Selective Inhibitors of Helicobacter pylori Methylthioadenosine Nucleosidase and Human Methylthioadenosine Phosphorylase

Article abstract of DOI:10.1021/acs.jmedchem.8b01642

Bacterial 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes adenine from its substrates to form S-methyl-5-thioribose and S-ribosyl-l-homocysteine. MTANs are involved in quorum sensing, menaquinone synthesis, and 5′-methylthioadenosine recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone pathway, making H. pylori MTAN a target for antibiotic development. Human 5′-methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes phosphorolysis of 5′-methylthioadenosine to salvage S-adenosylmethionine. Transition-state analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen unique transition-state analogues are described here and are used to explore inhibitor specificity. Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate nucleophile from the catalytic site.

Full text of DOI:10.1021/acs.jmedchem.8b01642

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Article
“
Selective inhibitors of H. pylori methylthioadenosine
nucleosidase and human methylthioadenosine phosphorylase”
Rajesh K. Harijan, Oskar Hoff, Rodrigo G. Ducati, Ross S. Firestone,
Brett M. Hirsch, Gary B. Evans, Vern L. Schramm, and Peter C. Tyler
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01642 • Publication Date (Web): 12 Mar 2019
Downloaded from http://pubs.acs.org on March 13, 2019
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Selective inhibitors of H. pylori methylthioadenosine nucleosidase and human
methylthioadenosine phosphorylase
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Rajesh K. Harijan , Oskar Hoff , Rodrigo G. Ducati , Ross S. Firestone , Brett M. Hirsch , Gary
b
a,*
b,*
B. Evans , Vern L. Schramm and Peter C. Tyler
a
Department of Biochemistry, Albert Einstein College of Medicine, New York 10461, USA.
Ferrier Research Institute, Victoria University of Wellington, Wellington 5040, New Zealand.
b
*
Corresponding authors: vern.schramm@einstein.yu.edu or peter.tyler@vuw.ac.nz
KEYWORDS: transition-state analogue; futalosine pathway; menaquinone; immucillins; S-
adenosylmethionine.
ABBREVIATIONS:
MTDIA: methylthio-DADMe-Immucillin-A; MTA: S-methyl-5'-thioadenosine; All: allyl; DQF-
COSY: double-quantum filtered correlation spectroscopy; Q-TOF: quadrupole time-of-flight;
MTAN: 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase; HpMTAN: Helicobacter
pylori MTAN; MTAP: 5'-methylthioadenosine phosphorylase; MTR: 5-methylthio-α-D-ribose 1-
phosphate; SAM: S-Adenosylmethionine; SAH: S-adenosylhomocysteine, SRH: S-
ribosylhomocysteine
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ABSTRACT
Bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes
adenine from its substrates to form S-methyl-5'-thioribose and S-ribosyl-L-homocysteine.
MTANs are involved in quorum sensing, menaquinone synthesis and 5'-methylthioadenosine
recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone
pathway, making H. pylori MTAN a target for antibiotic development. Human 5'-
methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes
phosphorolysis of 5'-methylthioadenosine to salvage S-adenosylmethionine. Transition-state
analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen
unique transition-state analogues are described here and are used to explore inhibitor specificity.
Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with
orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending
through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of
human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate
nucleophile from the catalytic site.
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INTRODUCTION
S-Adenosylmethionine (SAM) is involved in biological methylation reactions, in polyamine
biosynthesis and as a precursor of glutathionine.1,2 Two molecules of 5'-methylthioadenosine
(MTA) are formed from SAM in the synthesis of each spermine molecule (Scheme 1). In
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humans, MTA is metabolized only by methylthioadenosine phosphorylase (MTAP) to form 5-
methylthio-α-D-ribose 1-phosphate (MTR) and adenine. These are precursors for methionine and
ATP which can be recycled to SAM (Figure 1).3,4 Inhibition of MTAP in mammals causes
elevated MTA and decreased recycling of MTA to SAM.5,6 Synthetic-lethal genetic analysis of
MTAP-deleted cancer cell lines indicate sensitivity of these cell lines to pathways related to
SAM-related methyl transfer.7-10 It has also been proposed that inhibitors of MTAP may have
anti-cancer applications alone or in drug combinations, as they demonstrate anti-cancer
properties in mouse xenograft models.5,6
Most bacteria express methylthioadenosine/S-adenosylhomocysteine nucleosidase
(MTAN) instead of MTAP. The enzyme hydrolyzes MTA and S-adenosylhomocysteine (SAH)
to MTR or S-ribosylhomocysteine (SRH) and adenine, respectively (Scheme 1, Figure 1). The
MTAN reaction product, SRH, is used in the biosynthesis of homoserine lactones to form AI-2
quorum sensing molecules.11-13 MTAN is not essential in most bacteria, but a few species use the
unusual futalosine pathway for the biosynthesis of menaquinone, where MTAN plays an
essential role.14,15 These organisms include the pathogens Helicobacter pylori and
Campylobacter jejuni. Inhibition of this pathway is reported to be lethal to H. pylori.16,17
Inhibitors of MTAN are also expected to reduce polyamine biosynthesis and block production of
quorum sensing autoinducer (AI-2) molecules. For these reasons, human MTAP and bacterial
MTAN enzymes are of interest as drug targets.
