Structure‐Based Design of a New Scaffold for Cell‐Penetrating Peptidic Inhibitors of the Histone Demethylase PHF8

Abstract

The histone demethylase PHF8 catalyzes demethylation of mono- and di-methylated Lys9 on histone H3 (H3K9me1/2), and is a transcriptional activator involved in the development and cancer. Affinity and specificity of PHF8 towards H3K9me2 is affected by interaction with both the catalytic domain and a PHD reader domain. The latter specifically recognizes tri-methylated Ly4 on histone H3. A fragment of the histone H3 tail with tri-methylated Lys4 was used as a template for the structure-based design of a cyclic, cell-penetrating peptide that exhibits micromolar binding affinity to PHF8 in biochemical assays. The inhibitor has significantly lower affinity towards KDM2 enzymes (the phylogenetically closest subfamily), and to KDM3 and KDM6 subfamilies. Selectivity is only marginal towards an enzyme from the KDM4 family, which shares histone tail specificity with PHF8. It is a substrate of KDM5B, thus implying that the free N terminus is not part of the KDM5 enzyme substrate recognition machinery. The cyclic peptide's ability to penetrate cells is achieved by incorporation of a sequence derived from HIV Tat. The derived cyclic peptide can be used as a starting compound in the search for potent and selective PHF8 inhibitors.

Studies of cell-penetrating peptides by biophysical methods

“…By a similar approach, the degradation of TP10 CPP was also studied, showing that for the stability of the peptide Lys in the C-terminal part of the peptide is very important and also for the penetration into cells (Xue et al ., 2015). MALDI-TOF was used also for the observation of the inhibition of histone demethylase by TAT conjugated demethylase inhibitor that was able to penetrate the nucleus (Dorosz et al ., 2017).…”

Biology and targeting of the Jumonji-domain histone demethylase family in childhood neoplasia: a preclinical overview

“…A different KDM2/7 inhibitor was identified in a screen of analogues of the JmjC domain cofactor α-ketoglutarate 185 . A cyclic, cellpenetrating peptide inhibitor of PHF8 has also recently been developed 186 .…”

Proteomics contributions to epigenetic drug discovery

“…Genes encoding for histone modifying enzymes are frequently mutated in cancer [25], with 20%-90% of tumors showing at least one mutation in these genes [13]. Targeting epigenetic modifiers to revert the epigenetic aberrations found in diseased cells has been the object of intense drug discovery efforts during the last decades, particularly for what concerns lysine methylation [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] (Figure 1A) and acetylation [43][44][45][46][47][48][49][50][51] (Figure 1B). Currently, several inhibitors targeting DNMTs, HDACs, and EZH2 have been approved by the FDA for the treatment of different malignancies, mostly hematological.…”