Organic azide inhibitors of cysteine proteases

Article abstract of DOI:10.1021/ja0637649

Cysteine proteases are crucial regulatory enzymes in human physiology and disease. Inhibitors are usually designed with reactive electrophiles to covalently bond to the catalytic cysteinyl sulfur, and consequently they also indiscriminately interact with biological thiolates and other nucleophiles, leading to toxic side effects in vivo. Here we describe an alternative to using reactive electrophiles, demonstrating the use of a much less reactive azidomethylene substituent (-CH₂-N₃) that confers potent inhibition of cysteine proteases. This new approach resulted in potent, reversible, competitive inhibitors of caspase-1 (IC₅₀ < 10 nM), with significant advantages over aldehydes such as high stability in vitro to thiols (10 mM dithiothreitol (pH 7.2), 20 mM glutathione (pH 7.2, 9, 11)) and aqueous media, as well as some highly desirable druglike features. It was also demonstrated that azides can be incorporated into inhibitors of other caspases (e.g. 3, 8) and cathepsins (e.g. K, S, B), indicating the versatility of this valuable new approach to cysteine protease inhibition. Copyright

Full text of DOI:10.1021/ja0637649

Published on Web 09/02/2006
Organic Azide Inhibitors of Cysteine Proteases
Giang Thanh Le, Giovanni Abbenante, Praveen K. Madala, Huy N. Hoang, and David P. Fairlie*
Centre for Drug Design and DeVelopment, Institute for Molecular Bioscience, UniVersity of Queensland, Brisbane,
Qld 4072, Australia
Received June 4, 2006; E-mail: d.fairlie@imb.uq.edu.au
Table 1. Azidomethylene Based Inhibitors of Caspase-1
Cysteine proteases account for 26% of human endopeptidase
enzymes and play crucial roles in diseases, immune defense,
inflammation, apoptosis, and bone resorption.1 When overexpressed
or unregulated in diseased states, cysteine proteases are validated
therapeutic targets,2 but their inhibitors have not yet succeeded in
the clinic,2 mainly because of a common flaw in their design.
Inhibitors of cysteine proteases usually employ a reactive electro-
philic group (alkylating agent, aldehyde, nitrile, ketone, R-keto-
amide, halo-ketone, vinyl sulfone, etc.)3 to covalently bond to the
enzyme’s catalytic cysteinyl sulfur. Although useful tools in vitro,
such inhibitors are not druglike4 because of indiscriminate compet-
ing reactions in vivo with other nucleophiles (thiols, amines, etc),
resulting in low bioavailability, metabolic instability, and side-
effects.4 Electrophilic isosteres are still thought to be necessary for
potent inhibition of cysteine proteases, especially caspases.5 We
now report an alternative use of much less reactive alkyl azide
isosteres to confer potent inhibition of cysteine proteases. Inhibition
of a proinflammatory interleukin converting enzyme, caspase-1
(ICE),7 is shown to be competitive, reversible, and selective, relying
upon multiple noncovalent interactions for enzyme binding. The
new azido isostere is also shown to be effective for inhibiting
cathepsins (B, K, S) and other caspases (3, 8), suggesting its use
for inhibiting cysteine proteases in general.
Table 1 shows that replacing the C-terminal aspartic aldehyde
in the known caspase-1 inhibitor 18 by the azidomethylene
derivative of aspartic acid (X) maintains sub-µM inhibition (2, Table
1) of caspase-1. The reduced potency was recovered through
sequential variation at P3 and P4 positions (3-5, Table 1);
compound 5 with a 2-naphthoyl substituent at P4 being only 2-fold
less potent than 1. Substitution of Val and Ala in 5 with more
hydrophobic amino acids gave 6 (Table 1), with more druglike
characteristics (MW ) 524sethyl ester, logP ) 2.8, polar surface
area ) 143 Å2, H-bond donors/acceptors ) 3/10, 13 rotatable
bonds)12 and less susceptibility to proteolytic degradation. By
contrast, the nitrile analogue 7 (even at 50 µM) did not inhibit
caspase-1.
a log P (pH 7.4) refers to the mono/diethyl ester derivatives of the P1/
P3 Asp/Glu side-chain carboxlates.
