Transition State Analogue Inhibitors of 5′-Deoxyadenosine/5′-Methylthioadenosine Nucleosidase from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.biochem.7b00576

Mycobacterium tuberculosis 5′-deoxyadenosine/5′-methylthioadenosine nucleosidase (Rv0091) catalyzes the N-riboside hydrolysis of its substrates 5′-methylthioadenosine (MTA) and 5′-deoxyadenosine (5′-dAdo). 5′-dAdo is the preferred substrate, a product of radical S-adenosylmethionine-dependent enzyme reactions. Rv0091 is characterized by a ribocation-like transition state, with low N-ribosidic bond order, an N7-protonated adenine leaving group, and an activated but weakly bonded water nucleophile. DADMe-Immucillins incorporating 5′-substituents of the substrates 5′-dAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5′-Deoxy-DADMe-Immucillin-A was the most potent among the 5′-dAdo transition state analogues with a dissociation constant of 640 pM. Among the 5′-thio substituents, hexylthio-DADMe-Immucillin-A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state analogues differs from those of other bacterial homologues because of an altered hydrophobic tunnel accepting the 5′-substituents. Inhibitors of Rv0091 had weak cell growth effects on M. tuberculosis or Mycobacterium smegmatis but were lethal toward Helicobacter pylori, where the 5′-methylthioadenosine nucleosidase is essential in menaquinone biosynthesis. We propose that Rv0091 plays a role in 5′-deoxyadenosine recycling but is not essential for growth in these Mycobacteria.

Full text of DOI:10.1021/acs.biochem.7b00576

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Article
“Transition state analogue inhibitors of 5'-deoxyadenosine/5'-
methylthioadenosine nucleosidase from Mycobacterium tuberculosis”
Hilda A. Namanja-Magliano, Gary B. Evans, Rajesh Harijan, Peter C. Tyler, and Vern L. Schramm
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00576 • Publication Date (Web): 24 Aug 2017
Downloaded from http://pubs.acs.org on August 31, 2017
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Transition state analogue inhibitors of 5'ꢀdeoxyadenosine / 5'ꢀ
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methylthioadenosine nucleosidase from Mycobacterium tuberculosis
Hilda A. NamanjaꢀMagliano1, Gary B. Evans2,3, Rajesh K. Harijan1, Peter C. Tyler 2, and Vern
L. Schramm 1,*
1
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461, United States
2
The Ferrier Research Institute, Victoria University of Wellington , Lower Hutt, Wellington
5040, New Zealand
3The Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, New
Zealand
KEYWORDS
Rv0091, MTAN, ribocation analogues, Immucillins, DADMeꢀImmucillins
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ABSTRACT
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Mycobacterium tuberculosis 5'ꢀdeoxyadenosine/5'ꢀmethylthioadenosine nucleosidase (Rv0091)
catalyzes the Nꢀriboside hydrolysis of its substrates 5'ꢀmethylthioadenosine (MTA) and 5′ꢀ
deoxyadenosine (5'ꢀdAdo). 5'ꢀdAdo is the preferred substrate, a product of radical SAMꢀ
dependent enzyme reactions. Rv0091 is characterized by a ribocationꢀlike transition state, with
low Nꢀribosidic bond order, an N7 protonated adenine leaving group and an activated but weakly
bonded water nucleophile. DADMeꢀImmucillins incorporating 5'ꢀsubstituents of the substrates
5'ꢀdAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5'ꢀDeoxyꢀ
DADMe ImmucillinꢀA was the most potent among the 5'ꢀdAdo transition state analogs with a
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dissociation constant of 640 pM. Among the 5'ꢀthio substituents, hexylthioꢀDADMeꢀImmucillinꢀ
A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state
analogues differs from other bacterial homologues because of an altered hydrophobic tunnel
accepting the 5'ꢀsubstituents. Inhibitors of Rv0091 had weak cell growth effects on
Mycobacterium tuberculosis or Mycobacterium smegmatis, but were lethal towards Helicobacter
pylori, where the 5'ꢀmethylthioadenosine nucleosidase is essential in menaquinone biosynthesis.
We propose that Rv0091 plays a role in 5'ꢀdeoxyadenosine recycling, but is not essential for
growth in these Mycobacteria.
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INTRODUCTION
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5'ꢀMethylthioadenosine/Sꢀadenosylhomocysteine nucleosidases (MTANs) are absent in
human metabolism, however in bacteria, they are involved in pathways related to Sꢀ
adenosylmethionine (SAM) recycling, quorum sensing, methylation, menaquinone and
polyamine biosynthesis.1ꢀ3 The MTANs catalyze Nꢀribosidic bond hydrolysis of 5'ꢀ
methylthioadenosine (MTA), Sꢀadenosylhomocysteine (SAH), and 5'ꢀdeoxyadenosine (5'ꢀdAdo).
