ESI-MS assay of M. tuberculosis cell wall antigen 85 enzymes permits substrate profiling and design of a mechanism-based inhibitor

Article abstract of DOI:10.1021/ja204249p

Mycobacterium tuberculosis Antigen 85 enzymes are vital to the integrity of the highly impermeable cell envelope and are potential therapeutic targets. Kinetic analysis using a label-free assay revealed both mechanistic details and a substrate profile tha

Full text of DOI:10.1021/ja204249p

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ESI-MS Assay of M. tuberculosis Cell Wall Antigen 85 Enzymes Permits
Substrate Profiling and Design of a Mechanism-Based Inhibitor
Conor S. Barry,† Keriann M. Backus,†,§ Clifton E. Barry, III,*,§ and Benjamin G. Davis*,†
†Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
§Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, U.S. National Institute of Allergy and Infectious Disease,
Bethesda, Maryland, United States
S Supporting Information
b
ABSTRACT: Mycobacterium tuberculosis Antigen 85 en-
zymes are vital to the integrity of the highly impermeable cell
envelope and are potential therapeutic targets. Kinetic
analysis using a label-free assay revealed both mechanistic
details and a substrate profile that allowed the design and
construction of a selective in vitro mechanism-based inhibitor.
ycobacterium tuberculosis (Mtb), the causative agent of
tuberculosis, is among the foremost causes of death and
M
morbidity worldwide. It persists within the host with the aid of
a complex and highly impermeable cell envelope containing a
high content of long-chain fatty acids (mycolic acids) present as
mycolate esters of arabino-galactan and of trehalose 1.1ꢀ3
Trehalose di- and monomycolates (TDM 2a and TMM 3a,
respectively) are interconverted by the Antigen 85 a,b, and c
(Ag85) mycolyltransferase isoforms (Figure 1).4ꢀ6 The Ag85
enzymes play a vital role in the construction and maintenance of
the cell envelope,7 and have recently been shown to display
plasticity for substrates based upon the trehalose motif.8 Genetic
‘knockouts’ highlight Ag85c as the most active isoform; loss
causes a 40% decrease in mycolate content of the cell wall.9 These
enzymes are therefore suggested targets for novel antimycobac-
terial drugs,7 the development of which would benefit from a
rapid and accurate assay of Ag85 activity and a thorough under-
standing of enzyme mechanism through the development of
potential probes. However, despite the availability of several
crystal structures and KM estimates measured under pseudo-
single-substrate conditions or with heterogeneous substrates, no
detailed kinetic analysis or structureꢀactivity relationship (SAR)
studies have been reported. Current assays include nonspecific
radiometric procedures for the detection of generic mycolyl-
transferase activity and coupled enzymatic assays for Ag85c.4ꢀ6
Such methods preclude the possibility of readily automated
screening or do not allow complete dissection of the steps of
Ag85 acyl transfer, respectively. We report here the implementa-
tion of a label-free assay that allows full kinetic analysis as well as
the potential for moderate-throughput screening; a panel of
representative monosaccharides was used to map configurational
structureꢀactivity relationships. Together these data allowed the
design of a mechanism-based inhibitor probe of Ag85c.
Figure 1. Reaction timecourse catalyzed by Ag85. Conditions: 250 μM
1, 250 μM 2b, 2 μM Ag85c, 1 mM TEA buffer (pH 7.2), 37 °C. Full
kinetic analysis utilized the full range of conditions [Tre] = 5ꢀ125 μM,
[2b] = 25ꢀ600 μM, [Ag85c] = 10 nM.
simultaneously to give an unimpeded view of kinetic processes.10ꢀ13
Kinetic analysis and substrate/inhibitor profiling were enabled
by the development of a precise and quantitative MS assay of
Ag85 activity based on total ion counts (TICs) of ions corre-
sponding to trehalose, 6,60-dihexanoyltrehalose 2b (TDH), and
6-hexanoyltrehalose 3b (TMH) (Figure 1). This exploited our
previously described11 calibration approach with a pseudointernal
standard (here N-acetyl-D-glucosamine) injected concurrently with
the sample aliquot; calibration plots for each monitored mass
allowed quantitative measurements of concentration.
