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Mechanistic and structural requirements for active site labeling ofphosphoglycerate mutase by spiroepoxides{

Michael J. Evans,a Garrett M. Morris,b Jane Wu,c Arthur J. Olson,b Erik J. Sorensend andBenjamin F. Cravatt*a

Received 4th April 2007, Accepted 13th April 2007

First published as an Advance Article on the web 10th May 2007

DOI: 10.1039/b705113a

We recently reported the pharmacological screening of a natural products-inspired library of

spiroepoxide probes, resulting in the discovery of an agent MJE3 that displayed anti-proliferative

effects in human breast cancer cells. MJE3 was found to covalently inactivate phosphoglycerate

mutase-1 (PGAM1), a glycolytic enzyme with postulated roles in cancer cell metabolism and

proliferation. Considering that MJE3 is one of the first examples of a cell-permeable, small-

molecule inhibitor for PGAM1, we pursued a detailed examination of its mechanism and

structural requirements for covalent inactivation. MJE3 was found to label PGAM1 on lysine-

100, a conserved active site residue implicated in substrate recognition. Structural features of

MJE3 important for PGAM1 labeling included two key recognition elements (an indole ring and

carboxylic acid), the stereochemical orientation of the spiroepoxide, and presentation of these

various binding/reactive groups on a rigid cyclohexane scaffold. Modeling studies of the docked

MJE3–PGAM1 complex provide a structural rationale for these stringent requirements. Overall,

these studies indicate that a special combination of binding and reactive elements are united in the

MJE3 structure to inactivate PGAM1. More generally, our findings provide further evidence that

useful pharmacological tools can emerge from screening structurally diverse libraries of protein-

reactive probes.

Introduction

Advances in cell-based screening,1 global protein analysis

(proteomics),2 and small-molecule library synthesis3 have

sparked interest in the systematic discovery of chemical probes

to perturb cellular pathways. These pharmacological pursuits,

often referred to as chemical genetics, have engendered several

new compounds with interesting biological activities, including

agents that cause de-differentiation of lineage-committed

cells,4 activate novel pathways for cell death,5 overcome drug

resistance in cancer cells,6 and suppress the Dauer-forming

phenotype of daf-2 mutants in C. elegans.7

Determining the mechanism of action of chemical probes

requires identification of their molecular targets. This pursuit

is often confounded by the limited potency of lead compounds

that emerge from cell-based screens.8 We recently described a

strategy to directly couple cell-based screening with target

identification that employs small-molecule libraries predis-

posed to produce biological effects through covalent reactiv-

ity.9 By appending an alkyne handle onto protein-reactive

probes, in situ proteome reactivity profiles for pharmacologi-

cally active and inactive compounds can be compared after

click chemistry conjugation to an azide-containing reporter tag

(e.g., biotin, rhodamine).10,11 Protein targets selectively labeled

by active probes are then designated as likely candidates for

producing the biological effect of interest. The rationale for

this approach was based on the extraordinary number of

examples of natural products that produce their biological

effects through covalent inactivation of protein targets.12

Indeed, to further emulate the properties of natural products,

our first library of protein-reactive probes incorporated key

structural elements of the anti-angiogenic agent fumagillin,

which has been shown to covalently inactivate methionine

aminopeptidase-2 via a spiroepoxide reactive group.13

From a structurally diverse library of spiroepoxide probes

(Fig. 1), we identified an agent MJE3 (Fig. 1) that produced

anti-proliferative effects in human breast cancer cells.9 In situ

proteomic profiling showed that, among the spiroepoxide

probe set, MJE3 uniquely labeled a 26 kDa protein that was

aThe Skaggs Institute for Chemical Biology and Departments of CellBiology and Chemistry, The Scripps Research Institute, 10550 N. TorreyPines Rd., La Jolla, CA 92037, USA. E-mail: [email protected];Fax: +1-858-784-8023; Tel: +1-858-784-8633bThe Skaggs Institute for Chemical Biology and Department ofMolecular Biology, The Scripps Research Institute, 10550 N. TorreyPines Rd., La Jolla, CA 92037, USAcActivX Biosciences Inc., 11025 N. Torrey Pines Rd., La Jolla,CA 92037, USAdDepartment of Chemistry, Princeton University, Princeton, NJ 08544,USA{ Electronic supplementary information (ESI) available: Schemes forsynthesis of 11 and 14–23, general synthetic methods and character-ization data for 14–23 and MJE67, table with characterization data forspiroepoxide library members, figures showing 1H-NMR data fordetermining relative stereochemistry of 5, 1H-NMR data showinglong-range coupling in 12a, structures of amine substituents forspiroepoxide probes, MS fragmentation pattern for MJE probe,kinetic analysis of in situ proteome reactivity of MJE3, clusteringhistogram for docking results of MJE3–PGAM1 interactions, andimages of five lowest energy conformations of MJE3–PGAM1interaction. See DOI: 10.1039/b705113a

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

This journal is � The Royal Society of Chemistry 2007 Mol. BioSyst., 2007, 3, 495–506 | 495

subsequently identified as the glycolytic enzyme phosphogly-

cerate mutase-1 (PGAM1). MJE3 inhibited PGAM1 activity

in breast cancer cells with an IC50 value similar to its anti-

proliferative effects. Collectively, these findings point to

inhibition of PGAM1 as a likely mechanism for the anti-

proliferative effects of MJE3. Interestingly, MJE3 labeling

and inhibition of PGAM1 were observed in intact cells, but

not cell extracts, indicating that the MJE3–PGAM1 interac-

tion requires in situ factors that are disrupted upon cell

homogenization.

Several additional lines of evidence suggest that PGAM1

may be an attractive target for anti-cancer therapies.

First, cancer cells have long been recognized to depend more

greatly on glycolysis for viability than normal cells, a

phenomenon referred to as the Warburg effect.14 Within the

glycolytic pathway, PGAM1 appears to be a particularly

sensitive node in cancer cells. For example, peptide

inhibitors of PGAM1 phosphorylation and activity cause

growth arrest in cancer cells.15 Conversely, overexpression of

PGAM1 leads to the immortalization and indefinite growth of

mouse embryonic fibroblasts.16 Knockdown of PGAM1

expression in these cells by RNA interference caused a

senescent phenotype. Despite these findings, few small-

molecule inhibitors of PGAM1 have been described to date,

and most of these agents have only been shown to inhibit

PGAM1 in vitro.17 MJE3 thus could constitute a valuable

lead for the design of potent, cell-permeable inhibitors of

PGAM1. Toward this end, we describe here a detailed

investigation of the mechanism of inactivation of PGAM1

by MJE3. We show that MJE3 labels PGAM1 on a conserved

active site lysine residue implicated in substrate binding. A

combination of structure–activity and modeling studies

provided a rationale for the highly selective interaction of

PGAM1 with MJE3 relative to other members of the

spiroepoxide probe library.

Results and discussion

Synthesis of MJE3 and other members of the spiroepoxide probe

library

The common precursor molecule 10 for construction of the

spiroepoxide probe library was prepared following the general

route outlined in Scheme 1. The synthesis commenced by

Fig. 1 Structures of a spiroepoxide library of protein-reactive probes

(top) and specific library member MJE3 that exhibited anti-prolif-

erative effects in human cancer cells and inactivated the glycolytic

enzyme PGAM1.