Figure 1. (A) Modeled 5 docked in the active site of caspase-1 (pdb 1ICE);
(B) backbone (C, N, O atoms) superimposition of 5 (green) on enzyme-
bound structure13 of Ac-YVAD-H (red).
Azides 2-6 are competitive and reversible inhibitors of caspase-1
as shown by Lineweaver-Burk plots of 1/Vo versus 1/[S] at varying
[I] (Figure S1, Supporting Information (SI)). Km increased with [I]
but Vmax remained constant. Preincubation of, for example, 5 (50
nM) for 10 min with caspase-1 resulted in complete inhibition. Full
enzymatic activity was restored by adding 20× Km of substrate
(Ac-YVAD-AMC, Km ) 14 µM), establishing that inhibition by 5
is competitive and reversible (SI). Inhibitor 5 was also quite selec-
tive for caspase-1 (IC50 > 50 µM, caspase-3; 2.0µM, caspase-5).
Modeling of 5 in the substrate-binding active site of caspase-1
using GOLD (Figure 1) showed that the naphthoyl substituent (P4),
and side chains of Val (P3), Ala (P2), and pseudo-aspartate (P1)
of 5 occupied expected sites (Figure 1A: S4-S1, respectively) in
caspase-1 (crystal structure of (Ac-YVAD-H)-enzyme complex,
pdb: 1ICE13). Inhibitor 5 also occupies very similar conformational
space as 1 (Figure 1B), its backbone atoms (C, N, O) superimposing
well (rmsd 0.37 Å) on the corresponding atoms of 1 in its crystal
structure with caspase-1. The backbone adopts the classic extended
strand recognized by most proteases.14 Corresponding side chains
also align closely. The P1 carboxylate side chain of 5 is important,
as it is in 1,15 since conversion to the amide reduced caspase-1
inhibition 50-fold (IC50 230 nM). This supports analogous binding
for 5 and 1 at the S1 site in caspase-1.
By contrast, the azide of 5 is in a different position to the
aldehyde of 1. The azidomethylene carbon of 5 is 1.9 Å from the
aldehyde carbon of 1, and equidistant from the active site thiolate
(Cys 285-S...central azide nitrogen, 3.8 Å) and imidazole (His 237-
NH...terminal azide nitrogen, 3.5 Å). The azido substituent does
not extend to the S1′ subsite of enzyme. Thus the model supports
noncovalent interactions with enzyme. Given that azide has
9
12396
J. AM. CHEM. SOC. 2006, 128, 12396-12397
10.1021/ja0637649 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Azidomethylene Inhibitors of Other Cysteine Proteases
Acknowledgment. The Australian funding agencies ARC and
NHMRC are gratefully acknowledged for financial support.
Supporting Information Available: Synthesis and characterization
1
data (rpHPLC retention times, H NMR spectra, high-resolution MS
Figure 2. Selected 1H NMR signals for aldehyde 1 (left panel) and azide
5 (right panel) in H2O/D2O (9:1, phosphate buffer pH 7.4, 37 °C) at 5 min
(A), 3 h (B), 24 h (C) or with 10-fold Ac-Cys for 5 min (D), 3 h (E), or
3 weeks (F). New signals in traces B, C, and E were ascribed to respective
protons of D-isomer. 5 remained unchanged under all these conditions (right
panel).
data) for 1-12, enzyme assays, and stabilities. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Leung-Toung, R.; Li, W. R.; Tam, T. F.; Karimian, K. Curr. Med. Chem.
