Design, syntheses, and evaluation of Taspase1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.045

Taspase1 is a threonine protease responsible for cleaving MLL (Mixed-Lineage Leukemia) to achieve proper HOX gene expression. Subsequent studies identified additional Taspase1 substrates including Transcription Factor IIA (TFIIA) and Drosophila HCF. Taspa

Full text of DOI:10.1016/j.bmcl.2009.07.045

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5086–5090
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, syntheses, and evaluation of Taspase1 inhibitors
Jeong Tae Lee a, , David Y. Chen b, , Zhimou Yang a, , Alexander D. Ramos b,
,
a,c,
James J.-D. Hsieh b, , Matthew Bogyo
*
*
a Department of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA
b Molecular Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
c Department of Microbiology and Immunology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Taspase1 is a threonine protease responsible for cleaving MLL (Mixed-Lineage Leukemia) to achieve
proper HOX gene expression. Subsequent studies identified additional Taspase1 substrates including
Transcription Factor IIA (TFIIA) and Drosophila HCF. Taspase1 is essential for cell proliferation and is
overexpressed in many cancer cell lines. Currently no small molecule inhibitors of this enzyme have been
described. Here, we report the synthesis and evaluation of vinyl sulfone, vinyl ketone, epoxy ketone, and
boronic acid inhibitors designed based on the preferred Taspase1 cleavage site (Ac-Ile-Ser-Gln-Leu-Asp).
Specifically, we evaluated compounds in which the reactive warhead is positioned in place of the P1
aspartic acid side chain as well as at the C-terminus of the peptide. Interestingly, both classes of inhibitors
were effective and vinyl ketones and vinyl sulfones showed the greatest potency for the target protease.
These results suggest that Taspase1 has unique substrate recognition properties that could potentially be
exploited in the design of potent and selective inhibitors of this enzyme.
Received 30 April 2009
Revised 20 June 2009
Accepted 2 July 2009
Available online 10 July 2009
Keywords:
Taspase1
Protease inhibitor
Peptide vinyl sulfone
Epoxyketone
Boronate
Vinyl ketone
Ó 2009 Elsevier Ltd. All rights reserved.
Taspase1 is a highly conserved threonine protease that was ini-
tially purified based on its ability to cleave the MLL (mixed-lineage
leukemia) protein at conserved (QXD/G) sites.1 MLL encodes a
500kD nuclear coactivator that regulates embryogenesis, cell cycle
and stem cell growth.2 Deregulation of MLL by chromosome band
11q23 translocation leads to human leukemia with poor prognosis.
Key MLL targets include Hox and Cyclin genes.3 Proteolysis of MLL
leads to the formation of a stable heterodimer that localizes to the
nucleus where it acts as a histone H3 K4 methyl transferase (HMT).
Noncleavage of MLL results in a hypomorphic MLL with impairment
in its HMT activity.4 Taspase1 is the only protease in mammals capa-
ble of proteolytically activating MLL, as demonstrated by the inabil-
ity of Taspase1-deficient mice to cleave MLL resulting in homeotic
transformations.4 In addition to MLL, we have identified MLL2, TFIIA,
and Drosophila HCF as bona fide Taspase1 substrates.5 Taspase1 reg-
ulates cell cycle gene expression through cleavage-mediated sub-
strate activation and has been shown to be essential for cell
proliferation.4 Furthermore, Taspase1 is overexpressed in many can-
cer cell lines, and Taspase1-deficient cells are resistant to common
oncogenictransformation.4 Given thesefindings, chemically inhibit-
ing Taspase1 function may lead to anticancer therapeutics. How-
ever, Taspase1 has proven resistant to inhibition by general classes
of serine, cysteine and metallo protease inhibitors.1b
The activity of Taspase1 itself is regulated by proteolysis. It is ex-
pressed as a proenzyme that undergoes autoproteolysis to its active
form.1b The crystal structure of human Taspase1 revealed significant
conformational differences between the proenzyme and the active
conformer. The proenzyme starts as a homodimer that is hydrolyzed
into a 28 kDa
ramericactiveform of Taspase1.6 Interestingly, Taspase1onlyshows
homology to the -asparaginase_2 family of hydrolyases. However,
a and a 22 kDa b subunit that produce the hetero-tet-
L
unlike other members of this family, it has endopeptidase activity.
Taspase1 uses a threonine residue as its active site nucleophile to
cleave peptide bonds C-terminal to an aspartate residue.1b In addi-
tion, Taspase1 requires a glycine residue directly C-terminal to the
aspartate residue. Two Taspase1 cleavage sites have been identified
on MLL(CS1 andCS2). Theconservedsequencefor CS2is Ile-Ser-Gln-
Leu-Asp/Gly-Val-Asp-Asp, and CS1 is Glu-Gly-Gln-Val-Asp/Gly-Ala-
Asp-Asp, with the CS2 site being more optimal for cleavage.1b The
fact that Taspase1 has homology to asparaginases, enzymes that
hydrolyze the amide side chain of asparagine to generate aspartic
acid, suggests that it may also favor cleavage of isopeptide bonds
on a substrate. Furthermore, the requirement of a glycine at the P10
positionmaybe explainedbytheneedfor a smallresiduetofacilitate
peptide bond transfer from the main peptide backbone amide to the
aspartic acid side chain of a substrate. A possible substrate
rearrangement to produce two isoforms for cleavage by Taspase1
is illustrated (Fig. 1).
* Corresponding authors.
E-mail address: mbogyo@stanford.edu (M. Bogyo).
These authors contributed equally to this Letter.
In this Letter, we describe the design, synthesis, and evaluation
of Taspase1 inhibitors that contain a general scaffold based on the
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.045