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Transition-state analogue enzyme inhibitors have the potential to bind orders of
magnitude more tightly than substrates. Studies focused on N-ribosyltransferases have identified
18
transition states with ribocation character. MTAP and MTAN share MTA as a substrate and
form similar transition states, leading to similar interactions with transition-state analogues
(Figure 1).19-21 MTAP inhibitors have shown efficacy in animal models against human tumors,
while MTAN inhibitors influence bacterial quorum sensing and are antibiotics in organisms
using the futalosine pathway of menaquinone synthesis, notably, H. pylori. We have reported
transition-state analogues as inhibitors of human MTAP and several bacterial MTAN enzymes.16-
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Of several inhibitory chemical scaffolds, the DADMe-Immucillin structure exemplified by
MTDIA (1) is optimal for these enzymes (Scheme 1). Both enzymes tolerate substituents in the
'-position of the 3'-hydroxypyrrolidine ring – in particular for the bacterial MTAN enzymes.
4
Here we report new analogues to explore the structure-activity relationships for human MTAP
and HpMTAN.
NH2
NH2
N
NH2
N
H
NH2
N
N
O
NH2
N
N
N
N
N
N
1
N
N
MeS
MeS
HOOC
S
3
N
O
N
O
N
O
4
'
N
HOOC
HO
HO OH
MTA
HO OH
adenosylhomocysteine
HO OH
aminofutalosine
MTDIA (1)
Scheme 1.
The transition-state structures of MTAN and MTAP enzymes have been solved by kinetic
isotope effect measurements and quantum chemical calculations.19,21,27,28 Transition-state
analogues for these enzymes act as strong catalytic-site inhibitors of the enzyme.24-26 Two
inhibitors in particular, methylthio-DADMe-Immucillin-A (MTDIA) and parachloro-phenylthio-
DADMe-Immucillin-A (pCl-PhT-DADMe-ImmA), were slow-onset inhibitors with dissociation
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*
constants (K ) of 86 pM and 10 pM, respectively for human MTAP, and 86 and 570 pM,
i
respectively for HpMTAN.17,22 Inhibitors reported here provide insight into the 4'-substituent
inhibitor specificity for transition-state analogues of HpMTAN and human MTAP.
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RESULTS AND DISCUSSION
Synthesis of new transition-state analogue inhibitors
Modifications in the 9-deazaadenine moiety
The N3 of 1 makes no catalytic site contacts in MTAP or MTAN enzymes, motivating the
synthesis of a 3-deaza-analogue of 1.17,23,29 Treatment of 2-hydroxy-4-methyl-3-nitropyridine (2)
with Brederick′s reagent afforded enamine 3 (Scheme 2) which with zinc in acetic acid gave 3,9-
dideazahypoxanthine (4). This material was converted to the 6-chloro-derivative 5, which on
treatment with catalytic copper (I) chloride in aqueous ammonia afforded 3,9-dideazaadenine
(
6). Mannich reaction of 6 and pyrrolidine 7 gave the desired 3-deazaMTDIA 8.22
O
O
2
O
4
i
ii
H
O N
O N
2
2
N
NH
NH
NH
7
2%
76%
Me N
2
3
iii 56%
MeS
NH2
H
N
NH
N
X
H
HO
7
MeS
N
N
N
v, (52%)
HO
5
, X = Cl
iv
quant.)
8
(
6
, X = NH2
o
Reagents: (i) tBuOCH(NMe ) , DMF, 100 C; (ii) Zn, HOAc; (iii) POCl
2
2
3
o
o
o
1
00 C; (iv) aq NH , CuCl, 120 C (v) HCHO, aq EtOH, 80-100 C.
3
Scheme 2
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During transition-state inhibitor design work for purine nucleoside phosphorylase, 8-aza-
immucillins were found to be powerful inhibitors.26,30,31 The 8-aza-analogue of 1 was targeted for
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synthesis. Treatment of aldehyde 9 with pyrrolidine 7 and 2-methylpyridine borane complex
gave 10 (Scheme 3). Ammonolysis followed by deprotection afforded 8-aza-MTDIA 11.
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OMe
N
NH2
OMe
N
N
N
N
THP N
N
HN
N
i
ii, iii
MeS
MeS
THP N
OHC
N
N
N
N
71%
42%
9
HO
HO
10
11
o
Reagents: (i) Picoline borane, 7, MeOH; (ii) 7 N NH /MeOH, 120 C; (iii) aq HCl, MeOH.
3
Scheme 3
Modifications in the 5'-thio substituent
Both the human MTAP and HpMTAN enzymes tolerate diversity in the 5'-thio substituents –
particularly for the MTANs.24 With this in mind, we prepared 5'-substituted thio-MTDIA
analogues by coupling 5'-alkynyl derivatives with some azides using click chemistry.
