HDAC4 PROTACs as a potential palliative care therapy for Spinal Muscular Atrophy: Impact of the various linkers on enzymatic activity

HDAC4 is a class IIa member of histone deacetylases (HDACs), playing a role in regulating gene expression through chromatin remodeling, as well as in muscle alterations. It triggers the atrogin-1 and MuRF1 upregulation, muscle protein degradation, and slow atrophy progression, symptoms associated with Spinal Muscular Atrophy (SMA), for which it is considered an important target for corresponding palliative care therapeutic development.

In that sense, our interests were to develop selective PROTAC degraders of HDAC4 using known co-crystalized HDAC4 inhibitors (HDAC4Is) available at Protein Data Bank as starting points, particularly the truncated version of 6FYZ HDAC4I as a warhead stripped of the Cap moiety, as instructed by the generated preliminary Py-CoMFA 3-D QSAR model. The primary objective during the initial profiling phase was to investigate their interactions with HDAC4 and assess how structural modifications influence potency. To that, the PROTACs were initially autonomously designed by means of Python’s RDKit by merging the warhead with the in-house available linkers and either VHL-1 or CRBN E3 ligase ligands, respecting the desirable PK/PD profiles. Selected hits were promptly synthesized and submitted to enzymatic fluorogenic assaying, of which TG-49 and PJ594, as VHL-1-related PROTACs, showed remarkable IC₅₀s of 50 and 150 nM. Regarding the whole panel, interestingly, even minor changes in the linker structure resulted in significant variations in the inhibition of HDAC4, suggesting that the linker may play a critical role in mediating interactions with HDAC4. Yet, confirmation is pending with further ternary complex computational modeling and HDAC4 cellular degradation assays.