J. T. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5086–5090
5087
native cleavage site of MLL linked to a number of different prote-
ase-specific reactive functional groups. These inhibitors were de-
signed to determine both the optimal ‘warhead’ group as well to
determine if placement of this group at the Asp side chain en-
hanced reactivity (Fig. 2). We chose vinyl sulfones, expoxy ketones
and boronates because all of these functional groups have proven
to be efficient for inhibition of the catalytic threonine of the protea-
some.7 Initially we synthesized a vinyl sulfone (yzm16), vinyl ke-
tone (yzm19), epoxy ketone (yzm38) and boronic acid (yzm49)
at the side chain of the P1 aspartic acid. For the vinyl sulfone war-
head, we also synthesized a compound in which the reactive group
was placed in the main peptide backbone, as has been described
for standard protease inhibitors (yzm18).8 As control compounds,
we replaced aspartic acid with norleucine to generate Ac-Ile-Ser-
Gln-Leu-Nle-OH (yzm22) and Ac-Gln-Leu-Nle-OH (yzm23).
Schemes 1 and 2 outline the synthesis of yzm16 (Ac-Ile-Ser-
Gln-Leu-Asp(vs)-OH) and yzm18 (Ac-Ile-Ser-Gln-Leu-Asp-vs) from
Fmoc-Asp-OtBu and Fmoc-Asp(tBu)-OH respectively. The acids
were converted to Weinreb amides 2 and 7 by N,O-dimethylhydr-
oxyl-amine and then reduced to aldehydes 3 and 8 by DIBAL. Com-
pounds
3
and
8
were
coupled
with
diethyl
methylsulfonylmethylphophonate9 to produce the vinyl sulfone
compounds 4 and 9. After deprotection of the Fmoc group in a mix-
ture of diethylamine and acetonitrile, the resulting amines were
coupled with the protected peptide (Ac-Ile-Ser(tBu)-Gln(Trt)-Leu-
OH) using standard peptide coupling conditions. Finally, the pro-
tecting groups were removed in TFA/TIS/H2O mixture to yield the
desired products yzm16 and yzm18 that were further purified by
HPLC.
The vinyl ketone compound yzm19 was synthesized as shown
in Scheme 3. Homoserine was coupled with Ac-Ile-Ser(tBu)-
Gln(Trt)-Leu-OH to produce 12. The alcohol group was converted
to the aldehyde by Dess–Martin Periodinane and then reacted with
Figure 1. A potential peptide rearrangement to yield two substrate isoforms for
cleavage by Taspase1. The presence of an Asp-Gly sequence may facilitate internal
transfer of the peptide bond to the side chain of Asp. This would result in a substrate
that resembles asparagine and that would require hydrolysis at the side chain
amide, similar to how asparaginases function.
Figure 2. Taspase1 Inhibitors (yzm16, 18, 19, 38, 49) and control compounds (yzm22, 23) used in this study.