Scheme 1 Synthetic route for compound 10, the common precursor molecule for construction of the spiroepoxide probe library.

496 | Mol. BioSyst., 2007, 3, 495–506 This journal is � The Royal Society of Chemistry 2007

converting (1R)-(+)-nopinone (1, Aldrich, St. Louis, MO) to

the enantiopure diacetate 2 following a previously reported

protocol.18 The enol acetate functionality of 2 was then

oxidized to a diastereomeric mixture of a-hydroxyketones 3 via

Os(VI) catalyzed dihydroxylation,19 which were then protected

as tert-butyldimethylsilyl (TBDMS) ethers (4). Wittig olefina-

tion afforded compounds 5 and 5a in a 4 : 1 syn : anti mixture,

which could be separated by silica gel chromatography to yield

the desired syn compound in overall 42% yield (the orientation

of the substituents was verified by 1H NMR comparison of 5

after TBDMS deprotection to a previously reported com-

pound;20see ESI for details{).

Compound 5 was treated with K2CO3 in MeOH to cleave

the remaining acetate moiety. The resulting tertiary alcohol on

6 served as the site of attachment for the alkyne affinity

handle, which was achieved via esterification with 5-hexynoic

acid (7). After removal of the TBDMS protecting group (1 M

TBAF in THF), the secondary allylic alcohol on compound 8

was converted to the mixed carbonate 9 (N,N9-disuccinimidyl

carbonate, DMAP). Finally, the protein-reactive spiroepoxide

was installed with m-CPBA, and compound 10 was separated

from its diastereomer (10a) via silica gel chromatography.

To unequivocally assign the stereochemistry of the spiroep-

oxide on 10, we prepared sufficient quantities of 10a for

comparison by NMR. The synthesis of 10a was accomplished

by first converting the exocyclic alkene on 7 to an epoxide with

in situ-generated dimethyldioxirane (Scheme 2).21 The resulting

diastereomers 11 and 11a were readily separated via silica gel

chromatography, and each compound then treated with TBAF

(1 M in THF) to remove the TBMDS protecting groups to

provide 12 and 12a, respectively. Compound 12a was then

treated with N,N9-disuccinimidyl carbonate and DMAP to

generate 10a. NMR studies revealed long-range coupling

between a proton on the exo-methylene carbon of the

spiroepoxide, and a neighboring axial proton for compounds

10–12a, while no long-range coupling was observed between

the corresponding protons on 10–12 (as shown for 12 and 12a,

ESI Fig. 2{). Since this coupling relationship is established to

occur exclusively between protons on a pseudo-axial exo-

methylene carbon and an axial proton from the cyclohexyl

ring,22–25 we concluded that the major product of the m-CPBA

epoxidation was compound 10.

With a route to prepare 10, we explored strategies to

introduce chemical diversity to the oxa-spiro[2.5]octane core

scaffold. In general, we found that treating 10 with 1.0 equiv.

of a primary amine and 1.1 equiv. of a base (e.g., NEt3) in

MeCN at rt resulted in complete consumption of 10 within

1 hour (Scheme 3). After purification by silica gel chromato-

graphy, we isolated the corresponding epoxy carbamate in

yields suitable for biological studies (average yields of 53%).

Identification of K100 as the site of MJE3 labeling in PGAM1

Previous studies showed that MJE3 covalently labels and

inhibits PGAM1 in breast cancer cells.9 To determine the

precise site of probe modification in PGAM1, we subjected

MJE3-treated COS7 cells transfected with the human PGAM1

cDNA to click chemistry reaction with an azide–biotin tag

bearing a bridging TEV protease cleavage site.26 This azide–

TEV–biotin tag enables capture of probe-labeled proteins onto

avidin beads and, following trypsin digestion to remove non-

labeled peptides, release of probe-modified peptides by

treatment with TEV protease. TEV-cleaved peptides from

PGAM1-transfected cells were analyzed by LC-MS/MS,

resulting in the identification of a single probe-modified

Scheme 2 Synthesis of compounds used to assign the stereochemistry of the spiroepoxide probe library. See ESI Fig. 2{ for NMR data used to

assign the relative stereochemistry of spiroepoxide groups of 12 and 12a (similar data were obtained for 10/10a and 11/11a).

Scheme 3 Conversion of general precursor 10 to spiroepoxide library

members.


peptide in PGAM1 corresponding to amino acid (aa) residues

91–106 (Fig. 2A). This peptide was modified by the free acid

form of MJE3, suggesting that in situ hydrolysis of its benzyl

ester substituent may precede PGAM1 labeling. Consistent

with this premise, we previously found that the amide

analogue of MJE3 is not capable of labeling PGAM1 in cells.9

Further refinement of the site of MJE3 modification in

PGAM1 was accomplished by manual analysis of tandem MS

Fig. 2 Identification of K100 as the site of MJE3 probe labeling in PGAM1. (A) Structure of the covalent adduct between MJE3 and aa 91–106 of

PGAM1 (following click chemistry with an azide–TEV–biotin tag26 and TEV protease cleavage of the avidin-enriched MJE3–PGAM1 adduct).

Note that the free acid form of MJE3 is covalently bound to PGAM1, suggesting that the benzyl ester group of this probe is hydrolyzed in situ prior

to PGAM1 labeling (see Fig. 1 for parent structure of MJE3 probe). Dashed lines denote major probe fragmentation events that were observed in

tandem MS experiments (see ESI Fig. 4{ for structures of the major neutral loss fragments from the probe). (B) MS2 data for precursor ion m/z

809.8 (+3 charge state of MJE3-modified aa 91–106 peptide). (C) MS3 data for m/z 1015.2 (+2 charge state of MJE3-modified aa 91–106 peptide

minus the 397 probe fragment). For (B) and (C), b and y fragment ions are shown in red and blue, respectively. Ions listed in bold correspond to key

mass fragments used to identify K100 as the site of MJE3 labeling.


(MS2 and MS3) profiles of the probe-modified aa 91–

106 peptide. The MS2 spectrum of the m/z 809 precursor

ion, which constituted the +3 charge state of the probe-labeled

aa 91–106 peptide (Fig. 2B), contained several ions that

collectively placed the likely site of probe modification at

lysine-100 (K100). Prominent among these ions were the m/z

590 and 913 fragments, which corresponded to aa 91–99 and

101–106 peptides without probe modification. Conversely, an

m/z 1440 ion matched the predicted mass of the aa 91–

100 peptide modified by one molecule of MJE3 (minus loss of

397 Da corresponding to a prominent probe fragment; see

Fig. 2A and ESI Fig. 4{ for more details). An MS3 spectrum

was also generated for a prominent MS2 ion, m/z 1015, that

corresponded to the +2 charge state of the probe-modified aa

91–106 peptide minus loss of the 397 Da probe fragment

(Fig. 2C). This MS3 spectrum also provided clear evidence

that probe modification occurred at K100, including an m/z

1117 ion that matched the predicted mass of the probe-

modified aa 100–106 peptide (as well as the m/z 590 and 913

‘‘probe-free’’ ions mentioned above).

To provide further support that MJE3 reacted specifically

with K100 in the PGAM1 active site, we mutated this residue

to alanine and compared the probe labeling of this K100A

variant to wild type (WT) PGAM1 in transfected COS-7 cells.

