Phosphinic acid-based inhibitors of tubulin polyglutamylases

Article abstract of DOI:10.1016/j.bmcl.2013.05.069

Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglutamylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the function of microtubule associated proteins, neuronal development, and metastatic progression. This study describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against the polyglutamylase TTLL7 using tail peptides as substrates (100 μM) and the most potent inhibitor displayed an IC₅₀ value of 150 μM. The incorporation of these compounds into tubulin C-terminal tail peptides may lead to more potent TTLL inhibitors.

Full text of DOI:10.1016/j.bmcl.2013.05.069

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4408–4412
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phosphinic acid-based inhibitors of tubulin polyglutamylases
a,
Yanjie Liu a, Christopher P. Garnham b, Antonina Roll-Mecak b, , Martin E. Tanner
⇑
⇑
a Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
b Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke and National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglut-
amylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the func-
tion of microtubule associated proteins, neuronal development, and metastatic progression. This study
describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed
to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against
Received 26 March 2013
Revised 16 May 2013
Accepted 20 May 2013
Available online 30 May 2013
the polyglutamylase TTLL7 using tail peptides as substrates (100
played an IC50 value of 150 M. The incorporation of these compounds into tubulin C-terminal tail pep-
tides may lead to more potent TTLL inhibitors.
lM) and the most potent inhibitor dis-
Keywords:
Inhibitor
Tubulin
Post-translational modification
Ligase
l
Ó 2013 Elsevier Ltd. All rights reserved.
Phosphinic acid
The cytoskeleton of the eukaryotic cell plays key roles in deter-
mining cellular organization, shape, motility, and life cycle and is
therefore absolutely essential for cellular survival. The major struc-
tural components of the cytoskeleton are microtubules, which are
ATP-dependent amino acid ligases that are members of the
‘tubulin–tyrosine ligase-like’ (TTLL) family of enzymes.6 These en-
zymes belong to the ATP-grasp family of ligases that include the
prototypical member D-alanine–D-alanine ligase as well as tubu-
hollow cylindrical polymers formed by the self-assembly of
a- and
lin–tyrosine ligase (TTL).12,13 Of the 13 known TTLL enzymes in
the human genome, ten have been implicated as glutamylases.2
In vitro studies using recombinant enzyme have only been per-
formed on one of these, TTLL7, and it has been reported that this
enzyme is capable of catalyzing both initiation and elongation.14
As mentioned previously, this PTM is reversible and the enzymes
that remove the glutamate residues from tubulin have recently
been identified as members of the soluble cytosolic carboxypepti-
dase (CCP) family.15,16 Four CCP members have been implicated as
tubulin deglutamylases; however, in vitro activity has not yet been
demonstrated for most of them.
b-tubulin monomers.1 In the assembled microtubule, the C-ter-
mini, or tails, of the tubulin subunits are projected outward into
solution and contribute to defining the surface of the filament.
These tails are subject to reversible post-translational modifica-
tions (PTM’s) such as detyrosination, polyglutamylation, and poly-
glycylation.2,3 It is becoming increasingly clear that these
modifications affect both microtubule dynamics and interactions
with microtubule associated proteins (MAPS) in cells, and there-
fore serve as control elements in a variety of biological processes.
Tubulin polyglutamylation occurs at the C-termini of both
a
- and b-tubulin.4–7 This typically involves the addition of one to
Polyglutamylation has been shown to regulate the activity of
the microtubule associated molecular motors kinesin and dy-
nein.3,17,18 Not surprisingly, polyglutamylating enzymes are crucial
for normal neuronal development.19,5 Tubulin polyglutamylation
has also been implicated in positively regulating the activity of
the microtubule severing enzyme spastin,20 a protein that is mu-
tated in more than 40% of patients diagnosed with hereditary spas-
tic paraplegias.21 Loss of spastin function has been implicated in
defects in mitosis,22 late stage cytokinesis events,23 as well as den-
dritic arborization.24 Moreover, it has been found that prostate and
pancreatic cancer cells display higher levels of polyglutamylation
than normal cells.25,26 In particular, a recent study showed that
TTLL4 is highly expressed in pancreatic cancer cells and knock-
down of TTLL4 attenuated their growth,25 supporting the idea of
using TTLL enzymes as therapeutic targets for small molecule
six additional glutamate residues, and the overall extent of tubulin
polyglutamylation increases during development.8–11 The first glu-
tamate is added to the side chain of a main chain glutamate to form
an isopeptide bond in a process called initiation (Fig. 1). Subse-
quent glutamate residues could conceivably be added to either
the
a
-carboxylate or the
c
-carboxylate in elongation steps. HPLC
-elonga-
analyses using synthetic peptides have indicated that
a
tions predominantly occur during human brain tubulin polyglut-
amylation.8,10,11 These PTM’s are catalyzed by
a series of
⇑
Corresponding authors. Tel.: +1 301 402 7353; fax: +1 301 435 5666 (A.R.-M.);
tel.: +1 604 822 9453; fax: +1 604 822 2847 (M.E.T.).
E-mail addresses: Antonina@mail.nih.gov (A. Roll-Mecak), mtanner@chem.ubc.
ca (M.E. Tanner).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.069

Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4408–4412
4409
Figure 1. The initiation and elongation steps of tubulin polyglutamylation catalyzed by the TTLL enzymes.
A
inhibitors. Furthermore, hyperglutamylation has been linked to
neurodegeneration in mouse models and inhibition of the TTLL1
polyglutamylase reversed this neurodegenerative phenotype.15
Thus, potent inhibitors of the tubulin polyglutamylation cycle
could play key roles in understanding the structure and function
of these enzymes and could serve as lead compounds in the devel-
opment of therapies based on interfering with tubulin PTM levels.
Phosphinic acids are known to serve as effective inhibitors of
both ATP-dependent ligases and carboxypeptidases.27–38 The tetra-
hedral geometry and negative charge serves as an excellent mimic
of the tetrahedral intermediate formed in the ligase reaction (
Fig. 2). In some instances, the bound phosphinic acid may be phos-
phorylated to generate a tightly bound phosphorylated inhibi-
tor.13,33–35 The potency of these inhibitors is often quite
impressive and KI values in the low nanomolar range have been re-
ported for both the ligases and the carboxypeptidases.27,28,32,36,37
In order to target the tubulin glutamylation enzymes, three phos-
phinic acid inhibitors were designed (Fig. 3). Inhibitor 1 is expected
to mimic the tetrahedral intermediate formed in the initiation step
and inhibitors 2 and 3 are expected to mimic the intermediates
B
formed in the a- and c-elongation steps, respectively. These com-
pounds are expected to serve as inhibitors of the corresponding
deglutamylation enzymes as well. In this study we describe the
synthesis of inhibitors 1–3 and their inhibition of the TTLL7 reac-
tion using a tubulin tail peptide as substrate.
Inhibitors 1 and 3 resemble compounds previously prepared for
the inhibition of folylpoly-c-glutamate handling-enzymes and
were synthesized in an analogous fashion.39,27 The synthesis of
inhibitor 1 began with the treatment of the known alkene (S)-4
with ammonium hypophosphite and triethylborane to give the
mono-alkylphosphinic acid (Scheme 1).39,40 This acid was silylated
and added to dimethyl 2-methylenepentanedioate to generate the
di-alkylphosphinic acid.41,42 This was immediately methylated to
give compound 5 as a mixture of four diastereomers. The Cbz
and benzyl groups were removed by hydrogenolysis and the
resulting amino acid was acetylated and coupled to ethylamine
to give compound 6. Demethylation was performed with NaOH
to give inhibitor 1 as a mixture of two diastereomers. The
Figure 2. (A) The mechanism employed by the ATP-dependent amino acid ligases.
(B) The structure of phosphinic acid-based inhibitors and the potential phosphor-
ylation of the inhibitor within the ligase active site.
precursor to inhibitor 3, compound 7, was also prepared by the
hydrogenolysis of compound 5 followed by acetylation and meth-
ylation. Global demethylation using NaOH produced inhibitor 3 as
a mixture of two diastereomers. In order to prepare inhibitor 2, a
strategy reported by Georgiadis et al. was employed in which a
phenyl group is used as a carboxyl synthon (Scheme 2).41 The

4410
Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4408–4412
Figure 3. The structure of the potential intermediates involved in tubulin polyglutamylation and the corresponding inhibitors targeted against them.
Scheme 1.
known compound 8 was silylated and added to dimethyl 2-meth-
ylenepentanedioate to generate the di-alkylphosphinic acid. This
was immediately methylated to give compound 9 as a mixture of
eight diastereomers. Oxidation of the phenyl ring was performed
using RuCl3 and NaIO4 to give the carboxylic acid 10 which was
methylated to give compound 11. The Boc group was removed
with TFA and the free amine was acetylated with acetic anhydride
to give compound 12. Demethylation of the methyl esters by treat-
ment with NaOH gave inhibitor 2 as a mixture of four diastereo-
mers. With all three inhibitors, no attempts were made to
separate the diastereomers and the mixtures were used directly
in inhibition studies.
The activity of the enzyme was monitored using a synthetic N-
acetylated
bIVb-tubulin
C-terminal
peptide
(Ac-DATA-
EEEGEFEEEAEEEVA) as substrate (see Supplementary data for de-
tails). The peptide (100 lM) was incubated with recombinant
mouse TTLL7 in the presence of glutamate, ATP, Mg2+, and varying
amounts of inhibitors 1–3. Control reactions lacked ATP and gluta-
mate. Under these conditions, the predominant reaction product
consisted of mono-glutamylated peptide with small amounts

Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4408–4412
4411
Scheme 2.
(<20%) of di-glutamylated peptide (see Supplementary data
Fig. S1). The extent of reaction was determined using ESI TOF
LC–MS by comparing the decrease in intensity of the signal of
the unmodified peptide to that of the corresponding control reac-
tion. The most potent inhibition was observed with compound 2
150
l
M, whereas inhibitors 1 and 3 were only active in the milli-
-elongation
molar range. Inhibitor 2 was designed to mimic an
step of polyglutamylation. While TTLL7 has been reported to be
capable of catalyzing both initiation and elongation, it is not yet
known whether the elongation steps involve
It is likely that the stronger binding of inhibitor 2 reflects the abil-
ity of TTLL7 to catalyze -elongations. This is consistent with re-
ports that -elongation linkages have been observed in tubulin
a
a- or c
-additions.14
which showed an IC50 value of 150 lM (Fig. 4). Compounds 1
and 3 were weaker inhibitors and only showed inhibition at mM
concentrations. All three compounds were also tested for their
ability to inhibit the reaction catalyzed by the related enzyme,
tubulin–tyrosine ligase (TTL) but failed to show any inhibition at
mM concentrations.12 This indicates that the inhibition by inhibi-
tor 2 is specific for the glutamylase TTLL7 and does not interfere
with the activity of the closely related family member tubulin–
tyrosine ligase.
In summary, we have prepared a series of phosphinic acid pseu-
do-dipeptides designed to inhibit the enzymes of tubulin polyglut-
amylation/deglutamylation. Of these compounds, inhibitor 2 was
shown to inhibit the activity of mouse TTLL7 with an IC50 of
a
a
isolated from mouse brain.8,10,11 Alternatively, inhibitor 2 may
simply be acting as an initiation inhibitor and the additional nega-
tive charge (as compared to the designed initiation inhibitor 1) fa-
vors binding to an electropositive peptide-binding groove in the
enzyme (vide infra). The modest IC50 value observed with these
inhibitors likely reflects the fact that the true substrate is an intact
tubulin protein within a microtubule, as opposed to a dipeptide.9 It
is clear from this work, and that of others, that peptides containing
15 or more residues corresponding to the C-terminus of tubulin
can act as substrates for some polyglutamylases including TTLL7;
however, the minimal substrate length is not known for each iso-
zyme.43 In the case of the related enzyme tubulin–tyrosine ligase
(TTL), studies on substrate specificity with C-terminal peptides
have been performed.44 This ligase adds a single tyrosine to the
C-terminus of de-tyrosinated a-tubulin and it has been shown that
the activity drops off dramatically with peptides containing less
than 10 residues. An inspection of the structure of TTL explains this
phenomenon as an elongated stripe of conserved positively
charged residues connects the active site to the N-terminal do-
main.12 These residues interact with the highly negatively charged
tubulin tail and contribute significantly to peptide substrate bind-
ing. This is also thought to be the case with the TTLL enzymes and
can help to explain why dipeptide-based inhibitors are not able to
access much of the binding affinity. Similar observations have been
made with phosphinic acid inhibitors that target the amino acid li-
gases of peptidoglycan biosynthesis.28,31 The
ing enzyme MurD adds a -glutamate residue onto the alanine of
UDP-N-acetylmuramic acid- -Ala (UDP-MurNAc- -Ala). When the
phosphinic acid dipeptide corresponding to -Ala- -Glu was tested
as an inhibitor, an IC50 of >1 mM was observed. However, when the
D-glutamic acid-add-
D
L
L
Figure 4. A plot of normalized activity versus the concentration of inhibitor 2 (log
L
D
plot) for the inhibition of tubulin C-terminal peptide (100
TTLL7.
lM) glutamylation by

4412
Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4408–4412
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l
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of Canada (NSERC) (M.E.T.) and the intramural program of the
National Institutes of Health (NIH) (A.R.-M.).
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Supplementary data
Supplementary data (experimental and spectral details) associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2013.05.069.
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