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J. T. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5086–5090
Scheme 1. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, HBTU, DIPEA, DMF, rt; (b) DIBAL, THF, ꢀ10 °C; (c) diethyl methylsulfonylmethylphosph-
onate, LHMDS, THF, ꢀ10 °C; (d) diethylamine, acetonitrile, rt; (e) Ac-IS(tBu)Q(Trt)L-OH, HBTU, HOBt, DIPEA, DMF, rt; (f) TFA/TIS/H2O = 95/2.5/2.5.
Scheme 2. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, HBTU, DIPEA, DMF, rt; (b) DIBAL, THF, ꢀ10 °C; (c) diethyl methylsulfonylmethylphosph-
onate, LHMDS, THF, ꢀ10 °C; (d) diethylamine, acetonitrile, rt; (e) Ac-IS(tBu)Q(Trt)L-OH, HBTU, HOBt, DIPEA, DMF, rt; (f) TFA/TIS/H2O = 95/2.5/2.5.
Scheme 3. Reagents and conditions: (a) NHS, DIC, DCM, rt; (b) homoserine, NaHCO3, acetone/H2O, rt; (c) Dess–Martin Periodinane, DCM, rt; (d) 1-triphenylphosphos-
phoranylidene-2-propanone, DCM/ EtOAc, rt; (e) TFA/TIS/H2O = 95/2.5/2.5.
1-triphenyl-phosphoranylidene-2-propanone by Wittig reaction to
generate vinyl ketone compound 13. After deprotection of the
protecting groups, HPLC purification afforded the desired com-
pound yzm19.
Scheme 4 shows the synthesis of epoxy ketone compound
yzm38. It shares common intermediate with yzm16.
Compound 14 was generated by Grignard reaction with 3 and isop-
a
3
ropenylmagnesium bromide. The double bond was converted to an