Full bi-substrate kinetic analysis was performed by determin-
ing initial rates of TMH 3b formation at varied TDH 2b and
trehalose 1 concentrations.10ꢀ13 These data were fitted by non-
linear regression methods to a range of plausible kinetic models,
including modified rate equations that account for multiple acyl
transfer pathways.14 Together these and double-reciprocal anal-
ysis (see Supporting Information [SI]) ruled out simple (e.g.,
bi-bi ping-pong) mechanisms15,16 that are typical of other
Mass spectrometry (MS) can be readily automated, and
multiple natural substrates and products can be monitored
Received: May 9, 2011
Published: July 21, 2011
r
2011 American Chemical Society
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Table 1. Bi-substrate Kinetic Analysis of Ag85c
Table 2. Kinetic Parameters for Ag85 Isoforms Recorded
under Pseudo-Single-Substrate Conditions
Vt (μM/min)
Vh (μM/min)
0.70 ( 0.05
0.29 ( 0.08
kcat (acyl-transfer) (sꢀ1
kcat (hydrolysis) (sꢀ1
kcat/KM(TDH) (Mꢀ1 sꢀ1
kcat/KM(Tre) (Mꢀ1 sꢀ1
)
1.16
)
0.49
Ag85
varied
KM(app) kcat(app)
k
cat(app)/KM (app)
KM(TDH) (μM) 266.8 ( 41.2
)
4.35 ꢁ 103
isoform substratea
(μM)
(sꢀ1
)
(Mꢀ1 s 1)
KM(Tre) (μM)
Ki(TDH) (μM)
16.9 ( 5.3
)
6.88 ꢁ 104
a
b
c
1
1
1
175
112
62
0.014
0.003
0.182
82
30
355.3 ( 105.6
2952
a Assay conditions: [TDH] = 500 μM, [trehalose] = 10ꢀ250 μM,
[Ag85a/b] = 500 nM, [Ag85c] = 50 nM.
Figure 2. Mechanistic outline for Ag85.
acyltransferases. Nonetheless, crystal structure data show a clear
Ser-His-Asp catalytic triad at the trehalose binding site of Ag85,17
the serine of which has been mutated and nonspecifically
modified, suggesting a role as a nucleophile;18 it has also been
reported that Ag85 can form a mycolylꢀenzyme complex.6
Determination of the full kinetic parameters for Ag85c
(Table 1) including KM values for both substrates and separate
maximal reaction velocities for acyl transfer (Vt) and hydrolysis
(Vh), resolved this apparent conundrum and revealed consider-
able acylhydrolase activity (kcat 0.49 sꢀ1) in contrast with prior
reports.6,19 We reasoned that this previously unreported activity
of Ag85 was likely enzyme-mediated.20 Indeed, analysis using a
suitably modified ping-pong cycle gave an excellent correlation
with the data (R2 > 0.99). The implied mechanism (Figure 2) is
therefore one of ping-pong formation of a hydrolytically unstable
acyl enzyme intermediate that then releases catalyst through two
competitive pathways. Although rare, such mechanisms are not
entirely unprecedented.21ꢀ23 This insight informed later inhibi-
tor design (vide infra).
This precise dissection of different activities highlighted a
strong advantage compared with, for example, single-substrate
coupled assays that survey only one part of a given mechanistic
cycle.19 The value for KM(Tre) correlated well with the only
previous value of 8.3 μM, determined under pseudo-single-
substrate conditions.6 A higher KM value determined for TDH
may reflect the homogeneous, shorter lipid chain of 2b compared
with heterogeneous mycolic acid substrates. The relative acyl-
transferase activities of the three Ag85 isoforms were also
determined under pseudo-single-substrate conditions by mon-
itoring the production of both TMH and hexanoate concurrently
(Table 2), and confirmed the higher activity of Ag85c compared
to those of Ag85a or Ag85b.24
Figure 3. Substrate profiling screen and related compound structures.
Assay conditions: 500 μM TDH, 500 μM screen compound, 2 μM Ag85
incubated at 37 °C for 160 min before measurement of ν (product/
substrate peak ratio). νrel = 100 ꢁ (ν/νTre). See SI for Table S1.
With an accurate automated assay of Ag85 activity in place we
then probed substrate selectivity. We have previously shown the
utility of a semiquantititative green-amber-red (GAR) moderate-
throughput screen to discover novel substrates for other bi-
substrate (glycosyltransferase) enzymes.10ꢀ13 Relative reaction
velocity (νrel) was determined by MS-based GAR assay for a panel
of putative substrates (Figure 3, Table S1[SI]) to evaluate their
suitability as acyl acceptors. Both trehalose (1) itself and related
disaccharides 4 and 5 were found to act as substrates,8 although the
measured νrel for 5 was lower for Ag85a and b, indicating the
sensitivity of the enzymes to altering this scaffold configuration away
from that found in trehalose (here through inversion at C-4).