Mycobacterium tuberculosis (M. tuberculosis) Rv0091 was originally annotated as MTAN,
however recent studies demonstrates that it functions primarily as a 5'ꢀdAdo nucleosidase.4 5'ꢀ
dAdo is generated together with methionine as products of radical SAMꢀdependent enzyme
reactions (Scheme. 1).5 The M. tuberculosis genome encodes 21 open reading frames annotated
as radical SAM enzymes based on the sequence analysis identifying an ironꢀsulfur CX3CX2C
motif that has been linked to radical SAM function.6 Accumulation of 5'ꢀdAdo is reported to
block the activity of some SAMꢀrelated enzymes as a product inhibitor.3
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Scheme 1. Reaction catalyzed by radical SAM enzymes for the reductive cleavage of SAM with [4Fe –
4S]+ in the presence of substrate [AH] to give 5'ꢀdAdo and methionine
Tuberculosis (TB) is one of the most difficult bacterial infections as a result of the spread of
multidrug resistant strains. TB affects one third of the world population and is resistant to many
of the current antibacterial therapies.7 TB poses a worldwide threat and new antibiotics are
needed to treat the causative agent, M. tuberculosis. Here we explore the transition state
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analogue specificity of Rv0091 MTAN and test several inhibitors against cultured organisms as
potential antibacterials.
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Transition state analogues capture the geometry and electrostatic features of an enzymatic
transition state in chemically stable structures. The analogues have the potential to bind to the
enzyme much tighter than the substrate, with an upper limit of the factor equal to the catalytic
enhancement imposed by the enzyme, relative to the Michaelis complex.8ꢀ10 A perfect transition
state analogue is impossible to design, as the actual transition state structure consists of nonꢀ
equilibrium bond lengths and charge distributions that cannot be captured in a stable molecule.
The Immucillins and DADMeꢀImmucillins are stable transition state analogues of Nꢀ
ribosyltransferases developed from transition state analysis using kinetic isotope effects (KIEs)
and computational chemistry.11ꢀ13 Several bacterial MTANs are inhibited by these compounds
with potent dissociation constants (fM – pM range).14ꢀ17
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Previously, the transition state structure of M. tuberculosis Rv0091 was analyzed and was
found to resemble the DADMeꢀImmucillins.4 The DADMeꢀImmucillins contain a 5'ꢀthio group
to mimic the MTAN substrate MTA. A family of the DADMeꢀImmucillins inhibited Rv0091
with dissociation constants with pM to nM affinity. 5'ꢀdAdo was previously shown to be the
preferred substrate of Rv0091, therefore, we anticipated transition state analogues similar to the
transition state for 5'ꢀdAdo to have the best affinities. In this study, we incorporate features of 5'ꢀ
dAdo into transition state analogue design. Inhibition constants are established for several 5'ꢀ
deoxyalkylꢀDADMeꢀImmucillinꢀA compounds. Chemical synthesis and inhibition is described
for the novel compound 5'ꢀdeoxyꢀDADMeꢀImmA (4). The 5'ꢀdeoxyalkylꢀ and the 5'ꢀthio
containing DADMeꢀImmucillinꢀA compounds (MTA and SAH substrate features) displayed
dissociation constants in the pM to low nM range. The 5'ꢀdeoxyalkylꢀDADMeꢀImmucillin
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inhibitors show optimal inhibitory activity with short 5'ꢀsubstituents, while 5'ꢀthioalkylꢀDADMeꢀ
Immucillins bind most tightly with longer 5'ꢀsubstituents. This inhibitory pattern is not found in
other bacterial MTANs and is therefore unique to Rv0091. Molecular modeling of inhibitors into
the catalytic site of Rv0091 indicates an altered binding mode for 5′ꢀsubstituents compared to
other bacterial MTANs.
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Bacterial growth assays show that Rv0091 inhibitors have weak effects on the growth of M.
tuberculosis and Mycobacterium smegmatis (M. smegmatis). In contrast, the same inhibitors
displayed potent antibacterial activity against Helicobacter pylori (H. pylori) species. The
essential nature of MTAN in menaquinone synthesis for H. pylori is well documented.17 18 Thus,
the function of Rv0091 MTAN is not essential for laboratory growth in these Mycobacteria.
MATERIALS AND METHODS
General experimental approach for inhibitor synthesis. Compounds 5 – 20 were synthesized
as previously described.17, 19ꢀ23 Air sensitive reactions were performed under argon. Organic
solutions were dried over anhydrous MgSO4 and the solvents were evaporated under reduced
pressure. Anhydrous and chromatography solvents were obtained commercially and used
without any further purification. Thin layer chromatography (t.l.c.) was performed on glass or
aluminum sheets coated with 60 F254 silica gel. Organic compounds were visualized under uv
light or use of a dip of ammonium molybdate (5 wt %) and cerium(IV) sulfate 4 H2O (0.2 wt %)
in aq. H2SO4 (2 M), one of I2 (0.2 %) and KI (7%) in H2SO4 (1 M), or 0.1 % ninhydrin in EtOH.
Chromatography (flash column) was performed on silica gel (40ꢀ63 ꢁm) or on an automated
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system with a continuous gradient facility. H NMR spectra were measured in CDCl3, CD3OD,
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DMSO d6 (internal Me4Si, δ 0) or methanolꢀd4, and C NMR spectra in CDCl3 (centre line, δ
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77.0), CD3OD (centre line, δ 49.0),. Assignments of H and C resonances were based on 2D
(1Hꢀ1H DQFꢀCOSY, 1Hꢀ13C HSQC, HMBC) and DEPT experiments. Positive electrospray mass
spectra were recorded on a QꢀTOF Tandem Mass Spectrometer.