1
7
The thioacetate 12 was treated with sodium methoxide/methanol followed by either
propargyl bromide or 5-mesyloxy-pent-1-yne to give the alkynylthio-substituted compounds 13
and 14 (Scheme 4). These compounds, after acidic treatment to remove the Boc group followed
by a Mannich reaction with 9-deazaadenine, then afforded 15 and 16. The acetylenes 13 and 14
were individually treated with methyl iodide, allyl bromide, n-butyl bromide or benzyl bromide
along with sodium azide and catalytic copper (I) iodide affording the triazole ′click′ products 17-
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2
4. Deprotection, followed by a Mannich reaction with 9-deazaadenine then provided the 5'-
substituted thio- analogues 25-32. The 2-pyrimidinethio analogue 33 was prepared by steps v, ii
and iii (Scheme 4).
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NH2
H
N
N
AcS
S
i
n
ii, iii
S
NBoc
N
n
NBoc
N
HO
HO
12
HO
1
1
3, n=1 (95%)
4, n=3 (68%)
1
1
5, n=1, (45%)
6, n=3, (30%)
iv
R
R
NH2
N
N
N
N
N
H
N
N
v, ii, iii
N
(29%)
S
S
n
N
n
ii, iii
NBoc
N
HO
NH2
N
HO
H
25, R=Me, n=1, (35%)
26, R=Me, n=3, (43%)
17, R=Me, n=1, (27%)
18, R=Me, n=3, (65%)
19, R=All, n=1, (43%)
20, R=All, n=3, (54%)
21, R=nBu, n=1, (25%)
22, R=nBu, n=3, (39%)
23, R=Bn, n=1, (53%)
24, R=Bn, n=3, (66%)
N
N
2
7, R=All, n=1, (63%)
S
N
N
28, R=All, n=3, (23%)
29, R=nBu, n=1, (55%)
30, R=nBu, n=3, (32%)
31, R=Bn, n=1, (53%)
32, R=Bn, n=3, (41%)
N
HO
33
Reagents: (i) NaOMe, MeOH, 30min then 3-propargyl bromide or 5-mesyloxy-pent-1-yne; (ii) MeOH/aq
HCl(conc.) 3:1 v/v; (iii) 9-deazaadenine, formaldehyde, EtOH/water, 70-100°C (microwave), 2-6h; (iv) MeI,
allyl bromide, nBuBr or BnBr, NaN , CuI, MeOH; (v) NaOMe, MeOH, then 2-chloropyrimidine.
3
Scheme 4
Inhibition of MTAP and HpMTAN
Most of the compounds described here are structurally related to methylthio-DADMe-
Immucillin-A (MTDIA), a transition-state inhibitor of MTAP and MTANs. Here, the varied 5'-
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alkylthio groups yielded strong inhibitors of MTAP and HpMTAN (Figure 2) with dissociation
constants (K values) varying by over two orders of magnitude. Tight binding of these transition-
d
state analogues depends on the ribocation mimic of the transition state provided by the cationic
hydroxypyrrolidine and protonation of N7 in the 9-deazaadenine, a second important feature of
the transition state structure. The 6-amino group is essential, as its loss prevents binding (Figure
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).
Crystal structures of analogues with MTAP and HpMTAN
Structural analysis of inhibitors 15, 16, 30 and 32 bound to human MTAP and HpMTAN
correlated the catalytic site interactions with the binding affinity to these enzymes (Figure 2 and
Table 1). Human MTAP and HpMTAN were co-crystallized with the inhibitors and crystal
structures solved by molecular replacement using PHASER at high resolutions (Table 1).35
Structure analysis using MolProbity indicated that none of the amino acid residues are outliers in
the Ramachandran plots (Table 1).36
Human MTAP in complex with four inhibitors was solved at 1.62 to 1.99 Å resolutions
in different space groups using the apo human MTAP monomer as the initial phasing model in
PHASER. Human MTAP catalytic sites are located at the subunit interfaces of the trimer.
Asymmetric units of human MTAP crystals contain a monomer with 15 and 16; and a trimer
with 30 and 32. The trimer is the physiological state of human MTAP. Solvent accessible surface
2
area of the subunit-subunit interfaces is 1187 Å . The low RMSD for C (0.357 – 0.710 Å) of
the four inhibitor-bound structures indicate only minor structural differences. MTAP monomers
contain 10  sheets (residues are 1, 11-16; 2, 29-33; 3, 45-50; 4, 54-59; 5, 87-98; 6, 106-
1
7
10; 7, 112-116; 8, 161-164; 9, 165-172; 10, 210-220) with 6  helices (from residues 1,
3-84; 2, 146-159; 3, 180-189; 4, 200-208; 5, 233-259; 6, 264-274; Figure S1). The
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electron density map of the peptide backbone and amino acid side chains are clearly resolved.