Interestingly, 6, a known inhibitor of mycobacterial growth and
previously described as an in vitro inhibitor of Ag85c activity is also
a substrate for all Ag85 isoforms, indicating that it acts as a competitive
substrate and suggesting that the observed antibacterial activity may
arise from downstream events after Ag85-mediated mycolyltransfer or
through inhibition of other pathways.4,25
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Figure 4. Synthesis and evaluation of probe 19. (a) Lowest energy binding poses in the active site of Ag85c of trehalose 1 (cyan), TMH 2b (green), 17
(yellow) and 19 (magenta). The active site serine is shown with an arrow. Image produced using PyMol,26 and docking poses generated with AutoDock
Vina27 from pdb 1DQZ. (b) Synthesis of inhibitors 17 and 19. Reagents and conditions: i) hexyldichlorophosphate, pyridine, rt, 16 h; ii) 1:1 EtOH/H2O,
PdꢀC, H2, rt, 18 h; iii) BTFFH, DIPEA, DMF, rt, 18 h; iv) 1:1 EtOH/H2O, PdꢀC, H2, rt, 18 h. (c) Reaction of probe 19 with Ag85c indicates essentially
complete conversion to covalently modified Ser124 adduct by ES-MS.
Despite observed plasticity for variants of the trehalose
scaffold, truncated or more dramatically altered sugar variants
displayed considerably lower activity. Although trehalose is a 1,
1-diglucoside, methyl D-glucosides 9 and 10 were utilized less
efficiently. Interestingly, some low-level activity (up to ∼20%)
was also observed for the arabinofuranosides 7 and 8; this is
consistent with a possible role of Ag85 in mycolate scrambling
and mycolation of Mtb arabinogalactan. Notably, for these
glucosides and arabinosides differential anomeric selectivity
between enzyme isoforms was observed: R-anomers were pre-
ferred by Ag85a and b, with Ag85c showing little anomer
discrimination. In keeping with this observation of tolerance at
the anomeric center, Ag85c also showed the greatest tolerance
toward ketosides 4 and 5, which carry an additional methyl
substituent at C-1. As for the disaccharides, configurational
changes had a more dramatic effect. The very low νrel value
determined for D-galactosides 11 and 12 for all three Ag85
isoforms is clear additional evidence that the epimeric config-
uration at C-4 is a crucial selectivity determinant. Conversely, R-
methyl mannoside 13 was slowly utilized by Ag85c, indicating
some degree of flexibility with respect to C-2 configuration. As
expected, xyloside 14 was also poorly processed, confirming
that the majority of acyl transfer is OH-6. These differences
in substrate recognition stand in contrast to the conserved
active site of these three isoforms and suggest that the enzymes
may well have distinct physiological roles.18
intervention in the divergent pathways for deacylation
(Figure 2). Fluorophosphonate 19 (Figure 4b) was therefore
based upon (a) targeting the TI of acylation (TI1) through the
in situ formation of a tetrahedral mimic (Figure 2);28 (b)
targeting of the same TI by ensuring features that also mimic
both lipid and sugar moieties (Figure 2); (c) substrate preferences
that highlight trehalose as a superior sugar scaffold (Figure 3).
In silico evaluation supported another aspect of design with these
features: molecular docking27 was performed to compare binding
modes that would correspond to putative Michaelis complexes
(Figure 2, MC1 and Figure 4a). Lowest energy conformations of 19
(ꢀ7.3 kcal molꢀ1) overlaid closely with both TMH 2b (ꢀ7.0 kcal
molꢀ1) and also with trehalose (observed in the crystal structure of
Ag85b, pdb: 1F0P17), placing the acyl carbon (in 2b) and phos-
phoryl phosphorus (in 19) in close proximity to the targeted active
site serine 124.
19 was readily synthesized from hepta-benzyl-trehalose 158
(Figure 4b). Treatment of 16 with fluoro-N,N,N0,N0-bis-
(tetramethylene)formamidinium hexafluorophosphate (BTFFH)
afforded 18 in good yield; deprotection by catalytic hydrogenolysis
gave 19 with no concomitant hydrolysis of the phosphorylfluoride.
The inhibitory potential of 19 against Ag85c was assayed using
a saturating concentration of trehalose (>25ꢁ KM) to ensure
maximal turnover rate. Following incubation of Ag85c with 19
(150μM) activity was almost entirely ablated (<7%). Phospho-
nate 17, an unreactive analogue of 19 that cannot effectively
29
With this understanding of substrate tolerance, we sought to
rationally design an effective covalent inhibitor probe. Although
nonspecific approaches based simply on serine modification
might be considered,18 the true utility of this probe would be
vitally dependent on selectivity. Design was therefore essentially
informed by both mechanism (Figure 2) as well as by the
substrate screen. These dictated an inhibitor structure that would
mimic the tetrahedral intermediate (TI) formed in the first
unified ‘ping-to-pong’ acylation steps rather than through
mimic the targeted TI resulted in inhibition only to 46%. k /
obs
Ki was determined to be 1420 ((90) minꢀ1 mMꢀ1 (see SI); the
reactivity of 19 is such that saturation could not be achieved, as
has been reported for other covalent inhibitors.30 This marks 19
as a potent inhibitor in comparison with other fluorophos-
phonates.31,32With inhibitory potential confirmed, critical selec-
tivity of 19 for Ag85 over other enzymes that contain similar
catalytic triads was investigated. Serine protease subtilisin from
Bacillus lentus (SBL), lipase B from Candida antarctica (CalB),
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and Ag85c were incubated under identical conditions with 19.