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(3S,4R)ꢀtertꢀButylꢀ3ꢀ(bromomethyl)ꢀ4ꢀhydroxypyrrolidineꢀ1ꢀcarboxylate (2) synthesis.
Methanesulfonyl chloride (0.45 ml., 5.7 mmol) was added dropwise to a stirred solution of tertꢀ
butyl 3ꢀhydroxyꢀ4ꢀ(hydroxymethyl)pyrrolidineꢀ1ꢀcarboxylate (1) (1.13 g, 5.2 mmol) and 2,6ꢀ
dimethylpyridine (1.2 mL, 10.3 mmol) in acetone (20 mL) and the mixture left stirring for 24 h.
The resulting suspension was filtered to remove salt and then lithium bromide (2.25 g, 25.9
mmol) was added to the filtrate and the mixture refluxed for 3h at which time the reaction was
deemed complete. The crude reaction mixture was absorbed onto silica gel (5 g), concentrated in
vacuo and the resulting residue purified by chromatography (20%=>40%=>100% EA:PE) to
afford the title compound 2 (832 mg, 57%) as a syrup. 1H NMR (500 MHz, CDCl3) δ 4.06 (brs,
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1H), 3.67 (brs 2H), 3.47 – 3.36 (m, 2H), 3.28 – 3.18 (m, 2H), 2.50 (brs, 1H), 1.46 (s, 9H). C
NMR (125 MHz, CDCl3) δ 154.6, 79.9, (73.3, 72.5), (52.6, 52.3), (48.8, 48.3), (48.2, 47.6), 32.6,
28.5. HRMS (ESI) m/z calcd for C10H18NO3BrNa+ 302.0368, obsd 302.0364.
(3R,4S)ꢀ4ꢀMethylpyrrolidinꢀ3ꢀol (3) synthesis. A mixture of 2, triethylamine (1.85 mL, 13.2
mmol), and Perlman’s catalyst (150 mg, 1.0 mmol) in ethanol (20 mL) was stirred under an
atmosphere of hydrogen for 3h. The crude reaction mixture was then filtered through Celite and
concentrated in vacuo and the residue dissolved in methanol (2 mL) and conc HCl (2 mL) and
concentrated in vacuo. The resulting syrup was dissolved in additional conc HCl (2 ml) and
concentrated in vacuo. The residue was dissolved in MeOH and absorbed onto silica,
concentrated in vacuo and the resulting solid was purified by chromatography (30% 7N NH3 in
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MeOH:CHCl3) to afford the title compound 3 (501 mg, 97%) as an oil. H NMR (500 MHz,
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CDCl3) δ 4.24 (brs, 1H), 3.65 ꢀ 3.55 (m 2H), 3.25 (d, J = 15.0 Hz, 1H), 3.08 – 3.04 (m, 1H), 2.39
(brs, 1H), 1.09 (d, J = 10.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 75.5, 50.8, 50.2, 39.9, 15.1.
HRMS (ESI) m/z calcd for C5H12NO [MH]+ 102.0919, obsd 102.0914.
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(3R,4S)ꢀ1ꢀ[(9ꢀDeazahypoxanthinꢀ9ꢀyl)methyl]ꢀ3ꢀhydroxyꢀ4ꢀmethylpyrrolidine (4) synthesis.
Formaldehyde (0.41 mL 5.4 mmol, 37 mass% aq) was added to a stirred suspension of 3 (500
mg, 4.9 mmol,) and 9ꢀdeazaadenine (77 mg 5.4 mmol) in a water (2.5 mL) and ethanol (5 mL)
mixture and then left to stir at room temperature for 48 h. The crude reaction mixture was
absorbed onto silica, concentrated in vacuo and the solid residue purified by silica gel
chromatography to afford the title compound 4 (647 mg, 53%) as a solid. 1H NMR (500 MHz,
Methanolꢀd4) δ 8.18 (s, 1H), 7.49 (s, 1H), 3.87 – 3.74 (m, 2H), 3.37 (s, 1H), 3.05 (dd, J = 9.6, 7.7
Hz, 1H), 2.82 (dd, J = 10.4, 6.5 Hz, 1H), 2.70 (dd, J = 10.4, 4.3 Hz, 1H), 2.17 (dd, J = 9.6, 7.9
Hz, 1H), 2.04 (ht, J = 7.2, 3.5 Hz, 1H), 1.05 (d, J = 6.9 Hz, 3H).. 13C NMR (125 MHz,
Methanolꢀd4) δ 152.1, 151.0, 147.0, 130.1, 115.1, 112.6, 79.0, 62.1, 61.0, 49.2, 43.0, 17.8.
HRMS (ESI) m/z calcd for C12H18N4O [MH]+ 248.1511, obsd 248.1508.
Expression and purification of 5′ꢀdeoxyadenosine/ 5′ꢀmethylthioadenosine nucleosidase.
Enzyme expression and purification were done as described previously.4 Briefly, E. coli BL21
StarTM (DE3) plysS cell lines were employed for expression. A 25 mL initial culture with LB
medium (Gibco) supplemented with ampicillin (100 ꢁg/mL) and chloramphenicol (100 ꢁg/mL)
was incubated overnight at 37 °C. The following day, 6 mL of culture was added to 1 L of fresh
LBꢀampicillin (100 ꢁg/mL) and incubated at 37 °C to an OD600 of 0.6 to 0.8. Cells were induced
with 0.5 mM isopropylꢀDꢀthiogalactoside (IPTG) (Goldbio) and incubated overnight at 28 °C.