In situ treatment of transfected COS-7 cells with MJE3

resulted in strong labeling of WT-PGAM1, but not the

K100A mutant (Fig. 3). Western blotting confirmed that the

K100A mutant expressed to similar levels as WT-PGAM1.

These data support that K100 is the principal site of MJE3

labeling in PGAM1. Interestingly, K100 is a highly conserved

active site residue implicated in substrate binding,27 thus

providing a clear rationale for the inhibitory effects of MJE3.

Determination of the key structural features that dictate MJE3

labeling of PGAM1

We previously showed that labeling of PGAM1 was a unique

property of MJE3 compared to other members of the 50-

compound spiroepoxide probe library.9 To delineate further

the structural requirements for MJE3 labeling of PGAM1, we

synthesized a series of analogues and tested their in situ

reactivity with PGAM1 in the human melanoma cell line

MUM2B. MUM2B cells were selected because they displayed

strong levels of PGAM1 labeling and high sensitivity to the

anti-proliferative effects of MJE3 (Fig. 4).

Analogues of MJE3 included compounds with altered

presentations of the epoxide reactive group [the diastereomer

spiroepoxide (MJE55) and 2,2-disubstituted oxirane (MJE67)

analogues] and with binding groups (BGs) modified at the

a-carbon (MJE56–64) or O-alkyl/aryl ester (MJE52–54)

positions (Fig. 5A). We first performed a kinetic analysis of

the in situ labeling of PGAM1 by MJE3 in MUM2B cells,

which revealed that maximal labeling was achieved by 1 h (ESI

Fig. 5{). The efficiencies of probe labeling were compared at

this time point to facilitate the detection of partial labeling

events. Dramatic reductions in labeling of PGAM1 were

observed for both MJE55 and MJE67 (Fig. 5B), indicating

that the placement and relative orientation of the epoxide on

an entropically constrained ring system were critical features

for reactivity. Interestingly, many of the other protein targets

of MJE3 in cancer cells were labeled by MJE67 (Fig. 5B),

indicating that modification of PGAM1 was unusual in its

Fig. 3 WT-PGAM1 (PGAM1), but not the K100A mutant is labeled

by MJE3 in transfected COS-7 cells. PGAM1 proteins were expressed

in COS-7 cells as C-terminal Myc-tagged fusion proteins. Mock cells

were transfected with an empty vector for comparison. Probe labeling

was performed by treating COS-7 cells in situ with MJE3 (50 mM) for

1 h and detected by click chemistry with an azide–rhodamine tag, SDS-

PAGE, and in-gel fluorescence scanning (upper panel; fluorescent gel

shown in grayscale). Western blotting with anti-Myc antibodies

confirmed equivalent expression levels for the wild type and K100A

PGAM1 proteins (lower panel).

Fig. 4 In situ labeling of PGAM1 in human cancer cells by MJE3. (A)

MJE3 labeling of PGAM1 in human breast (MDA-MB-231) and

melanoma (MUM2B) cell lines. Upper panel, probe labeling was

detected by click chemistry reaction with an azide–rhodamine group

followed by SDS-PAGE and in-gel fluorescence scanning (fluorescent

gel shown in grayscale). Lower panel, Western blot using anti-PGAM1

antibodies. (B) Anti-proliferative effects of MJE3 in MDA-MB-231

and MUM2B cells. Data shown as percentage of control proliferation

observed in cells treated with vehicle (DMSO) control. The proteome

labeling and anti-proliferative effects of the pharmacologically inactive

probe MJE4, which possesses a tryptamine binding group, are shown

for comparison (see ESI Fig. 4{ for structure of MJE4). Data represent

the average ¡ standard deviation of four independent experiments

**p , 0.01 (planned comparison).


requirement for the spiroepoxide group. BG analogues bearing

substitutions at the a-carbon side chain showed weak or

negligible reactivity with PGAM1, underscoring the impor-

tance of the indole side chain of MJE3 for binding to the

PGAM1 active site. Inverting the stereochemical orientation of

this indole group (MJE56) also disrupted PGAM1 labeling.

Many of the side chain analogues of MJE3 maintained strong

labeling of additional protein targets in the cancer cell

proteome, indicating that the structure–activity relationship

(SAR) observed for labeling of PGAM1 was not simply a

reflection of the inherent protein reactivity of the probes. In

contrast to the negative impact of side chain alterations, all

three of the O-alkyl/aryl ester analogues MJE52–54 efficiently

labeled PGAM1, consistent with a model where these ester

groups are cleaved in situ to generate a common free acid

product that then binds and reacts with PGAM1.

Modeling the MJE3–PGAM1 interaction

The strict SAR observed for labeling of PGAM1 by MJE3

suggested that the probe makes highly specific contacts within

the enzyme active site. To explore this idea further, we

performed modeling studies of the covalent MJE3–PGAM1

complex. An adduct of MJE3 with the f-nitrogen and e-carbon

atoms of K100 was built using the Ghemical program28 and

energy minimized by molecular dynamics simulation to

provide a lowest energy conformation used for docking

experiments. Throughout most of the simulation, the cyclo-

hexane ring of MJE3 adopted a stable twist–boat conforma-

tion with all three bulky ring substituents in equatorial

positions. The MJE3–K100 adduct was set up for covalent

docking with the structure of the human PGAM1 from Protein

Data Bank entry 1YFK{27 (modified by deletion of the K100

f-nitrogen and e-carbon atoms).

The results from 200 independent dockings using AutoDock

329 were clustered using an RMSD tolerance of 3.0 A. The

lowest energy cluster at approximately 211 kcal mol21 was

well separated from the next lowest energy cluster by more

than 2 kcal mol21 (ESI Fig. 6{). The top five lowest energy

clusters were all observed with reasonable frequencies of

occurrence (16–38 members), with the remaining clusters being

Fig. 5 Structure–activity relationship (SAR) observed for PGAM1 labeling by spiroepoxide probe analogues of MJE3. (A) Structures of MJE3

and analogues. (B) In situ proteome reactivity profile for MJE3 and analogues in MUM2B cells (20 mM probe, 1 h labeling reaction). Upper panel,

probe labeling was detected by click chemistry reaction with an azide–rhodamine group followed by SDS-PAGE and in-gel fluorescence scanning

(fluorescent gel shown in grayscale). Lower panel, Western blot using anti-PGAM1 antibodies. (C) Quantification of the relative labeling intensities

of PGAM1 by MJE3 and analogues. Data represent the average ¡ standard deviation of three independent experiments.


sparsely populated (,10 members) (ESI Fig. 6). The lowest

energy conformation (cluster 1) is shown in Fig. 6, with the

other four well-populated low-energy conformations shown in

ESI Fig. 7. In all five conformations, the carboxylic acid and

indole groups of the side chain of MJE3 are deeply buried in

the PGAM1 active site. The acid group appears to make

contacts with R10 and R191 of PGAM1, while the indole ring

may form p–cation interactions with R116 and/or R117. The

main difference among the five lowest energy conformations

relates to the positioning of the cyclohexane ring. In cluster 1,

this ring is deeply buried in the PGAM1 active site, while in

clusters 2–5 it is more solvent exposed. A comparison of cluster

1 with the structure of PGAM1 bound to the substrate

analogue citrate27 reveals several sites of overlapping interac-

tion. For example, citrate was found to form hydrogen bonds

with R116, R117, and, perhaps most notably, K100, the site of

MJE3 labeling. These data thus provide a clear structural

model to explain how MJE3 labeling could effect PGAM1

inhibition by simple blockade of substrate binding.