J. T. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5086–5090
5089
Scheme 4. Reagents and conditions: (a) isopropenylmagnesium bromide, THF, ꢀ10 °C; (b) vanadyl acetylacetonate, t-butyl hydroperoxide, DCM, 0 °C; (c) Dess–Martin
Periodinane, DCM, rt; (d) diethylamine, acetonitrile, rt; (e) HBTU, HOBt, DIPEA, DMF, rt; (f) TFA/TIS/H2O = 95/2.5/2.5.
Scheme 5. Reagents and conditions: (a) LHMDS, THF, ꢀ78 °C; (b) 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, rt; (c) 0.2 N HCl; (d) (+)-pinanediol; (e) Fmoc-OSu,
acetone, rt; (f) TFA/DCM = 2/5; (g) SPPS; (h) TFA/DCM = 1/99; (i) TFA/TIS/H2O = 95/2.5/2.5.
epoxide by t-butyl hydroperoxide to produce 15. The racemic
alcohol group of 15 was transformed to ketone compound 16 by
Dess–Martin Periodinane. The Fmoc deprotected compound 17
was coupled with Ac-Ile-Ser(tBu)-Gln(Trt)-Leu-OH. The compound
was subsequently deprotected and HPLC purified to yield yzm38.
The boronic acid compound yzm49 was synthesized as shown
in Scheme 5. First, commercially available N-(diphenylmethyl-
ene)glycine t-butyl ester 18 was treated with LHMDS and then
1 h later addition of 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane10 to this mixture led to compound 19. The deprotec-
tion of diphenylmethylene group gave rise to the deprotection of
the boronic acid group. Without the protection of the boronic acid,
the coupling reaction with Ac-Ile-Ser(tBu)-Gln(Trt)-Leu-OH did not
occur. This problem was overcome by protection of the boronic
acid with (+)-pinanediol after removal of the diphenylmethylene
group generating compound 20.11 After Fmoc protection of the free
amine, the t-butyl group of 21 was removed and the resulting com-
pound 22 was loaded onto 2-chlorotrityl chloride resin. The pep-
tide sequence 23 was then prepared on resin using standard
SPPS. Cleavage from resin and deprotection of the protecting
groups afforded the desired compound yzm49.
fluorogenic coumarin (MCA) group and a 2,4-dinitrophenol (DNP)
quenching group. Upon Taspase1-mediated cleavage, the quencher
is removed, resulting in fluorescence. These results were further
confirmed by an in vitro cleavage assay utilizing a modified aa
2,500–2,800 fragment of MLL (Fig. 3). For both assays, yzm18
and yzm19 exhibited the strongest inhibitory activity (IC50
29.4
l
M, 63.4
lM, respectively), whereas yzm16 and yzm38 were
only minimally active (IC50 >100
l
M). The activity of the boronate
yzm49 could not be determined because its poor solubility pre-
Table 1
Inhibition IC50 values for yzm16, 18, 19, 22, 23, 38, 49 against recombinant Taspase1
as measured using a quenched fluorescent substrate
a
Compds
IC50
(
lM)
yzm16
yzm18
yzm19
yzm38
yzm49
yzm22
yzm23
>100
29.40 (1.04)
63.38 (1.65)
>100
n.d.a.
n.d.a.
n.d.a.
All these peptide derivatives were tested in vitro to determine
their activity against Taspase1. The IC50 values presented in Table
Compounds were added simultaneously with substrate to Taspase1 (see Supple-
mentary data).
1
were determined using a Fluorescence Resonance Energy
a
Values are means of three experiments, standard deviation is given in paren-
Transfer (FRET) based reporter assay. The FRET-based assay em-
ploys a reporter substrate (MCA-KISQLDGVDD-DNP) containing a
theses (n.d.a. = no detectable activity at max concentration (100 lM) used).

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J. T. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5086–5090
Figure 3. In vitro cleavage assay. A construct expressing a fragment of p45MLL from containing residues 2,500 to 2,800 with the CS1 site mutated to prevent taspase1
cleavage was used for in vitro translation. The resulting 35S-labled protein is cleaved from the p45 form to the p33 fragment upon addition of rTaspase1. For inhibition assays,
compounds indicated were preincubated with rTaspase1 at the indicated concentrations for 30 min prior to addition to the in vitro translated p45MLL. Inhibition was
observed as a loss of processing and accumulation of the full-length p45 fragment.
vented measurement of activity at concentrations over 100 lM.
Supplementary data
We also measured the inhibition rates of taspase1 by yzm18 after
different amounts of pre-incubation time and found that inhibition
was not time dependent. This result suggests that yzm18 is not
likely to be acting as a covalent irreversible inhibitor against this
target (see Supplementary Fig. 8). The vinyl sulfone is most active
when placed on the P1 side chain whereas the vinyl ketone showed
relatively strong activity when placed in the P1 side chain position.
Taken together, our study demonstrates that Taspase1 is capable of
binding inhibitors that contain reactive warheads on either the
main peptide backbone or in place of the P1 side chain. This sug-
gests that Taspase1 may be able to cleave substrates that have
undergone peptide backbone rearrangement. This rather unique
property may facilitate the design of highly selective inhibitors.
While the compounds presented here are not highly potent, they
serve as a starting point for further inhibitor design and may be
highly valuable for future structural studies of the enzyme with
inhibitors bound in the active site. We are currently working to
validate the selectivity of the most potent compound yzm18 in cell
culture models.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.045.
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