Cells were harvested and protein purified by NiꢀNTA (Qiagen) chromatography with a 30 to 250
mM imidazole buffer containing 20 mM TrisꢀHCl pH 7.4 and 300 mM NaCl. Fractions
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containing purified protein (greater than 90% by SDSꢀgel) were pooled, exchanged into 50 mM
HEPES, pH 7.4 and 10% glycerol and stored at –80 °C.
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Dissociation constants. Dissociation constants (Ki values) of Rv0091 inhibitors were determined
as previously described.24 Briefly, the 5′ꢀdAdo nucleosidase reaction was coupled with 1 unit
xanthine oxidase (SigmaꢀAldrich) in the presence of 1 mM 5′ꢀdAdo and varying concentrations
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of inhibitor in 50 mM HEPES, pH 7.4, at 25 °C. Formation of 2,8ꢀdihydroxyadenine was
monitored at 305 nm for 2 hours. The reactions were initiated by adding 10 nM of enzyme. The
Ki values were obtained from the rates with and without inhibitors (vi / v0) that were fitted using
the Morrison quadradic equation for tightꢀbinding inhibitors Eq. 1 on GraphPad Prism.25 The KM
for 5′ꢀdAdo is 10.9 ꢀM, E, I and S are enzyme, inhibitor and substrate concentrations,
respectively.4
Eq. 1

2


i
[S]
[E ]+[I]+K 1+
 − 4[E ][I]
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
v = v 1− E + I + K 1+
[ ]
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o
T
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KM
2[ET ]
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
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

Antibacterial testing. Mycobacterium smegmatis (M. smegmatis) strain mc2155 and M.
tuberculosis mc26230 (nonꢀpathogenic; pantothenate auxotroph) were cultured in Middlebrook
7H9 liquid media (Difco) to an OD600 = 0.6 and plated on Middlebrook 7H10 agar plates as
described previously.26, 27 Bacterial growth inhibition was determined by a drug diffusion
method. Test compounds were added to the center of discs and placed on M. smegmatis and M.
tuberculosis inoculated plates and allowed to grow for 96 hours at 37 °C.
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Antibacterial activity assay with H. pylori. H. pylori was grown under microaerophilic
conditions (5% O2, 10% CO2 and 85% N2) at 37 °C in brain heart infusion medium (Oxoid) with
10% fetal bovine serum (Gibco) and DENT supplement (Oxoid). HTꢀDADMe ImmA and
tetracycline (SigmaꢀAldrich) stocks were made in culture medium and MIC values were
determined by adding 50 ꢀL of varied drug concentrations in a 96 well plate format at 2X
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concentration in culture medium. Cultured H. pylori (50 ꢀL) was added to the medium with
inhibitor. After 72 hours incubation in a microaerophilic conditions at 37 °C, the OD at 600 nm
was determined on a SpectraMax plate reader and MIC50 values obtained using a nonꢀlinear
regression curve fit using GraphPad prism.
Homology modeling. An in silico model of Rv0091 for structure prediction was performed by
the IꢀTASSER, an online homology platform for automated protein structure prediction without
any additional templates.28 The structural comparisons of the Rv0091 model to MTAN structures
with close homology was accomplished with the secondary structure matching (SSM) algorithm
in COOT.29 The superposition of the binding site of Rv0091 was done in comparison to H. pylori
MTAN (HpMTAN; PDB ID: 4FFS) and E. coli MTAN (EcMTAN, PDB ID: 4WKC).
The Rv0091 model was analyzed by molecular docking with AutoDock Vina for binding
with the transition state analogues BTꢀDADMeꢀImmA (13) and HTꢀDADMeꢀImmA (14).30
Coordinates for BTꢀDADMeꢀImmA were obtained from BTꢀDADMeꢀImmA bound to
HpMTAN (PDB ID: 4FFS). Coordinates for HTꢀDADMeꢀImmA (14) were generated and
optimized using the eLBOW function of the PHENIX system for macromolecular structures.31
Molecular docking was performed using a grid box of the whole protein. The potential binding
site for BTꢀDADMeꢀImmA and HTꢀDADAMeꢀImmA was identified by applying the binding
mode of the inhibitors to the Rv0091 model. A second round of docking was done with the
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smaller grid (14 x 14 x 14 Å), centered on the binding site. The search completeness was
evaluated by the default value E = 8 during docking with AutoDock Vina. The binding site
analysis and comparisons were done with the published structures of HpMTAN (4FFS) and
EcMTAN (4WKC).17, 32 The figures were made by using PyMOL (The PyMOL Molecular
Graphics System, Version 1.3, Schrödinger, LLC).
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RESULTS AND DISCUSSION
DADMeꢀImmucillin mimics of the 5′ꢀsubstituent of Rv0091 substrates.