Conclusion

PGAM1 converts 3-phosphoglycerate to 2-phosphoglycerate

as part of the glycolytic pathway. Multiple lines of evidence

suggest a role for glycolysis, and PGAM1 in particular, in

supporting the pathogenic properties of cancer cells.14–16

Despite these findings, few inhibitors of PGAM1 have been

reported. We recently described the identification of an anti-

proliferative compound MJE3 from a library of natural

products-like spiroepoxide probes that covalently inhibited

PGAM1 in breast cancer cells.9 Here, we have examined the

mechanism and structural features required for inactivation of

PGAM1 by MJE3. Using a combination of MS analysis and

site-directed mutagenesis, we determined that MJE3 covalently

modified K100, a conserved residue in the PGAM1 active site

implicated in substrate binding. Cleavage of the benzyl ester

bond on MJE3 appears to precede PGAM1 labeling, a premise

that is supported by the nearly equivalent activity of analogues

bearing structurally distinct ester substituents.

In contrast to the permissive SAR observed for the ester

group of MJE3, modification of other portions of the probe

structure were found to disrupt PGAM1 labeling. In parti-

cular, the placement and relative orientation of the spiroep-

oxide on a cyclohexane scaffold were required for efficient

labeling of PGAM1. Likewise, substitutions made to the

indole portion of the BG generally disrupted PGAM1 labeling.

Collectively, these data indicate that multiple features of MJE3

are critical for preserving its specific interaction with PGAM1.

This premise was supported by computational studies where

MJE3 was docked into the PGAM1 active site as a covalent

adduct with K100. In this docked complex, key protein–small

molecule interactions were observed between multiple arginine

residues (R10, R116, R117, and R191) and the free carboxylic

acid and indole groups of MJE3. This model thus provides a

structural rationale to explain PGAM1’s selective reactivity

with MJE3 relative to other members of the spiroepoxide

probe library.

In summary, we have shown that the pharmacological

screening of a library of protein-reactive probes can identify

bioactive small molecules (e.g., MJE3) that possess unique

protein targets (e.g., PGAM1) in living systems. One potential

drawback of this approach is that the engendered chemical

probes may prove difficult to transform into ‘‘drug-like’’

molecules that operate by more selective, perhaps ideally, non-

covalent mechanisms. However, we believe that understanding

the detailed mechanism for probe inactivation of protein

Fig. 6 Modeling of the MJE3–PGAM1 adduct. (A) Molecular

surface of PGAM1 from PDB code 1YFK,{ showing the lowest

energy binding mode of the covalently-docked MJE3. K100 can be

seen covalently attached to MJE3 below the molecular surface. Note

that all the water molecules were removed from the protein structure

before docking. (B) Depiction of the lowest energy docked conforma-

tion of MJE3 in the PGAM1 active site. MJE3 is shown in ball-and-

stick format with its molecular surface colored grey, except where

atoms are in contact with the protein, in which case these parts of the

surface are colored by atom type. The side chains in the protein that

are within 5 A of MJE3 are shown as lines and labeled by residue name

and number. Note the carboxylate group of MJE3 binds between the

R10 and R191 guanidinium groups, while the indole ring binds next to

R117. The figure was created using AutoDockTools (ADT).


targets can serve as a valuable guide toward achieving this

objective. For example, by determining that K100 is the site of

MJE3 modification in PGAM1, we can now ask whether

substitution of the spiroepoxide group with a lysine-directed

binding element, such as an aldehyde or carboxylic acid, might

generate an MJE3 analogue with improved selectivity for

PGAM1. Finally, it is important to emphasize that certain

aspects of the MJE3–PGAM1 interaction still remain enig-

matic. Most notably, MJE3 and its active analogues are only

capable of labeling and inhibiting PGAM1 in living cells.9 It is

possible that PGAM1, as a result of post-translational

modification and/or protein–protein interactions, adopts a

structure in living systems that is conducive to binding and

reaction with MJE3. If these post-translational regulatory

events are disrupted upon cell homogenization, then so too

might be the PGAM1–MJE3 interaction. This curious

phenomenon does not, however, prevent the pursuit of more

potent and selective inhibitors of PGAM1 that act in a similar

manner to MJE3. Indeed, like other active site-directed

proteomic probes,30–33 MJE3 could itself form the basis for

an assay to screen for additional PGAM1 inhibitors, wherein

active compounds would be detected in competitive cell-based

assays by their ability to block MJE3 labeling of PGAM1.

Compounds that impair labeling of PGAM1, but not other

targets of MJE3, would be further interpreted to possess

improved selectivity and serve as leads for the future

development of potential anti-cancer agents.

Experimental procedures

Synthetic methods

2. Compound 2 was prepared according to the previously

reported protocol.18 To a solution of (1R)-(+)-nopinone

(1, 10.0 g, 72.3 mmol, 1.0 equiv.) and zinc acetate (13.7 g,

72.4 mmol, 1.0 equiv.) in acetic anhydride (100 mL) was added

boron trifluoride diethyl etherate (3.85 mL, 30.3 mmol,

0.42 equiv.) via syringe at 0 uC. The reaction was stirred for

18 h at 0 uC, and quenched by adding H2O (100 mL). After

stirring for 4 h, the reaction mixture was warmed to room

temperature, diluted with Et2O, and washed with water (1 650 mL) and aqueous saturated NaHCO3 (2 6 50 mL). The

organic layer was dried (Na2SO4), filtered, and concentrated

under reduced pressure with a rotary evaporator. The residue

was purified by flash chromatography (10–50% Et2O in

hexanes) to afford 11.1 g (64%) of 2 as a yellow oil. Rf =

0.25 (10% EtOAc in hexanes); [a]D = 247.0 (c = 1.0, CHCl3);1H NMR (400 MHz, CDCl3) d 5.32 (dd, 1H, J = 5.0, 2.4 Hz),

2.26 (m, 1H), 2.11–2.05 (m, 3H), 2.08 (s, 3H), 2.00–1.96 (m,

1H), 1.94 (s, 3H), 1.87–1.82 (m, 1H), 1.51–1.45 (m, 1H) 1.43 (s,

3H), 1.41 (s, 3H). The structure of 2 was verified by

comparison to the previously reported compound.18

3. A solution of 4-methylmorpholine N-oxide (6.8 g,

50.8 mmol, 1.1 equiv.) in H2O (35 mL) at rt was added to a

stirring solution of 2 (11.1 g, 46.2 mmol, 1.0 equiv.), citric acid

(17.7 g, 92.5 mmol, 2.0 equiv.) and K2OsO2(OH)4 (333 mg,

0.925 mmol, 0.020 equiv.) in 66% t-BuOH in H2O (105 mL

total).19 The reaction was stirred open to air for 30 min at rt.

After t-BuOH was removed under reduced pressure, the

solution was washed with Et2O (3 6 70 mL). The combined

organic extracts were dried (Na2SO4), filtered, and adsorbed

onto silica under reduced pressure. Flash chromatography

(10% EtOAc in hexanes) afforded 8.80 g (88%) of 3 as a

colorless oil. Rf = 0.55 (50% EtOAc in hexanes); 1H NMR

(500 MHz, CDCl3) d 4.33 (m, 1H), 2.55 (m, 2H), 2.42 (m, 2H)

2.05 (m, 1H), 1.97 (s, 3H), 1.44 (s, 3H), 1.52–1.36 (m, 5H). HR-

MS (MALDI-FTMS) Calcd for C11H18O4 (M + Na)+

237.1097, found 237.1102.