2002, 9, 979-1002.
resonance forms with positive and negative charge on central and
terminal nitrogens, respectively, noncovalent electrostatic interac-
tions between azide and cysteinyl-S or imidazole-N, might be
possible as proposed for sulfonyl azide inhibitors of COX-2.16
Azides are widely used in organic synthesis,9 but are remarkably
stable in biological environments. For example, azides are orthogo-
nal protecting groups in vivo,10 components of drugs, like the anti-
HIV drug AZT (only ∼1% is reduced to the amine metabolite by
cytochrome P450 after oral administration to rats11), and azido-sugars
are well tolerated in mice (300 mg/kg/day, 7 days).10 Azides 5 and
6 were completely stable in aqueous solutions (>1 month, 37 °C),
serum (24 h, 37 °C, pH 7.2), assay buffer (14 h, pH 7.2, 10 mM
dithiothreitol) ( enzyme, or 20 mM glutathione (pH 7.2, 9, 11), as
monitored quantitatively by LCMS. By contrast, aldehydes such
as 1 are unstable in aqueous solutions (Figure 2, left panel), existing
initially as a mixture of aldehyde and cyclic hemi-acetal (A)
followed by rapid aspartate racemization (B, C) in phosphate buffer
(t1/2 ) 4 h, 37 °C, pH 7.4). No aldehyde is detectable within minutes
in 10 mM Ac-Cys (D, E) or 20 mM glutathione. Azide 5 is very
stable under the same conditions (Figure 2, right panel). Under
harsher chemical, less biologically relevant, conditions, the azide
of 5 can be reduced (Pd/C/H2) to the amine, which is a 1000× less
potent caspase-1 inhibitor (IC50 ) 5 µM, SI).
Azides have not previously been reported as components of
protease inhibitors. Preliminary results here suggest that the
azidomethylene group can be used to produce potent inhibitors not
only of caspase-1, but also of other cysteine proteases. For example,
aldehydes Ac-DEVD-H17 and Cbz-Leu-Nle-H,18 were systematically
converted into nanomolar inhibitors (Table 2) of other caspases
(3, 8) and cathepsins (B, K, S) and are being further optimized for
potency, selectivity and bioavailability.
Many cysteine protease inhibitors with reactive electrophiles, like
aldehydes, ketones, and acyloxyketones, have failed clinical trials.2
Two caspase-1 inhibitors with a masked aldehyde, pralnacasan and
VX-765, have similarly faltered in clinical trials for arthritis. Very
few noncovalent inhibitors are known for cysteine proteases,6 so
this demonstration that the azidomethylene unit is much more inert
than aldehydes but still confers potent, competitive, and reversible
caspase-1 inhibition may be a valuable discovery toward new, less
reactive and/or noncovalent, inhibitors of caspases, cathepsins, and
other cysteine proteases.
(2) (a) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43,
305-341. (b) Abbenante, G.; Fairlie, D. P. Med. Chem. 2005, 1, 71-
104.
(3) (a) Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. ReV.
2002, 102, 4639-4750.(b) Otto, H.-H.; Schirmeister, T. Chem. ReV. 1997,
97, 133-171.
(4) (a) Rishton, G. M. Drug DiscoVery Today 2003, 8, 86-96. (b) Muegge,
I. Chem.sEur. J. 2002, 8, 1976-1981.
(5) Talanian, R. V.; Brady, K. D.; Cryns, V. L. J. Med. Chem. 2000, 43,
3351-3371.
(6) (a) Chowdhury, S. F.; Sivaraman, J.; Wang, J.; Devanathan, G.; Lachance,
P.; Qi, H. T.; Menard, R.; Lefebvre, J.; Konishi, Y.; Cygler, M.; Sulea,
T.; Purisima, E. O. J. Med. Chem. 2002, 45, 5321-5329. (b) Altmann,
E.; Renaud, J.; Green, J.; Farley, D.; Cutting, B.; Jahnke, W. J. Med.