The transition state structure of M. tuberculosis Rv0091 revealed a ribocationꢀlike ribose ring
with low C1′–N9 bond order, protonation of the adenine leaving group and weak participation of
the water nucleophile (Scheme. 2).4 The 5'ꢀalkylthioꢀDADMeꢀImmucillin inhibitors resemble the
Rv0091 transition state structure while incorporating features of the MTA substrate. MTꢀ
DADMeꢀImmA (11) incorporates the 5'ꢀmethylthio group of MTA and exhibits an inhibition
constant of 1.5 ± 0.4 nM (Figure 2, Table 1). Increased hydrophobicity induced by modifying the
5'ꢀsubstituent group to a 5'ꢀhexylthio (14) improved the dissociation constant to 87 ± 12 pM.4
Scheme 2. Reaction and transition state catalyzed by Rv0091 to give adenine and 5ꢀmethylthioribose
(MTR). The reaction goes through a transition state characterized by a ribocation character, protonation
of the adenine leaving group, weak but significant participation of the nucleophillic water and advanced
loss of C1'–N9 bond.4
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Enzyme catalytic theory posits that mimics of the transition state for the most proficient
substrate will provide the tightest binding transition state analogue.8 Values of kcat/KM for 5'ꢀ
dAdo, MTA and Sꢀadenosylhomocysteine (SAH) with Rv0091 are reported to be 4.4 x 104, 0.6 x
104 and 0.006 x 104 Mꢀ1sꢀ1, respectively. Therefore, transition state analogues of 5'ꢀdAdo would
be anticipated to have the highest affinity. The transition state structures of MTANs from E. coli
and S. pneumonia showed MTA hydrolysis to be a late dissociative process14, 15 and that of N.
meningitides to be an early SN1 mechanism.33 Analogues designed to mimic the early
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dissociative transition state are the immucillins, characterized by a cationic N4′ꢀimino group and
a single bond between the ribocation mimic and the 9ꢀdeazaapurine mimic of the leaving group.
An example is MTꢀImmA (19; Figure 2). The DADMeꢀImmucillins incorporate a methylene
bridge between the ribocation mimic and the adenine leaving group analogue to more accurately
mimic the transition state separation of these two groups. They are often near the fully
dissociated C1′–N9 bond distance of 3.0 Å at the transition state.13, 16 The Rv0091 transition state
has a C1′–N9 distance of 2.45 Å and a weakly bonded nucleophillic water, making it
intermediate between the early and late Nꢀribosyltransferase transition states.4 The geometric
preference for early or late transition state mimics was tested with the MTA transition state
mimics, MTꢀImmA (19) and MTꢀDADMeꢀImmA (11). The Ki values were 85 nM for (19) and
1.5 nM for (11), demonstrating the DADMeꢀimmucillin analogues to better mimic the Rv0091
transition state, consistent with the transition state analysis.4 Here we further explore the
DADMeꢀImmA analogues but extended their structural features to 5′ꢀdAdo, the preferred
substrate.
The catalytic activity of Rv0091 with 5′ꢀdAdo as a substrate is relatively inefficient. Like
many enzymes from M. tuberculosis, it displays a relatively low catalytic efficiency (kcat/KM) of
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4.4 x 104 Mꢀ1sꢀ1, however this is 7ꢀfold better than for MTA and 730ꢀfold better than for SAH. 5′ꢀ
AlkylthioꢀDADMeꢀImmucillinꢀA inhibitors mimic the MTA substrate at the transition state and
gave Ki constants in the pM to low nM range.4 Transition state analogues of the 5′ꢀdAdo
substrate were obtained by the synthesis of 5′ꢀdeoxyꢀDADMeꢀImmA (4) and its analogues
(Scheme 3). The most similar analogue of the 5′ꢀdAdo transition state (4) was a potent inhibitor
with a dissociation constant of 0.64 nM (Figure. 1).
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Scheme 3. Synthesis of 5′-deoxy-DADMe ImmucillinA (4). Reagents: (a) (i) MsCl, 2,6ꢀ
dimethylpyridine, acetone, room temp. (ii) NaBr, 57% for 2 steps; (b) (i) Pd(OH)2, H2 (g), EtOH, room
temp. (ii) cHCl, room temp. 97% yield for two steps. (c) 9ꢀDeazaadenine, 37% aq. formaldehyde, H2O,
EtOH, room temperature 53% yield.
Figure 1. Inhibition of Rv0091 by 5′-deoxy-DADMe-ImmucillinA (4). The reaction rate and inhibition
were monitored at 305 nm for the conversion of 5′ꢀdAdo to 2,8ꢀdihydroxyadenine in a reaction coupled to
xanthine oxidase as described in the methods. The Ki value was calculated from the fit to the Morrison
equation for tight binding inhibitors.25 The excess substrate (1.0 mM) relative to its Km value of 10.9 ꢀM
alters the apparent Ki by a factor of 93 for this competitive inhibitor. The true Ki of 0.64 nM is shown.
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Crystal structures of S. enterica34 and H. pylori17 MTANs contain hydrophobic tunnels
from the 5′ꢀposition of bound nucleosides, reaching to the solvent. This structure can
accommodate a variety of hydrophobic 5′ꢀsubstituents. We explored increasing hydrophobicity
at the 5′ꢀsubstituent position, as previously described 5′ꢀthioꢀDADMeꢀImmA analogues (11ꢀ15)
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showed favorable binding properties for this change. This is also the case for the 5′ꢀthioꢀ
DADMeꢀImmA analogues with Rv0091 MTAN. For example, HTꢀDADMeꢀImmA (14) bound
17 times tighter than MTꢀDADMeꢀImmA (11) (Table 1).4 However, the hydrophobicity of the
5′ꢀalkyl substituent did not result in improved inhibitor potency for Rv0091 (Figure 2, Table 1).
