Although noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. Here, we report a technique based on superinfection immunity of phages to enrich amber-containing clones, thus avoiding the observed bias that has hindered incorporation of ncAAs into phage libraries. We then take advantage of this technique for development of active site-directed ligand evolution of peptides, where the ncAA serves as an anchor to direct the binding of its peptides to the target’s active site. To demonstrate this, phage-displayed peptide libraries are developed that contain a genetically encoded butyryl lysine and are subsequently used to select for ligands that bind SIRT2. These ligands are then modified to develop low nanomolar inhibitors of SIRT2.

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded Nϵ‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded Nϵ‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (MPro) for replication and pathogenesis. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MPro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 reversibly covalent dipeptidyl MPro inhibitors and characterized them on in vitro enzymatic inhibition potency, structures of their complexes with MPro, cellular MPro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that MPro has a flexible S2 pocket that accommodates dipeptidyl inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro[4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have optimal characteristics. One compound MPI60 containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability and can be potentially advanced to further preclinical tests.

Siderophores are small molecules used to specifically transport iron into bacteria via related receptors. By adapting siderophores and hijacking their pathways, we may discover an efficient and selective way to target microbes. Herein, we report the synthesis of a siderophore-fluorophore conjugate VF-FL derived from vibrioferrin (VF). Using flow cytometry and fluorescence microscopy, the probe selectively labeled vibrios, including V. parahaemolyticus, V. cholerae, and V. vulnificus, even in the presence of other species such as S. aureus and E. coli. The labeling is siderophore-related and both iron-limited conditions and the siderophore moiety are required. The competitive relationship between VF-FL and VF in vibrios implies an unreported VF-related transport mechanism in V. cholerae and V. vulnificus. These studies demonstrate that the siderophore scaffold provides a method to selectively target microbes expressing cognate receptors under iron-limited conditions.

Phage‐assisted, active site‐directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon‐encoded noncanonical amino acid (ncAA) as an anchor to direct phage‐displayed peptides to a target for an enhanced ligand identification process. 2‐Amino‐8‐oxodecanoic acid (Aoda) is a ketone‐containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan‐inhibitor of Zn2+‐dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N𝜀‐acetyl‐lysyl‐tRNA synthetase in combination with tRNAPyl, we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon‐obligate phagemid library in E. coli encoding Aoda, we generated an Aoda‐containing phage‐displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7‐mer peptide GH8P01F1 with Aoda‐flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+‐chelating ncAA S‐2‐amino‐8‐hydroxyamino‐8‐oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8‐inhibition Ki value of 0.67 nM. GH8HA01 and its 5‐mer truncation analogue Ac‐GH8HA01Δ1Δ7 that has an HDAC8‐inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The novel helper phage allowed for a continuous amber codon enrichment strategy for two different libraries and demonstrated a 100-fold increase in packaging selectivity. CMa13ile40 was then used to create two peptide libraries containing separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine and Nϵ-allyloxycarbonyl-lysine, respectively. These libraries were used to identify peptide ligands that bind to the extracellular domain of ZNRF3. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 that was dependent upon the presence of the ncAA used for selection. Our results demonstrate that ncAAs in phages provide unique interactions for identification of unique peptides. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

Acute myeloid leukemia (AML) is the second most diagnosed and the deadliest subtype of leukemia. Recently genetic loss-of-function studies have demonstrated that a human YEATS domain-containing protein named eleven-nineteen-leukaemia (ENL) functions as a transcriptional coactivator and is essential for the proliferation of AML that harbours oncogenic multiple lineage leukemia (MLL) rearrangements. We previously synthesized a series of small molecule inhibitors (1,7-9,11-15and24) that displayed significant and specific inhibitory effects against the ENL YEAST domain. In the current work, we report the development of a novel NanoBRET system that allows the analysis of cellular permeability, potency, selectivity, and stability of synthesized ENL inhibitors for their prioritization for further characterizations. Followed by in vitro metabolic stability and cell growth inhibition studies, we narrowed down to a potent and specific ENL YEATS domain inhibitor13with both high in vitro metabolic stability and strong anti-proliferation ability on MLL-fusion leukemia cell lines. A mouse pharmacokinetic (PK) analysis showed that at an oral dose of 20 mg/kg compound13had 60.9% bioavailability and 2.6 h mean residence time. With these favorable PK characteristics, compound13is ready for efficacy studies in an animal model. Cumulatively, the current study has prioritized compound13as a promising drug candidate to disrupt the pathogenic functions of ENL for the AML treatment.