Chem. 2002, 45, 2352-2354. (c) Altmann, E.; Green, J.; Tintelnot-
Blomley, M. Bioorg. Med. Chem. Lett. 2003, 13, 1997-2001. (d)
Thurmond, R. L.; Beavers, M. P.; Cai, H.; Meduna, S. P.; Gustin, D. J.;
Sun, S. Q.; Almond, H. J.; Karlsson, L.; Edwards, J. P. J. Med. Chem.
2004, 47, 4799-4801. (e) Gustin, D. J.; Sehon, C. A.; Wei, J. M.; Cai,
H.; Meduna, S. P.; Khatuya, H.; Sun, S. Q.; Gu, Y.; Jiang, W.; Thurmond,
R. L.; Karlsson, L.; Edwards, J. P. Bioorg. Med. Chem. Lett. 2005, 15,
1687-1691.
(7) Braddock, M.; Quinn, A. Nat. ReV. Drug DiscoV. 2004, 3, 1-10.
(8) Rano, T. A.; Timkey, T.; Peterson, E. P.; Rotonda, J.; Nicholson, D. W.;
Becker, J. W.; Chapman, K. T.; Thornberry, N. A. Chem. Biol. 1997, 4,
149-155.
(9) Scriven, E. F. V.; Turnbull, K. Chem. ReV. 1988, 88, 297-368.
(10) (a) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873-
877. (b) Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R.
Glycobiology 2004, 14, 1073-1074.
(11) Pan-Zhou, X. R.; Cretton-Scott, E.; Zhou, X. J.; Yang, M. X.; Lasker, J.
M.; Sommadossi, J. P. Biochem. Pharmacol. 1998, 55, 757-766.
(12) (a) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K.
W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615-23. (b) Lipinski, C. A.
J. Pharmacol. Toxicol. Methods 2000, 44, 235-249. (c) Martin, Y. C. J.
Med. Chem. 2005, 48, 3164-3170.
(13) Wilson, K. P.; Black, J. A. F.; Thomson, J. A.; Kim, E. E.; Griffith, J. P.;
Navia, M. A.; Murcko, M. A.; Chambers, S. P.; Aldape, R. A.; Raybuck,
S. A.; Livingston, D. J. Nature 1994, 370, 270-275.
(14) (a) Tyndall, J. D. A.; Nall, T.; Fairlie, D. P. Chem. ReV. 2005, 105, 973-
1000. (b) Loughlin, W. A.; Tyndall, J. D. A.; Glenn, M. P.; Fairlie, D. P.
Chem. ReV. 2004, 104, 6085-6117. (c) Fairlie, D. P.; Tyndall, J. D. A.;
Reid, R. C.; Wong, A. K.; Abbenante, G.; Scanlon, M. J.; March, D. R.;
Bergman, D. A.; Chai, C. L. L.; Burkett, B. A. J. Med. Chem. 2000, 43,
1271-1281. (d) Tyndall, J.; Fairlie, D. P. J. Mol. Rec. 1999, 12, 363-
370.
(15) Sleath, P. R.; Hendrickson, R. C.; Kronheim, S. R.; March, C. J.; Black,
R. A. J. Biol. Chem. 1990, 265, 14526-14528.
(16) Habeeb, A. G.; Rao, P. N. P.; Knaus, E. E. J. Med. Chem. 2001, 44,
3039-3042.
(17) Rotonda, J.; Nicholson, D. W.; Fazil, K. M.; Gallant, M.; Gareau, Y.;
Labelle, M.; Peterson, E. P.; Rasper, D. M.; Ruel, R.; Vaillancourt, J. P.;
Thornberry, N. A.; Becker, J. W. Nat. Struct. Biol. 1996, 3, 619-625.
(18) Catalano, J. G.; Deaton, D. N.; Furfine, E. S.; Hassell, A. M.; McFadyen,
R. B.; Miller, A. B.; Miller, L. R.; Shewchuk, L. M.; Willard, D. H., Jr.;
Wright, L. L. Bioorg. Med. Chem. Lett. 2004, 14, 275-278.
JA0637649
9
J. AM. CHEM. SOC. VOL. 128, NO. 38, 2006 12397