Inhibitor binding is also well defined in the catalytic sites (Figure S2). The purine and
pyrrolidine rings of the four inhibitors bind in the same conformation but the 5'-alkylthio group
of 30 and 32 bind in a conformation different from 15 and 16 (Figure S3). The N1 of the
inhibitor forms a hydrogen bond with a structural water molecule while N6 forms hydrogen
bonds to the carboxyl oxygens of both Asp220 and Asp222. Asp220 also forms a hydrogen bond
interaction with N7, making it bidentate with respect to inhibitor binding. MTAP complexes of
1
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5 and 16 have the hydroxyl groups and N1' (corresponding to the C1' of the substrate) of the
pyrrolidine ring in hydrogen bond interactions with a phosphate oxygen and with Thr18 (Figure
). The candidate nucleophilic oxygen (O2, nearest to the reaction center) of phosphate is
3
hydrogen bonded with Thr93 (OG1), Thr197 (OG1) and a water molecule. The O3 of the
phosphate is in hydrogen bond interactions with peptide nitrogens of Thr18 and Ala94.
Phosphate O4 forms hydrogen bonds with Arg60 (NH1) and His61 (NE2). These interactions are
missing in MTAP complexes with 30 and 32, as phosphate is displaced in these structures.
Instead, a chloride ion is bound near the phosphate binding pocket and is coordinated with
Thr193 (OG1), Thr197 (OG1) and a water molecule. The 5'-alkylthio group binding mode of 15
and 16 is different from 30 and 32, where the extended chain folds into the phosphate binding
site (Figure 3; Table 2).
HpMTAN was co-crystallized with the same inhibitors and the structures solved at
resolutions of 1.45 to 1.62 Å (Table 1). Previous and current analysis indicates a functional
dimer for HpMTAN. The solvent accessible surface area of the dimer interface is 1654 Å2.
HpMTAN contains seven helices including one 3 -helix and ten -sheets, similar to other
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MTAN structures.37-39 They are arranged in three -layer structures with central mixed -
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sheets (Figure S4). The low RMSD (0.091 – 0.195 Å) of the inhibitor complexes indicate highly
similar C chains. Inhibitors were bound in both active sites of HpMTAN with low B-factors
and clear electron density maps (Figure S5). Except for the 5'-alkylthio groups, the binding
modes of the inhibitors are similar (Figure S3). Inhibitors with extended 5'-alkylthio groups fill
the full extent of the 5'-binding pocket while short 5'-alkylthio groups do not. The binding modes
of the purine and pyrrolidine rings of the inhibitors are the same. Hydrogen bond interactions to
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-deazaadenine include the Val154 nitrogen with N1, N6 with the carbonyl oxygen of Val154
and N7 with a carboxyl oxygen of Asp198 (OD2). N7 protonation is important to transition-state
formation during the MTAN hydrolysis reaction. The structural nucleophilic water oxygen is 2.7
Å from N1', in hydrogen bond contacts with Glu13 (OE2) and Arg194 (NH1). The 3'-hydroxyl
group of the pyrrolidine is hydrogen bonded with a carboxyl oxygen of Glu175 (Figure 4).
Inhibitor 5'-alkylthio groups occupy different parts of the 5'-alkylthio binding pocket, which
extends toward the solvent exterior (Table 2 and Figure 4). Inhibitors 15 and 16 fill the 5'-
alkylthio group binding pocket to engage the most interactions with the enzyme.
Structural comparisons
Crystal structures of human MTAP with inhibitors 30 and 32 (large 5'-alkylthio groups)
differ from those with 15 and 16 (smaller 5'-alkylthio groups). Binding of 30 and 32 caused
rearrangement of the loop between 1-2 (residues 17 to 28) to a more open conformation than
with 15 and 16 (Figure 5). In the complex with 15 and 16, this loop is closed. The rearrangement
of the 1-2 loop with 30 and 32 also alters the 4-1 loop conformation so His61 is displaced
slightly and His65 is flipped 180°. The hydroxyl group of the pyrrolidine ring is rotated 18°
towards the outside in 30 and 32 complexes. These conformational changes alter the phosphate
binding site which is occupied by the bulky 5'-alkylthio groups. For the structural comparisons,
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unliganded MTAP (PDB ID: 3OZE); and a complex with MTA and sulfate (PDB ID: 1CG6)
were compared.40,41 These structures are similar to that with inhibitors 15 and 16.
HpMTAN structures with the same four inhibitors were solved at high resolution in the
P4 2 2 space group (Table 1). In unliganded E. coli MTAN (PDB ID: 1Z5P), both catalytic sites
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are in an open configuration, but in unliganded HpMTAN (PDB ID: 3NM4) the binding of a Tris
buffer molecule induced a closed conformation in monomer-A with monomer-B in an open
conformation.39,42,43 The ligand-induced conformation change moves the 10-6 loop
approximately 7 Å closer to the binding site to form the closed state. When the HpMTAN bound
to 15, 16, 30 and 32 was compared to p-ClPh-Thio-DADMe-ImmA bound to HpMTAN and
resolved with neutron diffraction (PDB ID: 5K1Z); hydrogen bond contacts of the DADMe-
44
ImmA core were found to be intact, including the N7 hydrogen sharing with Asp198. All
HpMTAN-inhibitor complexes are in closed configurations but differ in the 5'-alkylthio group
binding pocket (Table 2; Figs. 4, 5).