The more hydrophobic 5'ꢀsubstituent compounds (5, 7 and 8) had slightly weaker inhibition
constants relative to the shorter 5'ꢀsubstituted 4. Compound 9 containing the longest, but
hydrophilic, substituent (PEG) was the weakest inhibitor, at greater than 300 nM Ki. In
comparison, 15 which contains a 5'ꢀthio PEG substituent had better potency of 8.2 ± 0.3 nM.
These results suggest that the 5'ꢀbinding site of Rv0091 is unlike other MTANs in that it prefers
5'ꢀalkyl substituents over 5'ꢀthio if the 5′ꢀsubstituent is short. When the substituent is longer, the
5'ꢀthio substituents are preferred.
Preference of (3R,4S)ꢀstereochemistry in the DADMeꢀImmucillin inhibitors.
The stereochemistry of the 3ꢀhydroxyꢀ4ꢀalkylpyrrolidine moiety on inhibitor potency was
explored. The transꢀracemate compound 5 was compared to the cisꢀracemate 6 and was shown to
be 13ꢀfold better in potency, demonstrating that the trans stereochemistry is preferred at the 3ꢀ
hydroxyꢀ4ꢀalkylpyrrolidine position. To evaluate the preference of (3R, 4S) in comparison to
(3S, 4R) stereochemistries, the enantiopure (3R,4S)ꢀMTꢀDADMeꢀImmA (11) was compared to
the enantiomer (3S,4R)ꢀMTꢀDADMeꢀImmA (20). 11 inhibits Rv0091 with good potency of 1.5
nM while compound 20 had no activity at concentrations to 400 µM. This observation is
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consistent with previous findings where (3R,4S) inhibitors displayed better potencies than their
enantiomeric counterparts towards other MTAN nucleosidases including that from E. coli.34
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Figure 2. DADMe-Immucillin and Immucillin-based inhibitors of Rv0091. 4,7,9ꢀ15, 19 and 20 are
enantiopure compounds ; 5, 6, 8, 16 – 18 are racemic mixtures at 3ꢀhydroxyꢀ4ꢀalkylpyrrolidine.
Abbreviated names for some of these compounds are provided in the text.
Table 1. Inhibition of Rv0091 with DADMe Immucillin analogues
.
Ki
Compound
(nM)a
Compound
Ki
(nM)a
4
5
6
7
8
0.64 ± 0.01
1.5 ± 0.1
18.9 ± 1.1
0.74 ± 0.05
1.64 ± 0.20
>300
5.0 ± 1.1
1.5 ± 0.44
13.9 ± 2.4
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19
20
1.3 ± 0.14
0.087 ± 0.0124
8.2 ± 0.3
6.7 ± 0.3
2.8 ± 0.2
13.5 ± 1.2
85 ± 114
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10
11
12
NIb
aDissociation constants are obtained from the xanthine oxidase
coupled assay at 25 ºC. The Ki value was calculated by using
Equation 1 for analysis of tight binding inhibitors.25 NI = Not
Inhbitor up pt 400 ꢁM.
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The affinities of these transition state analogues for Rv0091 (87 pM for the best
compound) and other bacterial MTANs, such as E. coli MTAN (fM range for best compounds)20,
35, establishes a reduced affinity for Rv0091. This difference can be attributed to relative
catalytic efficiencies of the E. coli and M. tuberculosis enzymes. Wolfenden postulated that the
binding of transition state analogues is associated with enzyme catalytic efficiency.9, 10 E. coli
MTAN has a high catalytic efficiency of 4.7 x 107 Mꢀ1sꢀ1 for MTA, approximately 1000ꢀfold
more efficient than Rv0091 for 5'ꢀdAdo.4 The E. coli MTAN is also more efficient for 5′ꢀdAdo
than is Rv0091 even though it is not a preferred substrate. Consequently, transition state
analogues mimicking both MTA and 5′ꢀdAdo substrate are known to bind tighter for the E. coli
enzyme by several orders of magnitude. This binding is also consistent with previous
observations for the inhibitor profiles of the slow S. pneumonia MTAN (SpMTAN).36
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Molecular modeling and putative binding site residues identification
The crystal structure of Rv0091 from Mycobacterium tuberculosis is not available. The
binding interactions of active site residues for Rv0091 were probed by docking the transition
state analogue inhibitors BTꢀDADMeꢀImmA and HTꢀDADMeꢀImmA to explain the difference
in 5'ꢀsubstituent interactions from the specificity known for H. pylori and E. coli MTANs. The
in silico model of Rv0091 was generated by IꢀTESSER server (Figure S1). The best structural
model of Rv0091 was obtained with the Cꢀscore of 0.43 (ideal Cꢀscore is between ꢀ5 and 2; a
score of 0.43 is an excellent match) and a TM score of 0.77 ± 0.10. The model has a similar
structural fold as seen in other MTANs with the exception of an extended loop (from residues
Leu148 to Pro162) unique to Rv0091 (Figure S1, S2). Rv0091 shares 29 and 32% sequence
identity with H. pylori and E. coli MTANs, respectively (Figure S2). Comparison of the Rv0091
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model with H. pylori and E. coli MTANs gave RSMD values between 0.96 to 1.03 Å without
and with the Rv0091 external loop, respectively.
