tools to better our knowledge about HAT biology or as potential chemotherapeutics
to target the numerous disorders where HATs seem significantly involved.
One of the main problems in HATi development is the structure of the target
itself: the highly conserved co-substrate-binding cleft and the shallow substratebinding pocket make attaining potency and/or selectivity for any specific isoform
very demanding. Another serious problem is the issue of the different results
obtained from diverse assay formats. The usage of different techniques, that provide
IC 50 values as results of different physical measurements, and the great variability of
types of buffer and substrate, enzyme sources, substrate/cofactor concentrations and
incubation times often lead to quite inconsistent and even contradictory results. For
this reason, the comparison of IC 50 values, even for similar assay settings, cannot be
reliable. The best solution to this issue is the standardization of the assay protocols,
the usage of reliable reference inhibitors and the comparison only of K i values after a
precise study of the catalytic mechanism of the diverse HAT enzymes and of their
inhibitors’ kinetics. Orthogonal assays are also vital to confirm initial results, and
counter screens against off-targets should be performed to establish selectivity of
hit/lead compounds.
Another problem in HATi development is that quite often their cellular effects do
not reflect the enzyme inhibitory activity measured in vitro. A possible explanation
of the evidence that potency and selectivity of HATi often vary between cellular and
enzymatic assays is that natively HATs are frequently part of multi-protein complexes, and their structural and/or catalytic functions can be modulated by the other
proteins within the complex depending on the specific cellular context. Moreover,
the majority of the HATi reported so far exhibit low potency, very scarce selectivity
and metabolic instability, which may be responsible of their low efficacy in cells and
in vivo.
The consequence of this situation is that currently, with the peculiar exception of
curcumin, there are no HATi in advanced clinical development. Even curcumin,
displaying an inhibiting activity against many other proteins in addition to p300, and
showing additional mechanisms of action that likely contribute to the pleiotropic
effects observed in cellular and animal models, when translated to human clinical
trials, is not as effective as predicted by both in vitro and cellular assays [91].
Nowadays, only two crystallographic structures of HATi in complex with a single
HAT enzyme are freely available: the complexes of p300 with 1 [73] and 17
[113]. Resolving this type of structures for other HATs, especially within their
native multicomponent protein complexes, will be of vital importance to improve
our understanding of crucial enzyme-inhibitor interactions, thereby providing precious information for the development of more potent and selective HATi. In fact,
supported by the new crystallographic data, computer-aided methods, in combination with the most recent approaches in the drug discovery field, will likely significantly increase our chances to developing HATi that could be used as potential
chemotherapeutics and/or at least as chemical probes for studying HAT biology.
114
D. Trisciuoglio and D. Rotili
to target the numerous disorders where HATs seem significantly involved.
One of the main problems in HATi development is the structure of the target
itself: the highly conserved co-substrate-binding cleft and the shallow substratebinding pocket make attaining potency and/or selectivity for any specific isoform
very demanding. Another serious problem is the issue of the different results
obtained from diverse assay formats. The usage of different techniques, that provide
IC 50 values as results of different physical measurements, and the great variability of
types of buffer and substrate, enzyme sources, substrate/cofactor concentrations and
incubation times often lead to quite inconsistent and even contradictory results. For
this reason, the comparison of IC 50 values, even for similar assay settings, cannot be
reliable. The best solution to this issue is the standardization of the assay protocols,
the usage of reliable reference inhibitors and the comparison only of K i values after a
precise study of the catalytic mechanism of the diverse HAT enzymes and of their
inhibitors’ kinetics. Orthogonal assays are also vital to confirm initial results, and
counter screens against off-targets should be performed to establish selectivity of
hit/lead compounds.
Another problem in HATi development is that quite often their cellular effects do
not reflect the enzyme inhibitory activity measured in vitro. A possible explanation
of the evidence that potency and selectivity of HATi often vary between cellular and
enzymatic assays is that natively HATs are frequently part of multi-protein complexes, and their structural and/or catalytic functions can be modulated by the other
proteins within the complex depending on the specific cellular context. Moreover,
the majority of the HATi reported so far exhibit low potency, very scarce selectivity
and metabolic instability, which may be responsible of their low efficacy in cells and
in vivo.
The consequence of this situation is that currently, with the peculiar exception of
curcumin, there are no HATi in advanced clinical development. Even curcumin,
displaying an inhibiting activity against many other proteins in addition to p300, and
showing additional mechanisms of action that likely contribute to the pleiotropic
effects observed in cellular and animal models, when translated to human clinical
trials, is not as effective as predicted by both in vitro and cellular assays [91].
Nowadays, only two crystallographic structures of HATi in complex with a single
HAT enzyme are freely available: the complexes of p300 with 1 [73] and 17
[113]. Resolving this type of structures for other HATs, especially within their
native multicomponent protein complexes, will be of vital importance to improve
our understanding of crucial enzyme-inhibitor interactions, thereby providing precious information for the development of more potent and selective HATi. In fact,
supported by the new crystallographic data, computer-aided methods, in combination with the most recent approaches in the drug discovery field, will likely significantly increase our chances to developing HATi that could be used as potential
chemotherapeutics and/or at least as chemical probes for studying HAT biology.
114
D. Trisciuoglio and D. Rotili