ABSTRACTAcute 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 byin vitrometabolic stability and cell growth inhibition studies, we narrowed down to a potent and specific ENL YEATS domain inhibitor13with both highin vitrometabolic 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.

Epigenetic reader proteins interpret histone epigenetic marks to regulate gene expression. Given their vital roles and the link between their dysfunction and various diseases, these proteins present compelling targets for therapeutic interventions. Nevertheless, designing selective inhibitors for these proteins poses significant challenges, primarily due to their unique properties such as shallow binding sites and similarities with homologous proteins. To overcome these challenges, we propose an innovative strategy that uses phage display with a genetically encoded noncanonical amino acid (ncAA) containing an epigenetic mark. This ncAA guides binding to the reader protein's active site, allowing the identification of peptide inhibitors with enhanced affinity and selectivity. In this study, we demonstrate this novel approach's effectiveness by identifying potent inhibitors for the ENL YEATS domain that plays a critical role in leukemogenesis. Our strategy involved genetically incorporating N-epsilon-butyryl-l-lysine (BuK), known for its binding to ENL YEATS, into a phage display library for enriching the pool of potent inhibitors. One resultant hit was further optimized by substituting BuK with other pharmacophores to exploit a unique pi-pi-pi stacking interaction with ENL YEATS. This led to the creation of selective ENL YEATS inhibitors with a K-D value of 2.0 nM and a selectivity 28 times higher for ENL YEATS than its close homologue AF9 YEATS. One such inhibitor, tENL-S1f, demonstrated robust cellular target engagement and on-target effects to inhibit leukemia cell growth and suppress the expression of ENL target genes. As a pioneering study, this work opens up extensive avenues for the development of potent and selective peptidyl inhibitors for a broad spectrum of epigenetic reader proteins.

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (M Pro ) for replication and pathogenesis. M Pro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as M Pro 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 M Pro inhibitors and characterized them on in vitro enzymatic inhibition potency, structures of their complexes with M Pro , cellular M Pro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that M Pro 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.

Main protease (M Pro ) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an M Pro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the M Pro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent M Pro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC 50 value in inhibiting SARS-CoV-2 from infecting ACE2 + A549 cells and a selectivity index of 875. The crystallography analyses of M Pro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent M Pro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 M Pro as therapeutics for COVID-19.

Eleven-nineteen leukemia protein (ENL) plays pivotal roles in the leukemogenesis. As a YEATS domain protein, ENL reads histone acylation marks and recruits key transcription factors to leukemic drivers such as HOXA9, MEIS1 , and MYB and therefore promotes leukemia development. The histone-reading function of ENL has been proven essential in the onset and progression of several acute leukemias, suggesting a putative therapeutic window for ENL inhibition. In this study, we developed a phage-assisted, active site-directed ligand evolution (PADLE) approach for the identification of potent and selective ENL inhibitors, where N ε -butyryl- l -lysine (BuK) that possesses known target-protein interactions with the ENL YEATS domain was genetically incorporated into a phage display library to serve as a warhead to direct displayed peptides to the active site of ENL YEATS for enrichment. Using this novel strategy in combination with structure-activity relationship that replaced BuK with other ncAAs for de novo π-π-π stacking interactions with two aromatic residues in ENL YEATS, selective and potent ENL inhibitors with a K d value as low as 2.0 nM were identified. One pentapeptide inhibitor tENL-S1f displayed selective inhibition of ENL over other YEATS domains as well as strong cellular target engagement and on-target effects in inhibiting leukemia cell growth and suppressing the expression of ENL target genes. As the first of its kind study, the current work opens a large avenue of research of using PADLE to develop selective and potent peptidyl inhibitors for a large variety of epigenetic reader proteins.

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.

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.

ABSTRACT In the past two decades, phage display has emerged as a powerful technique for the identification of antibodies and peptide ligands for therapeutic targets. Using the amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, we and others have shown that the chemical space in phage display can be significantly expanded for drug discovery. However, the use of amber codon in phages results in poor phage yields and requires tedious processes to enrich amber codon-containing (amber obligate) phage clones. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for rapid and continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by the insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The afforded novel helper phage allowed for a continuous amber codon enrichment strategy for two different phage display libraries and demonstrated a 100-fold increase in selectivity for packaging of library plasmids in comparison with original helper phage plasmids. To demonstrate the applicability of the system, CMa13ile40 was used to create two phage-displayed peptide libraries containing two separate ncAAs, N ε - tert -butoxycarbonyl-lysine (BocK) and N ε -allyloxycarbonyl-lysine (AllocK), respectively. These were then used to identify peptide ligands that bind to the extracellular domain of ZNRF3, a membrane-bound E3 ligase. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Using biolayer interferometry, enriched peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 and this affinity is dependent on the presence of the ncAA used for selection. Our results clearly show that ncAAs in phages provide unique interactions for selection of peptides that are different from each other and from canonical amino acids. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

Due to the great potentials of cyclic peptides as therapeutic agents, several phage-displayed peptide libraries in which cyclization is achieved by the covalent linkage of cysteines have been previously demonstrated to identify cyclic-peptide ligands for therapeutic targets. While problems remain in these cysteine conjugation strategies, 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 a potent ligand, CycH8a, for histone deacetylase 8 (HDAC8). We believe this approach will find broad applications in drug discovery.

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.

AbstractSuperior 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.

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.