De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains

Abstract

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogenous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

“…35 In brief, HeLa cells were cultured using DMEM supplemented with 10% FBS and 1% Pen/Strep and kept at 37ºC under 5% CO2. Cells were seeded in a 24-well plate (1×10 5 Figure 1; Y denotes a tyrosine with a chloroalkane tag coupled to its N-terminus; K denotes a lysine coupled to a chloroalkane tag; and K denotes a lysine coupled to an Alexa Fluor® 488 tag. Chloroalkane (ct) and Alexa Fluor® 488 (ax) tagged variants were used to study cellular uptake but only unlabeled peptides are shown here for brevity.…”

“…1,2 Recent interest in their use as therapeutics has been driven by breakthroughs in bioactive peptide discovery and optimization, which have led to the development of many peptides that can modulate protein targets with high in vitro potency and selectivity. [3][4][5][6] Despite these technological advances, targeting of intracellular protein-protein interactions remains a considerable challenge mainly because of the difficulty in delivery of drug leads into the cytosolic space. 7,8 The discovery of a short peptide from the human immunodeficiency virus (HIV)-1 three decades ago, called Tat, generated considerable excitement that it and other peptides with the ability to enter cells, called cell-penetrating peptides (CPPs), could be used to shuttle exogenous cargoes into the cytosol.…”

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

“…In 2017, Kawamura et al used Random nonstandard Peptide Integrated Discovery (RaPID) methodology to perform a virtual screening, and a series of selective and potent cyclic peptides were obtained as KDMs inhibitors (Figure ). Through a combination of total chemical synthesis of target proteins and screening against cyclic peptide library (RaPID system), a series of macrocyclic peptides with potent anticancer activities were discovered by Nawatha et al in 2019 …”

Small molecules that target the ubiquitin system

“…Although not drug-like, molecules called ubistatins have been shown to bind to ubiquitin in a chemical genetic screen [120,121], although these molecules have features also found in 'frequent hitters'. An additional advance in a similar direction came through the discovery of a cyclic peptide ubiquitin binder, by using the RaPID system [122]. These peptides have been shown to efficiently trigger apoptosis in cancer cells by binding tightly and with a high degree of specificity to K48-linked Ub chains, thus disrupting their interaction with the proteasome and their subsequent degradation.…”