Research Projects

Histones are decorated several PTMs including lysine methylation, acetylation, ubiquitination etc. Such PTM modified histones are substrates for histone readers e.g. bromodomains (BRD) and eraser enzymes e.g. lysine demethylases (KDMs), histone demethylases (HDACs) that dynamically regulate their epigenetic status. Their interactions with specific PTMs are regulated by peptide-protein interface ranging from vanderwaal, ionic to H-bonds. Small molecule inhibitors play an important role in understanding their fundamental role as well as inhibiting diseased histone readers or erasers. Over past decades several small molecule inhibitors of BRDs, KDMs and HDACs are reported and have gone through clinical trials. However, their success is limited by the unspecific off-target activity. Therefore, recently focus has been shifted to discover macrocyclic inhibitors that inhibit desired targets with unprecedented specificity. Towards this direction we propose to develop PTM specific cyclic peptide inhibitors of readers, writers and erasers. The cyclic peptide inhibitors will incorporate the “signature PTM” of respective reader or eraser proteins as determinant of specificity of the active site binding. We will utilize directed evolution platform of ribosome display to prepare genetically encoded semisynthetic cyclic peptide library. An split-intein mediated cyclization will be used to make cyclic peptides encoding such modifications. The cyclic peptides will be of semisynthetic origin comprising of a synthetic and recombinant part, which are ligated via protein trans-splicing by using a split-intein. The cyclization will be achieved by conjugation of an aryl fluorosulfate (AFS) warhead group with N- and C- terminal cysteine of synthetic and recombinant parts respectively. The synthetic peptide can contain one or multiple histone PTM amino acids along with other unnatural amino acids like with click handle for covalent pulldown of the inhibitory complex. The specific PTM AA will incorporated to the synthetic peptide via solid phase peptide synthesis (SPPS). This will enable targeting the cyclic peptide ligands to the active binding site of readers and erasers and at the same time developing high affinity cyclic peptide binders using a DNA randomized library of the genetically encoded part. Such dual functionalities of the cyclic peptide binders will potentially achieve both high specificity and affinity to a PTM specific reader or eraser. Furthermore, specificity can be potentially achieved by encoding the native flanking residues of the PTM-AA from histones into the cyclic backbone. The flanking residues will ensure further specificity of the cyclic peptide with respective reader, writer or eraser proteins. After binding of the cyclic peptide with the target protein, AFS warhead group will potentially crosslink covalently with the nucleophilic residues (e.g. Tyr, Lys, His) present around the binding site via proximity driven SuFEx chemistry.