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Crystal structures of H. pylori and E. coli MTAN in complex with BTꢀDADMeꢀImmA
are known.24, 32 AutodocꢀVina and the H. pylori structure with BTꢀDADMeꢀImmA (4FFS) was
used for the in silico docking of BTꢀDADMeꢀImmH and HTꢀDADMeꢀImmH. The best fit of the
interactions between inhibitor and Rv0091 was selected from the topꢀranked cluster by binding
affinities. Active site superimposition of BTꢀDADMeꢀImmA bound to HpMTAN and EcMTAN
with Rv0091 modeled with both BTꢀDADMeꢀImmA and HTꢀDADMeꢀImmA shows the
pyrrolidine and deazaadenine inhibitor groups to bind in the same orientation in all the MTANs
(Figure 3 and S3). The catalytic site geometry of 5'ꢀalkylthio inhibitor groups differ considerably
for Rv0091. The active site tunnel residue Ile52 (in HpMTAN) and Ile50 (at the same position in
EcMTAN) has nonꢀpolar interactions with the 5'ꢀalkylthio group of the BTꢀDADMeꢀImmA.
This interaction is disrupted by the larger Met52 residue in this position in Rv0091 MTAN. The
side chain of the Met52 creates a steric clash with 5'ꢀalkylthio group of the inhibitors to displace
it relative to its geometry in EcMTAN and HpMTAN (Figure 3).
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Inhibitors modeled into Rv0091 show the 3'ꢀhydroxyl group to be hydrogen bonded with
Glu197. The N1 and N3 of the 9ꢀdeazaadenine ring have hydrogen bonding interactions with the
backbone nitrogen of Leu176 and Glu195, respectively. The N6 amino and N7 of the inhibitors
form hydrogen bond interactions with both the peptide bond NH of Leu176 and with side chain
hydroxyl group of Asp220 (Figure 3 and S3). These groups are conserved between Rv0091,
HpMTAN and EcMTAN, supporting the major divergence of Rv0091 being related to the
hydrophobic region in the catalytic site that accommodates the 5'ꢀalkylthio groups. The
dissociation constant for BTꢀDADMeꢀImmA is 1.3 nM, which is 15 times weaker binding than
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HTꢀDADMeꢀImmA (Kd = 87 pM). HTꢀDADMeꢀImmA has two additional carbons at the 5'ꢀ
alkylthio group that make additional hydrophobic contacts with the enzyme and contributes to
the higher affinity of HTꢀDADMeꢀImmA. From the model, the butylthio group of BTꢀDADMeꢀ
ImmA predicts van der Waals interacts with eight amino acids, Ile11, Met52, Met 196, Pro119,
Tyr113, Val104, Tyr175 and Phe230. These plus two additional contacts with Glu108 and
Val105 are predicted for the hexylthio group of HTꢀDADMeꢀImmA, consistent with its tighter
binding.
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Figure 3. Stereoꢀview superimposition of the active sites of H. pylori and E. coli MTAN in complex with
BTꢀDADMeꢀImmA compared with Rv0091. The interactions of binding site residues of Rv0091 modeled
with BTꢀDADMeꢀImmA (yellow, panelꢀA) and with HTꢀDADMeꢀImmA (green, panelꢀB) are highlighted.
The BTꢀDADMeꢀImmA of H. pylori and E. coli are shown in blue and pink, respectively.
Antibiotic activity against Mycobacterium species and H. pylori.
The antibacterial activity of DADMeꢀImmucillin compounds was investigated in a discꢀ
diffusion assay against M. tuberculosis mc26230 and M. smegmatis mc2155 strains.37 HTꢀ
DADMeꢀImmA (14) is the most potent inhibitor towards Rv0091 and displayed a small zone of
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clearance at 1000 ꢀg/mL in both Mycobacterium species (Figure. 4A and 4B). Kanamycin as an
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antibiotic control displayed large zones of clearance at 100 ꢀg/mL. Other DADMeꢀImmucillin
compounds showed similar results to HTꢀDADMeꢀImmA (14) indicating that the function of
Rv0091 is not essential for Mycobacteria growth. This finding is in agreement with previous
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gene deletion experiments in M. tuberculosis, where Rv0091 was found to be nonꢀessential for
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growth or for infection.38,
Unlike Mycobacteria, MTAN has an essential function in
menaquinone synthesis in H. pylori.17 We tested HTꢀDADMeꢀImmA (14) in a cultured cell assay
using H. pylori with tetracycline as a control. HTꢀDADMeꢀImmA (14) exhibited antibacterial
activity against H. pylori with an IC50 of 13.0 ± 1.8 ng/mL (equivalent to 35 ± 5 nM), which is
six times more potent than tetracycline as an antibiotic (Figure 4C).
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Figure 4. Antibacterial activity. Transition state analogue HTꢀDADMeꢀImmA (14) and antibiotic control
were tested for growth inhibition against (A) M. smegmatis mc2155 and (B) M. tuberculosis mc26230 in a
disc diffusion assay by growth for 96 hours 37 °C on agar plates. (C) Inhibition of Helicobacter pylori (J99)
growth at 37 °C in liquid culture by HTꢀDADMeꢀImmA (14) and by tetracycline. Growth was evaluated at
OD = 600 nm and MIC50 values were calculated by nonꢀlinear regression using Graphpad prism 7.
