2 Hydroxamic Acid: A Transition State Mimic
Zn
2+ -dependent HDACs catalyze deacetylation through the following mechanism:
first, Kac is accommodated in the catalytic pocket favoring coordination of the PTM
to the Zn
2+ ion, then, a water molecule present in the active site performs a
nucleophilic attack on the carbonyl group, and, finally, acetate is released
[27]. The reaction is possible because coordination to Zn
2+ enhances electrophilicity
of the acetamido group, which favors the nucleophilic attack, and it also stabilizes
the hydrated transition state of the reaction via chelation to the metal ion. In addition,
the interactions between the PTM and Zn
2+ ensure the right conformation for the
reaction with the water molecule. All this has been proposed with the support of
kinetic studies and several crystal structures of inactive HDAC8 mutants (Fig. 2a, b)
[27, 28]. Interestingly, the hydroxamic acid group of HDAC inhibitors binds to the
active site in analogy to the transition state of the reaction, forming two oxygenmetal bonds with Zn
2+ and, presumably, interacting with the water molecule
(as shown in a crystal structure of HDAC2 with compound 1.2, PDB code:
4LXZ). Hydrolysis, though, is not favorable for this moiety due to the electronic
characteristics of the carbonyl group. Moreover, the nitrogen and two oxygen atoms,
and their spatial distribution, allow for additional interactions with the catalytic
pocket that further stabilize the interaction (Fig. 2d) [29, 30].
In general, aliphatic hydroxamic acids are neutral at physiological pH, since their
pK a is 9.4 in aqueous solution. However, it has been argued whether coordination to
Zn
2+ in HDACs can lead to deprotonation due to a decrease in pK a influenced by the
biological environment. Likewise, a monodentate or bidentate character of the
interaction between this functional group and the metal ion has been debated.
Inhibitors with a bulky warhead such as phenylhydroxamic acids have been shown
to prefer a monodentate hydroxamate-Zn
2+ interaction in the active site of zebrafish
HDAC6 [31]. On the other hand, quantitative structure-activity relationship (QSAR)
studies, together with X-ray crystal structures, have demonstrated that the protonated
and more stable form of the hydroxamic acid generally coordinates in a bidentate
fashion to the catalytic Zn
2+ in class I HDACs [32–39]. The chelating effect, together
with multiple hydrogen bonds established with side chains and, sometimes, the
water molecule in the active site, explains why this moiety contributes so significantly to the binding affinity of HDAC inhibitors, especially when compared to other
Zn
2+ -binding groups.
Many of its properties support hydroxamic acid as the ideal warhead for the
design of HDAC inhibitors, but its versatility has been discussed in terms of two
major disadvantages: promiscuity and mutagenicity. The fact that inhibitors bearing
strong chelating groups are able to target most metalloenzymes is widespread, and it
has been argued as an explanation for the amount of side effects associated with
them [40]. HDAC inhibitors such as SAHA frequently present such promiscuity
toward several HDAC isotypes in vitro [24]. On the other hand, a number of in vitro
studies have supported the inability of SAHA to inhibit other metalloenzymes than
HDACs [41, 42], as well as to alter the concentration of free Zn
2+ in living cells
Hydroxamic Acid-Containing Peptides in the Study of Histone Deacetylases
33
Zn
2+ -dependent HDACs catalyze deacetylation through the following mechanism:
first, Kac is accommodated in the catalytic pocket favoring coordination of the PTM
to the Zn
2+ ion, then, a water molecule present in the active site performs a
nucleophilic attack on the carbonyl group, and, finally, acetate is released
[27]. The reaction is possible because coordination to Zn
2+ enhances electrophilicity
of the acetamido group, which favors the nucleophilic attack, and it also stabilizes
the hydrated transition state of the reaction via chelation to the metal ion. In addition,
the interactions between the PTM and Zn
2+ ensure the right conformation for the
reaction with the water molecule. All this has been proposed with the support of
kinetic studies and several crystal structures of inactive HDAC8 mutants (Fig. 2a, b)
[27, 28]. Interestingly, the hydroxamic acid group of HDAC inhibitors binds to the
active site in analogy to the transition state of the reaction, forming two oxygenmetal bonds with Zn
2+ and, presumably, interacting with the water molecule
(as shown in a crystal structure of HDAC2 with compound 1.2, PDB code:
4LXZ). Hydrolysis, though, is not favorable for this moiety due to the electronic
characteristics of the carbonyl group. Moreover, the nitrogen and two oxygen atoms,
and their spatial distribution, allow for additional interactions with the catalytic
pocket that further stabilize the interaction (Fig. 2d) [29, 30].
In general, aliphatic hydroxamic acids are neutral at physiological pH, since their
pK a is 9.4 in aqueous solution. However, it has been argued whether coordination to
Zn
2+ in HDACs can lead to deprotonation due to a decrease in pK a influenced by the
biological environment. Likewise, a monodentate or bidentate character of the
interaction between this functional group and the metal ion has been debated.
Inhibitors with a bulky warhead such as phenylhydroxamic acids have been shown
to prefer a monodentate hydroxamate-Zn
2+ interaction in the active site of zebrafish
HDAC6 [31]. On the other hand, quantitative structure-activity relationship (QSAR)
studies, together with X-ray crystal structures, have demonstrated that the protonated
and more stable form of the hydroxamic acid generally coordinates in a bidentate
fashion to the catalytic Zn
2+ in class I HDACs [32–39]. The chelating effect, together
with multiple hydrogen bonds established with side chains and, sometimes, the
water molecule in the active site, explains why this moiety contributes so significantly to the binding affinity of HDAC inhibitors, especially when compared to other
Zn
2+ -binding groups.
Many of its properties support hydroxamic acid as the ideal warhead for the
design of HDAC inhibitors, but its versatility has been discussed in terms of two
major disadvantages: promiscuity and mutagenicity. The fact that inhibitors bearing
strong chelating groups are able to target most metalloenzymes is widespread, and it
has been argued as an explanation for the amount of side effects associated with
them [40]. HDAC inhibitors such as SAHA frequently present such promiscuity
toward several HDAC isotypes in vitro [24]. On the other hand, a number of in vitro
studies have supported the inability of SAHA to inhibit other metalloenzymes than
HDACs [41, 42], as well as to alter the concentration of free Zn
2+ in living cells
Hydroxamic Acid-Containing Peptides in the Study of Histone Deacetylases
33