Histone deacetylase 6 (HDAC6) is
involved in multiple cellular
processes such as aggresome formation, protein stability, and cell
motility. Numerous HDAC6-selective inhibitors have been developed
as cellular chemical tools to elucidate the function of HDAC6. Since
HDAC6 has multiple domains that cannot be studied by HDAC6-selective
inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate
the nonenzymatic functions of HDAC6. However, these genetic methods
have many limitations. Proteolysis targeting chimera (PROTAC) is an
emerging technology for the development of small molecules that can
quickly remove the entire protein in cells. We previously developed
multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3
ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6
but also neo-substrates of CRBN. They are excellent candidates for
the development of anticancer therapeutics, but the multifunctional
nature of the CRBN-based HDAC6 degraders has limited their utility
as specific chemical probes for the study of HDAC6-related cellular
pathways. Herein we report the development of the first cell-permeable
HDAC6-selective degraders employing Von Hippel–Lindau (VHL)
E3 ubiquitin ligase, which does not have any known neo-substrates.
The DC50’s of the most potent compound 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, respectively.
The D
max’s of 3j in
these two cell lines are 90% and 57%, respectively.
Proteolysis targeting chimeras (PROTACs)
are molecules that induce
protein degradation via formation of ternary complexes between an
E3 ubiquitin ligase and a target protein. The rational design of PROTACs
requires accurate knowledge of the native configuration of the PROTAC-induced
ternary complex. This study demonstrates that native and non-native
ternary complex poses can be distinguished based on the pose occupancy
time in MD, where native poses exhibit longer occupancy times at both
room and higher temperatures. Candidate poses are generated by MD
sampling and pre-ranked by classic MM/GBSA. A specific heating scheme
is then applied to accelerate ternary pose departure, with the pose
occupancy time and fraction being measured. This scoring identifies
the native pose in all systems tested. Its success is partially attributed
to the dynamic nature of pose departure analyses, which accounts for
entropic effects typically neglected in the faster static scoring
methods, while entropy plays a greater role in protein–protein
than in protein–ligand systems.
Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that can induce the ubiquitination of targeted proteins via the formation of ternary complexes between an E3 ubiquitin ligase and a target protein. The poly-ubiquitinated target protein will be escorted to the proteasome for degradation. Rational design of PROTACs require knowledge of an accurate configuration of the PROTAC induced ternary complex. This study demonstrates that native ternary poses can be distinguished by scoring candidate poses based on the pose residence time. The scoring is essentially heat-and-dissociate trials of candidate poses sampled by MD and pre-ranked by the classic MM/GBSA method. It is practical, simple to use and self-intuitive, relying on the observation that the assumed more stable native crystal ternary poses maintained a longer residence time than non-native ones at both room and higher temperatures. A time score and temperature score were generated from multiple replicate trajectories. These scores were able to correctly identify the native pose from non-native ones in all the systems examined. The absolute numbers were comparable across different systems in all currently available VHL and CRBN-containing ternary crystal structures. Therefore, it is also possible to provide an empirical criteria for unresolved ternary structures that under the conditions of this study. If a ternary pose is stable up to over a certain threshold score, it is likely a native pose. The success of the method is in part attributed to the dynamic nature of the pose change analysis which naturally involves entropic effects, one that is intrinsically unavailable with faster static scoring methods that consider molecular mechanical energy only. Protein-protein binding entropy is much more significant than in protein-ligands binding. The success is also attributed to the fact that the protein structures themselves were all stable in the short heating trials.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.