4. To a 250 mL round bottomed flask was added 3 (8.00 g,

37.3 mmol, 1.0 equiv.), imidazole (15.0 g, 220 mmol,

6.0 equiv.), and tert-butyldimethylsilyl chloride (17.0 g,

112 mmol, 3.0 equiv.). The flask was purged of oxygen, and

DMF (160 mL) was added via cannula. The reaction was

stirred at rt for 18 h. DMF was removed under reduced

pressure with a rotary evaporator, and the residue was

dissolved in EtOAc (50 mL). The solution was washed with

aqueous saturated NH4Cl (1 6 20 mL) and with H2O (1 620 mL). The organic layer was dried (Na2SO4), filtered, and

concentrated under reduced pressure. The crude residue was

purified with flash chromatography (20% EtOAc in hexanes)

to afford 10.7 g (88%) of 4. Rf = 0.7 (33% EtOAc in hexanes);1H NMR (500 MHz, CDCl3) d 4.22–4.18 (dd, 1H, J = 6.6,

12.1 Hz), 2.64 (m, 1H), 2.45 (m, 1H), 2.3 (m, 1H), 2.18 (m, 1H),

1.99–1.97 (m, 1H), 1.98 (s, 3H) 1.62 (m, 1H), 1.48 (m, 1H), 1.45

(s, 3H), 1.44 (s, 3H), 0.91 (s, 9H), 0.11 (s, 3H), 0.06 (s, 3H).

HR-MS (ESI-TOF) Calcd for C17H32O4Si (M + Na)+

351.1962, found 351.1959.

5. Methyltriphenylphosphonium bromide (36.0 g, 101 mmol,

3.0 equiv.) and potassium tert-butoxide (11.4 g, 101 mmol,

3.0 equiv.) were added to a 250 mL three-necked flask, which

was then fitted with a reflux condenser. The reaction was

purged of oxygen, and THF (140 mL) was added via cannula.

The solution was refluxed for 2 h, at which time a solution of 4

(11.7 g, 35.3 mmol, 1.0 equiv.) in THF (10 mL) was added. The

reaction was refluxed for an additional 2 h, and cooled to rt.

The reaction was quenched with H2O (500 mL), washed with

EtOAc (2 6 300 mL), dried (Na2SO4), and filtered. The

organic extract was adsorbed onto CeliteTM. Purification by

silica gel chromatography (10% EtOAc in hexanes) afforded

4.50 g (42%) of 5 (syn) and 1.12 g (11%) of 5a (anti) as colorless

oils. 5: Rf = 0.3 (10% EtOAc in hexanes); [a]D = 216.0 (c = 1.0,

CHCl3); 1H (500 MHz, CDCl3) d 4.98 (s, 1H), 4.66 (s, 1H),

4.01 (dd, 1H, J = 11.0, 4.8 Hz), 2.43 (m, 1H), 2.24 (m, 1H),

1.97 (s, 3H), 1.98–1.94 (m, 3H), 1.74 (m, 1H), 1.39 (s, 3H), 1.38

(s, 3H), 1.25–1.04 (m, 3H), 0.92 (s, 9H), 0.07 (s, 3H), 0.06 (s,

3H). HR-MS (ESI-TOF) Calcd for C18H34O3Si (M + Na)+

349.2169, found 349.2173. 5a: Rf = 0.25 (10% EtOAc in

hexanes). HR-MS (ESI-TOF) Calcd for C18H34O3Si (M +

Na)+ 349.2169, found 349.2170. The structure and stereo-

chemistry of compound 5a was verified by comparison of

the 1HNMR spectra to the structurally-related methane

monoterpene.21

6. To a solution of 5 (5.0 g, 15.3 mmol, 1.0 equiv.) in

methanol (100 mL) and water (100 mL) was added K2CO3


(6.2 g, 45.9 mmol, 3.0 equiv.). The reaction was stirred at 60 uCfor 8 h. Methanol was removed under reduced pressure, and

the residue was dissolved in 50 mL EtOAc, separated from the

aqueous layer, and washed with H2O (1 6 20 mL). The

organic extracts were dried (Na2SO4), filtered, and concen-

trated under reduced pressure. Flash chromatography (10%

EtOAc in hexanes) purified 3.90 g (91%) of 6. Rf = 0.1 (10%

EtOAc in hexanes); [a]D = 211.0 (c = 1.0, CHCl3); 1H NMR

(500 MHz, CDCl3) d 4.97 (s, 1H), 4.72 (s, 1H), 4.02, (dd, 1H,

J = 5.2, 11.1 Hz), 2.44 (m, 1H), 2.06 (m, 1H), 1.97 (m, 1H),

1.55 (m, 1H), 1.17 (s, 3H), 1.16 (s, 3H), 1.19–1.13 (m, 2H), 1.06

(m, 1H), 0.92 (s, 9H), 0.07 (s, 6H). HR-MS (ESI-TOF) Calcd

for C16H32O2Si (M+Na)+ 307.2064, found 307.2068.

7. To a solution of 6 (3.70 g, 13.0 mmol, 1.0 equiv.), DMAP

(1.60 g, 13.0 mmol, 1.0 equiv.), diisopropylcarbodiimide

(6.06 mL, 39.0 mmol, 3.0 equiv.) in toluene (100 mL) was

added 5-hexynoic acid (4.30 mL, 39.0 mmol, 3.0 equiv.). The

reaction was stirred for 3 h at rt, at which time the reaction was

concentrated under reduced pressure, and resuspended in

EtOAc (50 mL). The organic extracts were washed with

aqueous 10% HCl (1 6 20 mL), and saturated aqueous NaCl

(1 6 20 mL). The organic extracts were dried (Na2SO4),

filtered, and concentrated under reduced pressure. The

resulting residue was purified with flash chromatography

(5% EtOAc in hexanes) to afford 4.45 g (90%) of 7. Rf = 0.8

(20% EtOAc in hexanes). [a]D = 217.5 (c = 1.0, CHCl3); 1H

NMR (500 MHz, CDCl3) d 4.98 (s, 1H), 4.72 (s, 1H), 4.01 (dd,

1H, J = 11.4, 4.8 Hz), 2.41 (dt, 1H, J = 12.8, 3.6 Hz), 2.36 (t,

2H, J = 7.35 Hz), 2.24 (td, 2H, J = 7.0, 2.6 Hz), 2.2 (m, 1H),

2.0–1.94 (m, 1H), 1.96 (t, 1H, J = 2.5 Hz), 1.80 (m, 2H), 1.42

(m, 2H), 1.41 (s, 3H), 1.38 (s, 3H), 1.18 (q, 1H, J = 23.4,

11.7 Hz), 1.08 (m, 1H), 0.91 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H).

HR-MS (ESI-TOF) Calcd for C22H38O3Si (M + Na)+

401.2482, found 401.2476.

8. To a solution of 7 (1.3 g, 3.4 mmol, 1.0 equiv.) in THF

(6.8 mL) was added TBAF (6.8 mL, 1 M in THF, 2.0 equiv.).

