)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
0.4
[Ap [Ap4A] 4A]
-0.5
-1
-1-1 (mM (mM ))
r2
3.6
0.988
3.0
0.978
2.4 1.8
0.992 0.993
1.0
0.994
0
0.998
6.0 4.5 3.0 1.5
0.6
[2] (mM)
0.0
0.5
1.0 -1 -1
1.5
2.0 -1 [Ap
[Ap4A] [NJP09] (mM ) 4A]
19
ACS Paragon Plus Environment
2.5
3.0
(mM-1)
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Paragon Plus Environment
Page 20 of 20