Structure-inhibition relationship
Inhibitors 15 and 16 gave K values of 0.63 and 0.94 nM, respectively, for human MTAP,
d
slightly better than 30 and 32, with K values of 1.3 and 1.4 nM, respectively. 9-Deazaadenine of
d
the inhibitors is co-located in the catalytic site for the four inhibitors, while the 3'-hydroxyl group
of the pyrrolidine of 30 and 32 is shifted upward relative to 15 and 16 (Figs. S1 and S3). The 5'-
alkylthio groups of the inhibitors, important for the potency, are bound in different
conformations. Inhibitors 15 and 16 occupy the hydrophobic binding site normally occupied by
the 5'-methylthiol or 5'-homocysteinyl groups of the natural substrates. In contrast, 30 and 32
induced 1-2 loop rearrangement by exceeding the size of the 5'-alkylthio binding pocket.
Upon 30 and 32 binding, the inhibitor triazoles reposition under the 3'-hydroxypyrrolidine to
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occupy the phosphate binding site. As 30 and 32 occupy both nucleoside and phosphate binding
sites, phosphate is not bound in the 30 and 32 MTAP complexes. Ion pair formation between the
cationic 3'-hydroxypyrrolidine ring and anionic phosphate is part of the transition-state
ensemble, contributing to tight binding of structurally compatible analogues. Remarkably, most
of the binding affinity is retained in 30 and 32 without bound phosphate.
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Enzyme inhibition experiments revealed that 15, 16, 30 and 32 are 26 to 55 picomolar
inhibitors of HpMTAN (Figure 2). The 5'-alkylthio binding site accommodates groups
approximately 10 Å extending from the 4'-carbon of the pyrrolidine group and longer groups are
accommodated as the binding channel opens to solvent. The optimal fit of 16 to the site explains
its high binding affinity, but even relatively small 5'-alkylthio groups (15) or bulky and
hydrophilic triazole groups (30 and 32) are picomolar inhibitors. In the HpMTAN-15 complex,
the unfilled space in the 5'-alkylthio binding pocket is occupied by an ethylene glycol molecule
interacting with solvent molecules and a carboxyl oxygen Asp209 (OD1). The groups of 30 and
3
2 are larger, forcing them into the solvent space beyond the organized binding site. The distal
atoms of 30 and 32 are disordered, resulting in decreased affinity. Despite these differences, the
sum of the interactions provides favorable binding energy (Table 2; Figure 4).
Inhibitor catalytic site contacts in both HpMTAN and MTAP show no interactions with
the N3 atom of 9-deazaadenine. In contrast, both enzymes have hydrogen bond interactions with
the protonated N7. As protonation of N7 is a feature of the transition state, this is an important
characteristic of high affinity inhibitors. A 3,9-dideazaadenine inhibitor (8) retains the desired
protonation at N7 in a scaffold otherwise identical to MTDIA (1). However, (1) is an 86 pM
inhibitor for both HpMTAN and MTAP while the dissociation constant for (8) increased by
>100-fold to 10 and 32 nM for HpMTAN and MTAP, respectively, indicating weaker N7 to
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enzyme interactions because of the loss of electron contribution from N3. The N6 exocyclic
amino group is doubly hydrogen-bonded at the catalytic sites and its replacement by O6
eliminates binding to the catalytic sites at concentrations of 5 M (34, 35).
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Conclusions
Human MTAP is a validated drug target. Its genetic deletion in 15% of all human cancers
makes those malignancies more susceptible to inhibitors of PRMT5, MAT2A or RIOK1.7-10
HpMTAN catalyzes an essential step in menaquinone synthesis in H. pylori but not in common
gut bacteria, making it a species-specific target in the treatment of peptic ulcers. New transition-
state analogues of HpMTAN and MTAP were synthesized to explore variations of the MT-
DADMe-Immucillin-A chemical scaffold and the structure-function relationship to these target
enzymes. The 4'-position of the 3'-hydroxypyrrolidine ring of DADMe-Immucillin-A was varied
by click-chemistry addition of varied substituents. Inhibition constants indicated the best
compounds (15 and 16) to be picomolar inhibitors against both MTAP and HpMTAN, with 12 to
3
2
6-fold preference for the HpMTAN. Bulky 4'-substituents (eg. 25 – 29 and 33) showed a 92- to
43-fold preference for HpMTAN. The hydrophobic pocket accepting the 4'-substituent in
HpMTAN can accommodate methylthio (in MTA), homocysteine (in SAH) and the bulky side
chain of aminofutalosine. It does so in a hydrophobic tunnel that opens towards solvent. Human
MTAP has a more restricted 4'-binding pocket that accepts only the methylthio and
homocysteine groups. Larger substituents fold under the 3'-hydroxypyrrolidine ring and occupy
the phosphate binding site, while retaining other contacts to the 9-deazaadenine and
hydroxypyrrolidine that dominate the transition state binding energy. Four transition-state
analogues were co-crystallized with MTAP and HpMTAN. Structural analysis revealed that
binding of 30 and 32 to human MTAP caused the structural rearrangement to displace phosphate
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and accommodate long and bulky 5'-alkylthio groups. With HpMTAN, inhibitor 16 takes full
advantage of the HpMTAN binding pocket, and thus binds more tightly than other inhibitors.
