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Main protease (MPro) 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 MProsubstrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MProcatalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MProinhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50value in inhibiting SARS-CoV-2 from infecting ACE2+A549 cells and a selectivity index of 875. The crystallography analyses of MProbound 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 MProinhibitors 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 MProas therapeutics for COVID-19.

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. MProis a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MProinhibitors. 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 MProinhibitors and characterized them onin vitroenzymatic inhibition potency, structures of their complexes with MPro, cellular MProinhibition potency, antiviral potency, cytotoxicity, andin vitrometabolic stability. Our results indicated that MProhas 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 highin vitrometabolic stability and can be potentially advanced to further preclinical tests.

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 asHOXA9, MEIS1, andMYBand 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, whereNε-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 aKdvalue as low as 2.0 nM were identified. One pentapeptide inhibitortENL-S1fdisplayed 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.

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 aCandidatus Methanomethylophilus alvuspyrrolysyl-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,Nε-tert-butoxycarbonyl-lysine (BocK) andNε-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.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (M

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M

As an essential enzyme to SARS-CoV-2, main protease (M (Pro) ) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N -terminal protection group, we synthesized a series of M (Pro) inhibitors that contain β -(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC (50) values that range from 4.8 to 650 nM. The determined IC (50) values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro M (Pro) inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the α -carbon, and the inhibition potency is sensitive toward the N -terminal protection group. X-ray crystal structures of M (Pro) bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the M (Pro) active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In M (Pro) , large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of M (Pro) in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O - tert -butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O - tert -butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of M (Pro) . This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M (Pro) ) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert -butyl-glycine, and a P4 N -terminal tert -butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M (Pro) inhibitors including PF-07321332 and characterized their M (Pro) inhibition potency in test tubes ( in vitro ) and human host cells ( in cellulo ). Crystal structures of M (Pro) bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a β-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N -terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the M (Pro) active site cysteine. The P1 opal residue, P2 dimethylcyclopropylproline and P4 N -terminal tert -butylcarbamide make strong hydrophobic interactions with M (Pro) , explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains an P4 N -terminal isovaleramide. In its M (Pro) complex structure, the P4 N -terminal isovaleramide is tucked deep in a small pocket of M (Pro) that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed M (Pro) in human 293T cells. All inhibitors including PF-07321332 with a P4 N -terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N -terminal cap is changed to a carbamate. The installation of a P3 O-tert -butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N -terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC (50) values around 1 μM. A control compound with a nitrile warhead and a P4 N -terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N -terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

As one of the most valuable tools for genetic code expansion, pyrrolysyl-tRNA synthetase (PylRS) is structurally related to phenylalanyl-tRNA synthetase (PheRS). By introducing mutations that mimic ligand interactions in PheRS into PylRS, we designed a PylRS mutant. This mutant, designated as oClFRS, recognizes a number of o-substituted phenylalanines for their genetic incorporation at amber codon. Its efficiency in catalyzing genetic incorporation of o-chlorophenylalanine (o-ClF) is better than that for N(ε)-tert-butyloxycarbonyl-lysine catalyzed by PylRS. The crystal structure of oClFRS bound with o-ClF shows that o-ClF binds deeply into a hydrophobic but catalytically inactive pocket in the active site and involves two halogen bonds to achieve strong interactions. The shift of o-ClF to a catalytically active position in the oClFRS active site will be necessary for its activation. This is the first reported aminoacyl-tRNA synthetase that involves two halogen bonds for ligation recognition and might represent an alternative route to develop aminoacyl-tRNA synthetase mutants that are selective for noncanonical amino acids over native amino acids.

As an essential enzyme of SARS-CoV-2, main protease (M

The rapid spread of COVID-19 has caused a worldwide public health crisis. For prompt and effective development of antivirals for SARS-CoV-2, the pathogen of COVID-19, drug repurposing has been broadly conducted by targeting the main protease (M