The reaction was stirred for 2 h at ambient temperature, after

which time EtOAc (20 mL) was added, and the reaction was

washed with H2O (1 6 50 mL) and aqueous saturated NaCl


filtered, and concentrated under reduced pressure. Flash

chromatography (30% EtOAc in hexanes) afforded 0.9 g

(91%) of 8 as a colorless oil. Rf = 0.5 (30% EtOAc in hexanes).

[a]D = 210.5 (c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) d

4.92 (d, 1H, J = 1.4 Hz), 4.77 (d, 1H, J = 1.8 Hz), 4.06 (dd, 1H,

J = 11.4, 4.8 Hz), 2.44 (dt, 1H, J = 12.8, 3.6 Hz), 2.35 (t, 2H,

J = 7.35 Hz), 2.24, (td, 2H, J = 7.0, 2.6 Hz), 2.19 (m, 1H), 2.11

(m, 1H), 2.01 (td, 1H, J = 13.2, 4.0 Hz), 1.97 (t, 1H, J = 2.5 Hz),

1.90 (br, 1H), 1.78 (m, 2H), 1.74 (m, 1H), 1.41 (s, 3H), 1.40 (s,

3H), 1.44–1.36 (m, 1H), 1.10 (m, 1H). HR-MS (ESI-TOF)

Calcd for C16H24O3 (M + Na)+ 287.1618, found 287.1606.

9. To a solution of 8 (0.77 g, 2.90 mmol, 1.0 equiv.) and

DMAP (0.71 g, 5.80 mmol, 2.0 equiv.) in MeCN (3.0 mL) was

added N,N9-disuccinimidyl carbonate (3.7 g, 14.6 mmol,

5.0 equiv.). The reaction stirred at rt for 2 h, and diluted with

EtOAc (10 mL). The solution was washed with aqueous

saturated citric acid (2 6 10 mL) and aqueous saturated NaCl


filtered, and adsorbed onto silica. Flash chromatography (30%

EtOAc in hexanes) afforded 1.1 g (91%) of 9 as a yellow oil.

Rf = 0.5 (50% EtOAc in hexanes); [a]D = 214.0 (c = 1.0,

CHCl3); 1H (500 MHz, CDCl3) d 5.10 (dd, 1H, J = 11.3,

4.4 Hz), 4.91 (s, 1H), 4.85 (s, 1H), 2.82 (s, 4H), 2.49 (dt, 1H, J =

12.8, 3.6 Hz), 2.36 (t, 2H, J = 7.35 Hz), 2.23 (td, 2H, J = 7.0,

2.6 Hz), 2.26 (m, 1H), 2.20 (m, 1H), 2.04 (td, 1H, J = 13.5,

4.0 Hz), 1.97 (t, 1H, J = 2.9 Hz), 1.78 (m, 2H), 1.75 (m, 1H),

1.43 (s, 3H), 1.40 (s, 3H), 1.37 (m, 2H). HR-MS (ESI-TOF)

Calcd for C21H27NO7 (M + Na)+ 428.1680, found 428.1676.

10. To a 5 mL round bottomed flask was added 9 (0.030 g,

0.074 mmol, 1.0 equiv.) and m-CPBA (0.015 g, 0.088 mmol,

1.2 equiv.). The flask was purged of oxygen, and CH2Cl2(0.74 mL) was added via syringe. The reaction was stirred at rt

for 5 h, and was adsorbed onto silica. Flash chromatography

(30% EtOAc in hexanes) afforded 0.021 g (67%) of 10 and

0.0020 g (6%) of 10a. 10: Rf = 0.4 (50% EtOAc in hexanes);

[a]D = 27.5 (c = 1.0, CHCl3); 1H (500 MHz, CDCl3) d 4.99

(dd, 1H, J = 12.0, 7.6), 3.03 (d, 1H, J = 4.1 Hz), 2.82 (s, 4H),

2.68 (d, 1H, J = 4.1 Hz), 2.38 (t, 2H, J = 7.3 Hz), 2.24 (td, 2H,

J = 7.0, 2.6 Hz), 2.26–2.20 (m, 1H), 2.15 (m, 1H), 1.96 (t, 1H,

J = 2.9 Hz), 1.93 (td, 1H, J = 13.5, 4.0 Hz), 1.79 (m, 2H), 1.69

(m, 2H), 1.52–1.40 (m, 2H), 1.46 (s, 3H), 1.44 (s, 3H). HR-MS

(ESI-TOF) Calcd for C21H27NO8 (M + Na)+ 444.1629, found

444.1635. (See below for characterization data for 10a.)

11, 11a. A solution containing, 7 (1.1 g, 3.0 mmol,

1.0 equiv.), NBu4?HSO4 (0.26 g, 0.80 mmol, 0.25 equiv.) and

acetone (6.8 mL, 90 mmol, 30 equiv.) in CH2Cl2 (15 mL) was

prepared and cooled in an ice bath. OXONETM

(5.7 g,

9.1 mmol, 3.0 equiv.) and NaHCO3 (3.4 g, 40 mmol, 13 equiv.)

were weighed in separate vials and each dissolved in H2O

(12.5 mL). The solution containing NaHCO3 was added

dropwise via syringe over 10 min to the solution containing 7.

The solution containing OXONETM

was then added dropwise

via syringe to initiate the formation of dimethydioxirane

in situ,21 and the reaction cocktail was allowed to warm to rt.

After 18 h, the reaction was complete, and CH2Cl2 (15 mL)

was added. The organic layer was separated, washed (1 610 mL H2O), dried (Na2SO4), filtered, and concentrated under

reduced pressure. The residue was purified with flash

chromatography (6% EtOAc in hexanes) to afford 0.82 g

(70%) and 0.26 g (22%) of 11 and 11a, respectively. 11: Rf = 0.5

(25% EtOAc in hexanes); [a]D = 25.5 (c = 1.0, CHCl3); 1H

NMR (400 MHz, CDCl3) d 3.86 (dd, 1H, J = 11.4, 4.4 Hz),

3.04 (d, 1H, J = 5.5 Hz), 2.55 (d, 1H, J = 5.5 Hz), 2.38 (t, 2H,

J = 7.3 Hz), 2.24 (td, 2H, J = 7.0, 2.6 Hz), 2.26–2.20 (m, 1H),

2.15 (m, 1H), 1.96 (t, 1H, J = 2.9 Hz), 1.93 (td, 1H, J = 13.5,

4.0 Hz), 1.79 (m, 2H), 1.69 (m, 2H), 1.52–1.40 (m, 2H), 1.46 (s,

3H), 1.44 (s, 3H) 0.88 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H). HR-

MS (ESI-TOF) Calcd for C22H38O4Si (M + H)+ 395.2612,

found 395.2615. 11a: Rf = 0.55 (25% EtOAc in hexanes); [a]D =

26.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) d 3.77 (m,

1H), 2.95 (m, 1H), 2.41 (d, 1H, J = 5.5 Hz), 2.38 (t, 2H, J =

7.3 Hz), 2.24 (td, 2H, J = 7.0, 2.6 Hz), 2.26–2.20 (m, 1H), 2.15

(m, 1H), 1.96 (t, 1H, J = 2.9 Hz), 1.93 (td, 1H, J = 13.5, 4.0 Hz),


1.79 (m, 2H), 1.69 (m, 2H), 1.52–1.40 (m, 2H), 1.46 (s, 3H),

1.44 (s, 3H) 0.88 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H). HR-MS

(ESI-TOF) Calcd for C22H38O4Si (M + H)+ 395.2612, found

395.2614.