MATERIALS AND METHODS
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Chemical synthesis of transition-state analogues
The MTDIA chemical scaffold was explored by synthesizing a new generation of
transition-state analogues for HpMTAN and MTAP. The 4'-position of the 3'-hydroxypyrrolidine
ring of MTDIA was varied by click-chemistry. All reactions were performed under an argon or
nitrogen atmosphere, unless water was used as solvent or the reaction mixture was heated above
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00 °C. All final compounds gave satisfactory purity (≥95%) by HPLC and by H and C NMR.
Details of the chemical synthesis are provided in the supplementary information.
Expression and purification of human MTAP
4
1
Human MTAP was prepared as previously described with some modifications. In brief,
a plasmid containing the coding region for MTAP was transformed into BL21-CodonPlus(DE3)-
RIPL E. coli chemically competent cells. Nucleotide sequencing validated the DNA sequence for
MTAP. The culture was grown at 37 ˚C and 200 rpm in LB medium containing 100 µg/mL
ampicillin. Heterologous protein expression was induced when OD600 reached 0.6-0.8 by
addition of 1 mM IPTG (final concentration). After 8 h induction at 37 ˚C and 200 rpm, the cells
were harvested by centrifugation (5,000 x g for 20 min) and stored at -80 ˚C. All subsequent
steps were carried out at 4 ˚C, unless stated otherwise.
The pellet was suspended in lysis buffer (50 mM HEPES-NaOH, 5 mM imidazole at pH
7
.0) (2.5 mL/g of cell pellet) with addition of protease inhibitor cOmplete Mini EDTA-free (one
tablet per 20 g of cell pellet; Roche) and homogenized by stirring for 30 min. A spatula tip of
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lysozyme (Sigma) and DNAse I (Sigma) was added to the mixture and, after 30 min stirring,
cells were disrupted by sonication (15 sec on, 15 sec off, at 30% amplitude for 30 min) and
centrifuged (20,000 x g for 20 min) to remove cell debris. The recombinant MTAP contains 14
additional amino acids at N-terminus of the native enzyme, including a His tag (and a TEV
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protease cleavage site). The supernatant was incubated with Ni-NTA agarose (1.0 mL of slurry/g
of cell pellet; Qiagen) for 45 min with rocking, and the mixture was poured into an empty
column and washed with 12 column volumes of cell lysis buffer. The collection of 4 column
volume fractions from a 50 to 500 mM imidazole stepwise elution gradient gave proteins
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (200 V
and 185 mA for 60 min in MOPS running buffer) analysis, and the fractions containing the target
protein with purity over 95% (150 to 500 mM imidazole) were pooled. Purified MTAP contains
adenine which was removed by dialysis against 50 mM HEPES-NaOH at pH 7.0 with 0.2%
(m/v) activated charcoal (Sigma) overnight using 10 kDa dialysis cassettes (Thermo Scientific).
Adenine analysis in MTAP involved denaturation with 10% (v/v) perchloric acid. Denatured
protein was removed by centrifugation and the concentration of adenine in the supernatant was
tested by spectrophotometry. Adenine-free MTAP was concentrated to approximately 300 μM or
-1
-1
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0 mg mL (extinction coefficient is estimated to be 30.94 mM cm at 280 nm), and aliquots
were frozen in liquid nitrogen and stored at -80 ˚C. A total of 21 mg of protein was obtained
from 10 L of culture.
Expression and purification of HpMTAN
HpMTAN was prepared as previously described with some modifications.16,17 Briefly,
the plasmid containing His-tag HpMTAN gene was transformed into BL21 (DE3) E. coli
chemically competent cells. Nucleotide sequencing validated the DNA sequence for HpMTAN.
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The culture was grown at 37 ˚C and 200 rpm in LB medium containing 50 µg/mL ampicillin,
and heterologous protein expression was induced when OD600 reached 0.6-0.8 by addition of 0.5
mM IPTG (final concentration). Temperature was lowered to 30 ˚C upon addition of IPTG, and
the culture was grown an additional 20 h. Cells were harvested by centrifugation (5,000 x g for
20 min) and stored at -80 ˚C. All subsequent steps were carried out at 4 ˚C, unless stated
otherwise. The purification of HpMTAN was the same as described above for MTAP. A total of
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10 mg of protein was obtained from 2 L of culture.
Inhibition assays of MTAP
MTAP catalytic activity was measured using the absorbance difference between MTA
41
and adenine. A second assay followed the conversion of MTA to 2,8-dihydroxyadenine based
on the oxidation of adenine by xanthine oxidase to give an absorbance change at 305 nm (ε305
=
-1
-1
15.5 mM cm ). Inhibition constants were analyzed by fitting rate data to the Morrison quadratic
45
equation. Reactions in a 1 mL cuvette containing 100 mM K PO , 1 mM DTT, 800 μM MTA,
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nM MTAP, 1 unit of xanthine oxidase, and varying concentrations of each inhibitor.
Equilibrium dissociation constants were determined from reaction rates inhibition following
*
slow-onset binding (K ). The rates of each reaction were taken 40 min after initiation of the
i
reaction, a time when slow-onset equilibrium had occurred.