Detailed analysis by MS revealed essentially complete protein
modification of Ag85c at Ser124 (Figure 4c and SI), while
no detectable modification or inhibition of either SBL or CalB
was observed.
(4) Belisle, J. T.; Vissa, V. D.; Sievert, T.; Takayama, K.; Brennan,
P. J.; Besra, G. S. Science 1997, 276, 1420–1422.
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MS has provided an accurate and automated method to
determine the full kinetic parameters of Ag85c, which revealed
that the enzyme displays a combination of acyltransferase and
acylhydrolase activities. Substrate profiling revealed that,
although the Ag85 enzymes show promiscuity for trehalose-
based substrates,8 selectivity for differing monosaccharides and
anomeric configurations is marked: gluco- and arabinofurano-
sides are preferred over galacto-, manno-, or xylopyranosides.
These data provide additional evidence that Ag85 is responsible
for both trehaloseꢀmycolate scrambling and mycolylation of the
mycobacterial arabinogalactan. They also suggest that regions
distal to the active sites influence activity of the three isoforms.
The screen data indicate that disaccharides are better substrates
than the constituent monosaccharides, which suggests an ex-
tended active site. This in turn suggests that the optimal starting
point for rational drug design may be the trehalose scaffold that
was used here to design a tailored fluorophosphonate 19. This
compound was a potent and highly selective covalent inhibitor
probe of Ag85c that shows no reactivity toward other serine
acyltransferases. The selectivity of this molecule may allow
interrogation of the importance of Ag85 activity in vitro or in
infected macrophages. We anticipate that this and related
‘tagged’ compounds may find use as activity-based probes33 of
Ag85 function and might serve as a starting point for the design of
novel anti-mycobacterial drugs.
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(14) See SI for derivation.
(15) See SI for details of double reciprocal analysis of Ag85c.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental methods and
b
(23) The divergent deacylation pathways may also be related to the
nature/identity of the fatty acyl chain.
supporting figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
(24) νtransfer =(νTMH ꢀ νhydrolysis)/2
(25) Kalscheuer, R.; Syson, K.; Veeraraghavan, U.; Weinrick, B.;
Biermann, K. E.; Liu, Z.; Sacchettini, J. C.; Besra, G.; Bornemann, S.;
Jacobs, W. R. Nat. Chem. Biol. 2010, 6, 376–384.
(26) The PyMOL Molecular Graphics System, Version 0.99rc6:
Schr€odinger, LLC: http://pymol.org/pymol.
’ AUTHOR INFORMATION
Corresponding Author
cbarry@niaid.nih.gov; Ben.Davis@chem.ox.ac.uk
(27) Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455–461.
(28) Bachovchin, D. A.; Ji, T. Y.; Li, W. W.; Simon, G. M.; Blankman,
J. L.; Adibekian, A.; Hoover, H.; Niessen, S.; Cravatt, B. F. Proc. Natl
Acad. Sci. U.S.A. 2010, 107, 20941–20946.
’ ACKNOWLEDGMENT
(29) See SI for details.
(30) McCarter, J. D.; Withers, S. G. J. Am. Chem. Soc. 1996, 118,
241–242.
(31) Diisopropylfluorophosphonate and soman have kobs/Ki values
of 140 and 9200 minꢀ1 mMꢀ1, respectively, against human acetylcho-
line esterase.
(32) Worek, F.; Thiermann, H.; Szinicz, L.; Eyer, P. Biochem.
Pharmacol. 2004, 68, 2237–2248.
This work was funded by the Intramural Research Program of
the National Institutes of Health, National Institute of Allergy
and Infectious Disease (C.E.B.), the Rhodes Trust (K.M.B.), the
Biotechnology and Biological Sciences Research Council (C.S.B.),
and the Bill and Melinda Gates Foundation through the TB Drug
Accelerator Program (C.E.B, B.G.D.). B.G.D. is a Royal Society
Wolfson Research Merit Award recipient. We thank Colorado
State “TB Vaccine Testing and Research Materials” contract for
providing Antigen 85 plasmids.
(33) Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106, 3279–3301.
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