12. To a solution of 11 (0.13 g, 0.33 mmol, 1.0 equiv.) in

THF (3 mL) was added TBAF (0.70 mL, 1 M in THF, 2.1 eq)

via syringe. The reaction was stirred at rt for 1 h, at which time

EtOAc (5 mL) was added. The reaction was washed with H2O

(1 6 10 mL) and aqueous saturated NaCl (1 6 10 mL). The

organic extracts were dried (Na2SO4), filtered, and concen-

trated under reduced pressure. Flash chromatography (40%

EtOAc in hexanes) afforded 0.038 g (42%) of 12 as a colorless

oil. Rf = 0.2 (25% EtOAc in hexanes); [a]D = 215.0 (c = 1.0,

CHCl3); 1H NMR (400 MHz, CDCl3) d 3.78 (m, 1H), 3.12 (d,

1H, J = 4.6 Hz), 2.60 (d, 1H, J = 4.6 Hz), 2.37 (t, 2H, J =

7.3 Hz), 2.24 (td, 2H, J = 7.0, 2.6 Hz), 2.24 (m, 1H), 2.10 (m,

1H), 1.96 (t, 1H, J = 2.9 Hz), 1.93 (td, 1H, J = 13.5, 4.0 Hz),

1.79 (m, 2H), 1.78–1.69 (m, 2H), 1.64 (m, 1H), 1.52–1.47 (m,

1H), 1.46 (s, 3H), 1.44 (s, 3H), 1.38 (m, 1H). HR-MS (ESI-

TOF) Calcd for C16H24O4 (M + Na)+ 303.1567, found

303.1569.

12a. Compound 11a (0.046 g, 0.12 mmol, 1.0 equiv.) in THF

(1.5 mL) was treated with TBAF (0.2 mL, 1M in THF,

2.1 equiv.) and compound 12a (0.024 g, 73%) was isolated as a

colorless oil using the protocol outlined for compound 12. Rf =

0.25 (25% EtOAc in hexanes); [a]D = 211.0 (c = 1.0, CHCl3);1H NMR (400 MHz, CDCl3) d 3.81 (m, 1H), 3.07 (m, 1H),

2.55 (d, 1H, J = 4.6 Hz), 2.37 (t, 2H, J = 7.3 Hz), 2.26 (td, 2H,

J = 7.0, 2.6 Hz), 2.20 (m, 1H), 2.17 (m, 1H), 2.04 (br, 1H), 1.97

(t, 1H, J = 2.9 Hz), 1.88 (m, 2H), 1.80 (m, 2H), 1.61 (br, 1H),

1.45–1.41 (m, 2H), 1.46 (s, 3H), 1.44 (s, 3H) 1.28 (m, 1H), 1.24

(m, 1H). HR-MS (ESI-TOF) Calcd for C16H24O4 (M + Na)+

303.1567, found 303.1568.

10a. To a solution of 12a (0.076 g, 0.27 mmol, 1.0 equiv.)

and DMAP (0.066 g, 0.54 mmol, 2.0 equiv.) in MeCN (1.0 mL)

was added N,N9-disuccinimidyl carbonate (0.34 g, 1.4 mmol,

5.0 equiv.). The reaction was stirred at rt for 4 h, and diluted

with EtOAc (10 mL). The solution was washed with aqueous

saturated citric acid (2 6 10 mL) and aqueous saturated NaCl


filtered, and adsorbed onto silica. Flash chromatography (30%

EtOAc in hexanes) afforded 0.079 g (69%) of 10a as a colorless

oil. Rf = 0.2 (25% EtOAc in hexanes); [a]D = 21.8 (c = 1.0,

CHCl3); 1H (500 MHz, CDCl3) d 4.97 (m, 1H), 3.04 (m, 1H),

2.81 (s, 4H), 2.57 (d, 1H, J = 4.6 Hz), 2.37 (t, 2H, J = 7.3 Hz),

2.32 (m, 1H), 2.25 (td, 2H, J = 7.0, 2.6 Hz), 2.16 (m, 1H), 2.17

(m, 1H), 2.04 (m, 1H), 1.98 (t, 1H, J = 2.9 Hz), 1.90 (m, 1H),

1.80 (m, 2H), 1.64 (br, 1H), 1.45–1.41 (m, 2H), 1.46 (s, 3H),

1.44 (s, 3H) 1.33 (m, 1H), 1.26 (m, 1H). HR-MS (ESI-TOF)

Calcd for C21H27NO8 (M + Na)+ 444.1629, found 444.1634.

MJE3. To a stirring solution of 10 (0.0040 g, 0.0095 mmol,

1.0 equiv.) in MeCN (0.45 mL) was added L-tryptophan benzyl

ester (0.0030 g, 0.0095 mmol, 1.0 equiv.) and MP-

CarbonateTM polystyrene resin (0.0036 g, 0.010 mmol,

1.1 equiv.). The reaction was stirred at rt, and reaction

progress was monitored by TLC. After 30 min, the reaction

was complete, and the solution was removed from the reaction

vessel with a syringe, and transferred to a fresh vial. The

solution was concentrated under a stream of nitrogen. The

resulting residue was purified with flash chromatography (40%

EtOAc in hexanes) to afford 0.0040 g of MJE3 (70%). Rf =

0.65 (50% EtOAc in hexanes). 1H NMR (500 MHz, CDCl3) d

8.01 (br, 1H), 7.52 (d, 1H, J = 8.2 Hz), 7.34 (m, 1H), 7.33 (m,

3H), 7.22 (m, 2H), 7.18 (m, 1H), 7.10 (m, 1H), 6.79 (d, 1H, J =

2.0 Hz), 5.34 (d, 1H, J = 8.2 Hz), 5.09 (s, 2H), 4.99 (dd, 1H,

J = 12.0, 7.3 Hz), 4.73 (m, 1H), 3.28 (m, 2H), 2.64 (d, 1H, J =

4.4 Hz), 2.52 (d, 1H, J = 4.4 Hz), 2.35 (t, 2H, J = 7.6 Hz), 2.24

(td, 2H, J = 7.0, 2.6 Hz), 2.12 (td, 1H, J = 13.5, 4.0 Hz), 1.96 (t,

1H, J = 2.6 Hz), 1.99 (m, 1H), 1.94 (m, 1H), 1.80 (m, 2H), 1.71

(m, 1H), 1.61 (br, 1H), 1.54–1.40 (m, 2H), 1.46 (s, 3H), 1.45 (s,

3H). HR-MS (ESI-TOF) Calcd for C35H40N2O7 (M + Na)+

623.2728, found 623.2725.

Synthesis of other probe library members followed a similar

protocol; more details can be found in the ESI.{

In situ proteome reactivity profiling. MDA-MB-231 and

MUM2B cells were counted and seeded at 2.25 6 106 cells per

10 cm2 dish under the conditions previously reported.9 After

10 h, media was exchanged, and 20 mM of probe was added

directly to 5 mL medium in the plate. After incubating for 0,

15, 30, 90, and 120 min, the medium was aspirated, and the

cells were washed 3 times in PBS and harvested by scraping.