Inhibition assays of HpMTAN
Inhibition constants were determined as above. Reactions (1 mL) contained 100 mM
HEPES pH 7.2, 1 mM DTT, 100 mM NaCl, 1 mM MTA, 0.6 nM HpMTAN, 1 unit of xanthine
oxidase, and varying concentrations of each inhibitor (0 to 100 μM). Reactions were monitored
as described above for MTAP to obtain the inhibition constants following slow-onset binding
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when appropriate (K ). The rates of each reaction were taken 40 minutes after initiation of the
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reaction, to a time when slow-onset equilibrium had occurred.
Co-crystallization with transition-state inhibitors
Co-crystallization of MTAP and HpMTAN with four tight binding transition-state
inhibitors (15, 16, 30, and 32) used sitting drop vapor diffusion at 22 ˚C. MTAP or HpMTAN (5
mg/ml) was mixed with inhibitors in a 1:2 molar ratio and incubated for two hours on ice. The
proteins were screened for crystal-forming conditions with the Microlytic (MCSG1-4) and
Hampton (crystal screenHT) kits. Crystallization trials were in 96-well INTELLI plates using the
CRYSTAL-GRYPHON crystallization robot (ART ROBBINS). Crystallization drops contained
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.5 μL of enzyme-inhibitor mixture and 0.5 µL of well solution. The volume of the well solution
was 70 µL. Good quality crystals were obtained in one week (Table 1).
Data Collection and Processing
Diffraction data were collected at the LRL-CAT beam line (Argonne National
Laboratory, Argonne, IL) at a wavelength of 0.97931 Å (Table 1). Data were processed using the
iMOSFLM program and scaled by the AIMLESS program in the CCP4 suite, using the
appropriate space group (Table 1).46,47 Data quality was analyzed using the SFCHECK and
XTRIAGE.47,48 Matthews coefficient (V ) calculations indicated the number of monomers
m
present in the unit cells.
Structure Determination and Refinement
Crystal structures of MTAP and HpMTAN in complex with transition-state analogue
3
5
inhibitors were solved by molecular replacement using PHASER. Chain-A of wild-type MTAP
(PDB ID: 5TC6) and HpMTAN (PDB ID: 4WKP) structures were used as the initial phasing
model. The model obtained from PHASER was manually adjusted and completed using the
4
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graphics program COOT. Structure refinement was performed with REFMAC5, using standard
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protocols for the NCS refinement. Inhibitor molecules were deleted from the models to initiate
the refinement. After water was added, inhibitor molecules were fitted into their electron
densities (Table 1).
Structure Analysis
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Crystal structures of unliganded wild-type MTAP (PDB ID: 3OZE, chain: B), HpMTAN
(
PDB ID: 3NM4, chain: B) and EcMTAN (PDB ID: 1Z5P, chain: A) were used for structure
comparisons. The MTA complex with MTAP (PDB ID: 3T94) and p-ClPh-Thio-DADMe-ImmA
complex with HpMTAN (PDB ID: 5K1Z) were also used in the structural comparisons. All
structural superimpositions used the SSM protocol of COOT and the geometry analyses of the
36
final model used MolProbity. Additional structure analyses, including the calculation of the B-
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factor profiles used BAVERAGE of the CCP4 suite. Structural figures were generated with the
molecular graphics program PyMOL. For MTAP and HpMTAN structures, subunit-A was used
for all the structural analyses and comparisons.
ACKNOWLEDGMENTS
This project was supported by research grant GM041916 from the National Institutes of
Health and the New Zealand Foundation for Research Science and Technology contract
C08X0701. The Albert Einstein Crystallographic Core X-Ray diffraction facility is supported by
NIH Shared Instrumentation Grant S10 OD020068. Data collection also involved resources of
the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-
AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-
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CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly
Company, which operates the facility.
AUTHOR INFORMATION
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Corresponding Author
* E-mail: vern.schramm@einstein.yu.edu or peter.tyler@vuw.ac.nz
ORCID
Rajesh K. Harijan: 0000-0003-1503-5057
Rodrigo G. Ducati: 0000-0002-8783-8847
Peter C. Tyler: 0000-0002-3151-6208
Gary B. Evans: 0000-0002-6973-2002
Vern L. Schramm: 0000-0002-8056-1929
Oskar Hoff: 0000-0002-7716-7242
NOTES
The authors declare no competing financial interest.
AUTHOR CONTRIBUTIONS
V.L.S. and P.C.T. supervised the project. O.H. synthesized the transition-state analogues.
R.G.D., R.S.F. and B.M.H expressed, purified the enzymes and determined the enzyme kinetics.
R.K.H. conducted the structure determinations and characterizations. R.K.H., R.G.D, P.C.T and
V.L.S. wrote the paper. All the authors were involved in reviewing the data and manuscript
drafts.
STRUCTURAL DATA
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PDB ID CODES: 6DYZ, 6DZ0, 6DZ3, 6DZ2, 6DYU, 6DYV, 6DYY, 6DYW. Authors will
release the atomic coordinates and experimental data upon article publication.