Genomic context of Rv0091.
MTAN in E. coli has been reported to be involved in SAH and MTA recycling and in
quorum sensing pathways.2 BTꢀDADMeꢀImmA (13) inhibited production of the quorum sensing
signaling molecule autoinducer (AIꢀ2), but was not toxic to E. coli or Vibrio cholera growth,
providing support for its role in quorum sensing.1 Comparison of the gene maps for Rv0091
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(NCBI gene ID: 886953), EcMTAN (NCBI gene ID: 948542) and HpMTAN (NCBI gene ID:
900251) indicate no organized operon function. In E. coli, the open reading frame for MTAN is
adjacent to btuF, expressing a periplasmic B12 binding protein and dgt, known to express a dGTP
triphosphohydrolase (Figure S4). In H. pylori, the adjacent Hp0090 is a malonylꢀCoA: acyl
carrier protein transacylase. Near the Rv0091 locus in M. tuberculosis, a nearby open reading
frame, Rv0089 shares 41% identity in amino acids 32 to 146 (of 197) with the E. coli BioC
protein, a SAMꢀdependent malonylꢀacyl carrier protein methyltransferase, an early step in biotin
synthesis.40 In E. coli, the BioC protein is expressed from the bioC gene within the bioABFCD
operon.41 Based on the local gene organization near Rv0091, no insight is provided to its
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biological function, supporting a role in removing the 5’ꢀdAdo product from the 21 potential
radical SAM proteins in M. tuberculosis.
Conclusions.
Transition state analogues resembling the geometry and charge of the transition state for
hydrolysis of 5'ꢀdAdo by Rv0091 were synthesized and found to bind with pM to nM
dissociation constants. The analogue that best captures the features of the preferred Rv0091
substrate 5'ꢀdAdo and those containing different 5'ꢀsubstituents were found to bind with pM to
nM dissociation constants. (3R,4S)ꢀ3ꢀHydroxyꢀ4ꢀalkylpyrrolidine stereochemistry is preferred
for these compounds, consistent with findings for related bacterial MTANs.34 Comparison of 5'ꢀ
alkylthioꢀDADMeꢀImmA inhibitors that mimic the MTA substrate provided insight into the
relative specificity for 5'ꢀdAdo and 5'ꢀmethylthio adenosine substituents for Rv0091. Shortꢀchain
5'ꢀsubstituents are preferred for the 5'ꢀdeoxyalkylꢀinhibitors while longer hydrophobic chains
displayed tighter binding for the 5'ꢀalkylthioꢀinhibitors. In both cases, substitution of polar atoms
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into the 5'ꢀsubstituent, i.e. CH2ꢀ to Oꢀ reduces potency of the inhibitors. In silico docking of BTꢀ
DADMeꢀImmA (13) and HTꢀDADMeꢀImmA (14) with a model of Rv0091 showed similarity in
binding to E.coli and H. pylori MTAN of the pyrrolidine and deazaadenine moieties, however
differed at the 5'ꢀalky or 5'ꢀalkylthio groups. This difference is due to a methionine sterric clash
near the 5'ꢀsubstituent in the catalytic site of Rv0091. Isoleucine is present at this position in the
MTANs from E. coli or H. pylori. Furthermore, weak antibacterial activity of these inhibitors
against Mycobacteria indicates that Rv0091 functions in a nonꢀessential pathway for growth.
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SUPPORTING INFORMATION
Figure S1: Homology model of Rv0091 and superposition with HpMTAN and EcMTAN.
Figure S2: Comparison of primary sequence of Rv0091, HpMTAN and EcMTAN.
Figure S3: Superimposition of active site of HPMTAN and EcMTAN complex with BTꢀ
DADMeꢀImmA; with Rv0091 modeled with BTꢀDADMeꢀImmA and HTꢀDADMeꢀImmA.
Figure S4: Analysis of neighboring genes of Rv0091, HpMTAN and EcMTAN.
AUTHOR INFORMATION
Correspondence author
*To whom correspondence should be addressed: vern.schramm@einstein.yu.edu
Author contributions
HAN and VLS designed inhibitors. GBE and PCT designed and performed the chemical
synthesis of compounds. HAN designed and performed inhibitor experiments. RKH performed
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molecular modeling and docking. VLS and HAN analyzed data. All authors contributed to
writing the manuscript.
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ACKNOWLEDGEMENTS
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We acknowledge Drs. Scott Cameron (Einstein) for valuable discussions and A. Malek
(Einstein) for help with the discꢀdiffusion assays. M. smegmatis mc2155 and M. tuberculosis
mc26230 strains were gifts from the laboratory of Dr. William Jacobs (Einstein).
NOTES
Authors declare no conflict of interest with the contents in this article.
ABBREVIATIONS
5′ꢀmethylthioadenosine/Sꢀadenosylhomocysteine
nucleosidases
(MTANs),
5′ꢀ
methylthioadenosine (MTA), Sꢀadenosylhomocysteine (SAH), 5′ꢀdeoxyadenosine (5′ꢀdAdo),
polyethelene glycol (PEG), Sꢀadenosylmethionine (SAM), Kinetic isotope effect (KIE)
FUNDING
This work was supported by NIH research grant GM041916 and by the Michael Price Family
Foundation.
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