Soluble proteome extracts were prepared by homogenization

of cell pellets in PBS and centrifugation at 100 000g (super-

natant) for 1 h and were normalized for protein concentration

(Dc protein assay kit; Bio-Rad). Detection of probe-labeled

proteins was carried out using previously described click

chemistry methods. Labeled proteins were separated by 1D

SDS-PAGE (13 mg protein per gel lane) and visualized using a

Hitachi FmBIO IIe flatbed laser-induced fluorescence scanner

(MiraiBio, Alameda, CA). Integrated band intensities were

calculated for PGAM1 for probe treated proteomes (n = 3),

and reported as average values (¡SEM) with the PRISM

software (GraphPad, San Diego). Equivalent levels of PGAM1

expression was assessed by Western blot analysis with a

polyclonal antibody to PGAM1 (Abcam, Cambridge, MA).

Antiproliferative studies in human cancer cell lines. Human

cancer cell lines (MDA-MB-231, MUM2B) were plated in

50 mL media (10 000 cells per well) and treated with 50 mL of

media containing 50 mM probe for 12 h (1% final DMSO

concentration). Proliferation was measured with the XTT

colorimetric assay (Roche) following manufacturer’s guide-

lines (excitation 450 nm; reference, 650 nm). Reported values

were derived from duplicate treatments from two independent

trials (n = 4).

Mass spectrometry analysis of the site of MJE3 modification

on PGAM1. COS-7 cells were transiently transfected with a

pcDNA3.1-myc/His vector containing the PGAM1 cDNA.9

Transfected cells were treated in situ with MJE3 (50 mM) for

1 h, isolated by scraping, homogenized in PBS, and centrifuged

at 100 000g to provide a soluble proteomic extract (super-

natant; 1.3 mg protein mL21). The soluble proteomic fraction


was passed over a PD-10 size exclusion column (GE

Healthcare) and the eluent reacted under click chemistry

conditions with a biotin–TEV–azide tag.26 The click chemistry

and avidin purification protocols were carried out as

described.11 The avidin-enriched sample was subjected to on-

bead trypsin digestion and the resulting peptide mixture

analyzed by LC/LS-MS/MS as described26 using a quaternary

Agilent 1100 series HPLC directly coupled to an LTQ ion trap

mass spectrometer equipped with a nano-LC electrospray

ionization source (ThermoElectron). A second proteolysis was

then performed using the TEV protease to release probe-

labeled peptides form the beads, which were analyzed by LC-

MS/MS as described.26

Using the Sequest algorithm, tandem mass spectra from the

whole protein trypsin digest was searched against a human.

fasta database from the European Bioinformatics Institute

(EBI, www.ebi.ac.uk) as described.26 PGAM1 was identified

with y74% sequence coverage, with one conspicuous region

being absent – aa 91–113 (HYGGLTGLNKAETAAKH-

GEAQVK). A manual search of the TEV MS1 data set for

candidate probe-modified peptides within this region identified

masses corresponding to the +2 through +5 charge states for

the free acid form of the MJE3 probe adducted to aa 91–106

(HYGGLTGLNKAETAAK). The MS2 data from the

+3 charge state ion (m/z 809.8) and MS3 data from a major

fragment ion of m/z 809.8 (m/z 1015.2) were manually analyzed

to identify K100 as the site of MJE3 modification on the aa

91–106 peptide.

Generation of point mutants to confirm the site of labeling.

The K100A point mutant was generated using the full length

PGAM1 construct subcloned into pcDNA3.1 myc/His B9 with

the Quikchange PCR kit (conditions: 30 s at 94 uC, 1 min at

50 uC, 7 min at 68 uC; 40 cycles). The primers used were

59-CAC TAT GGG GGT CTA ACC GGT CTC AAT GCC

GCA GAA ACT GCT GCA AAG CAT GGT GAG GCC-39

(forward), and 59-GGT CTC ACC ATG CTT TGC AGC

AGT TTC TGA GGC ATT GAG ACC GGT TAG ACC

CCC ATA GTG-39 (reverse). The wild type, K100A, and

mock constructs were transiently transfected into COS-7 cells

using Lipofectamine as described previously,1 and labeled with

50 mM MJE3 in situ for 2 h. Cells were harvested, and labeled

proteins were visualized as described above. Equivalent levels

of wild-type and mutant PGAM1 was verified by Western blot

analysis with a polyclonal antibody recognizing the C-terminal

myc tag on protein (Invitrogen).

Molecular modeling of the MJE3-PGAM1 interaction. The

adduct of MJE3 with the f-nitrogen and the e-carbon atoms of

the side chain of K100 was built using Ghemical GMS 2.10

(University of Iowa, http://www.uiowa.edu/yghemical).

Energy minimization was performed until the DE termination

criterion was reached. This was followed by a molecular

dynamics simulation, consisting of 5000 steps of heating,

5000 steps of equilibration, 180 000 steps of simulation and

2000 steps of cooling, using a time step of 0.5 fs, at a

temperature of 300 K. Every 25 000th step from the simulation

stage was selected in turn and subjected to energy minimiza-

tion until the DE termination criterion was reached. The lowest

energy conformation was found after energy minimizing the

conformation from the 100 000th step, and this was

the structure used for subsequent dockings. It was notable

that the cyclohexane ring adopted a very stable twist–boat

conformation for most of the simulation, with all three bulky

substituents adopting equatorial positions.

The MJE3-adduct was set up for covalent docking using

AutoDockTools (ADT) version 1.4.4 (http://autodock.scripp-

s.edu/resources/adt), the graphical user interface to

AutoDock;29 13 rotatable bonds were defined. The e-carbon

atom in the adduct was changed to a covalently-binding atom

type. The structure of human B type phosphoglycerate mutase

from Protein Data Bank entry 1YFK{ was modified by

deleting the f-nitrogen and the e-carbon from the side chain of

K100. The citrate and all water molecules were also deleted.

Atomic affinity maps, an electrostatic potential map and a

covalent affinity map were computed using AutoGrid.29 The

covalent affinity map was calculated using a spherically-

symmetric inverted Gaussian-shaped potential energy with a

half-width of 5.0 A and an energy penalty of 1000.0 kcal

mol21, with the minimum energy value centred on the

coordinates of the e-carbon atom (i.e. 21.766, 23.232,

53.765). In subsequent dockings, the covalently-binding atom

type in the adduct seeks out this position, effectively binding

the ligand to the receptor.

AutoDock 329 was used to perform 100 independent

dockings, each one starting from random initial positions.

The Lamarckian Genetic Algorithm was used with a popula-

tion size of 150, and a maximum number of energy evaluations

of 25 million, while default values were accepted for all the

other parameters. To investigate the effect of sampling on the

results, a second set of 100 independent dockings was

performed, this time using a maximum number of 100 million

energy evaluations. The results of the 200 dockings were

reclustered using ADT, using an RMSD tolerance of 3.0 A.

Acknowledgements

We thank Alan Saghatelian for assistance with the develop-

ment of synthetic routes for the spiroepoxide probe library and

the Cravatt group for helpful discussions. This work was

supported by the National Institutes of Health grant

CA087660 (to B.F.C.), the California Breast Cancer

Research Foundation (B.F.C.), and the Skaggs Institute for

Chemical Biology.

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mechanistic and structural requirements for active site ...after tbdms deprotection to a previously...

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