SUPPORTING INFORMATION CONTENT
Page S1: Title page and abbreviations
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Page S2: Index to Supplementary Information Content
Page S3: Figure S1. Human MTAP subunit ribbon structure comparing free and liganded
enzyme.
Page S4: Figure S2. Electron density omit maps for MTAP-inhibitor complexes of 15, 16, 30,
32.
Page S5: Figure S3. Inhibitor geometry at the catalytic sites of human MTAP and HpMTAN.
Page S6: Figure S4. HpMTAN subunit ribbon structures comparing free and liganded MTANs.
Page S7: Figure S5. Electron density omit maps for HpMTAN-inhibitor complexes of 15, 16,
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Page S8: Table S1. Crystallization and crystal handling
Pages S9 – 33: Details of chemical synthesis
Page S34: SMILES formula data for all inhibitions indexed by compound number
Pages S35 – 47: NMR spectra of all inhibitors
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ABBREVIATIONS: MTDIA: methylthio-DADMe-Immucillin-A; MTA: S-methyl-5'
thioadenosine; All: allyl; DQF-COSY: double-quantum filtered correlation spectroscopy; Q-
TOF: quadrupole time-of-flight; MTAN: 5'-methylthioadenosine/S-adenosylhomocysteine
nucleosidase; HpMTAN: Helicobacter pylori MTAN; MTAP: 5'-methylthioadenosine
phosphorylase; MTR: 5-methylthio-α-D-ribose 1-phosphate; SAM: S-Adenosylmethionine;
SAH: S-adenosylhomocysteine, SRH: S-ribosylhomocysteine; NMR: nuclear magnetic
resonance; HPLC: high performance liquid chromatography; RT: room temperature; t-Bu: tert-
butyl; DMF: dimethylformamide; aq: aqueous; Et: ethyl; Me: methyl; THP: tetrahydropyranyl;
BOC: tert-butyloxycarbonyl; Ac: acyl; n-Bu: n-butyl; Bn: benzyl; PRMT5: protein arginine
methyltransferase 5; MAT2A: S-adenosylmethionine synthetase 2A; HSQC: heteronuclear single
quantum coherence spectroscopy; DEPT: distortionless enhancement by polarization transfer;
APT: Attached proton test
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0. Appleby, T. C.; Erion, M. D.; Ealick, S. E. The Structure of Human 5'-Deoxy-5'-
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FIGURE LEGENDS
Figure 1. The transition states and reactions catalyzed by MTAP and HpMTAN. (A) MTAP catalyzes the reaction
via a ribocationic transition state and phosphate as the nucleophile. Adenine and methylthio-α-D-ribose 1-phosphate
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are the products. Bonds to the leaving group and the attacking nucleophile are weak, less than 0.1 Pauling bond
N N
order, making the reaction more S 1 than S 2 in character. (B) HpMTAN also catalyzes its reaction via a
ribocationic transition state. Water acts as the nucleophile. Adenine and methylthio-D-ribose are the products.
Figure 2. Transition-state analogue inhibitors of MTAP and HpMTAN. Inhibitors were ranked based on their K
d
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d
value for MTAP. ND means inhibition not detected at 5 μM inhibitor concentration.The specificity ratio (K Hs/Hp)
is the affinity for human MTAP relative to HpMTAN.
Figure 3. Stereoview of the binding sites of MTAP in complex with transition-state analogue inhibitors. The
inhibitor complexes of 15, 16, 30 and 32 are shown in panels A, B, C and D, respectively. The residues interacting
with inhibitors from monomer-A are shown in green and from the neighboring subunit are shown in light blue.
Selected hydrogen bond interactions are shown in orange dotted lines.
Figure 4. Stereoview of the binding sites of HpMTAN in complex with transition-state analogue inhibitors. The
inhibitor complexes of 15, 16, 30 and 32 are shown in panel A, B, C and D, respectively. The residues interacting
with inhibitors from monomer-A are shown in yellow and from monomer-B are shown in light blue. Selected
hydrogen bond interactions are shown in orange dotted lines.
Figure 5. Catalytic site conformations of apo- and ligand-bound MTAP and HpMTAN. (A) Stereoview
superposition of unliganded MTAP (PDB ID: 3OZE; green) with four inhibitor-bound structures including MTAP-
1
5 (PDB ID: 6DYZ; cyan), MTAP-16 (PDB ID: 6DZ0; yellow), MTAP-30 (PDB ID: 6DZ3; blue) and MTAP-32
PDB ID: 6DZ2; light pink), are overlapped. The 1-2 loop (highlighted with red star) is altered substantially on
the binding of the 30 and 32. (B) The superposition of an unliganded MTAN binding site (E. coli MTAN PDB ID:
Z5P; blue) with four inhibitor bound complexes of HpMTAN including MTAP-15 (PDB ID: 6DYU; brick red),
(
1
MTAP-16 (PDB ID: 6DYV; cyan), MTAP-30 (6DYY; yellow) and MTAP-32 (6DYW; red). The helix 6 and
associated loop of the unliganded MTAN changes conformation to elongate helix 6 as a result of inhibitor binding
(highlighted with a red star).
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