Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors.

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 Nε-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 π-π-π stacking interaction with ENL YEATS. This led to the creation of selective ENL YEATS inhibitors with a KD 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.

Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors.

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

SARS-CoV-2, the COVID-19 pathogen, 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 dipeptidyl MPro inhibitors and characterized them on 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 to accommodate 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 favorable 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.

An amber-encoding helper phage for more efficient phage display of noncanonical amino acids.

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.

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease.

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.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

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.

Repurposing Halicin as a potent covalent inhibitor for the SARS-CoV-2 main protease.

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 (MPro), a key enzyme responsible for the replication of virus inside the host. In this study, we evaluate the inhibition potency of a nitrothiazole-containing drug, halicin, and reveal its reaction and interaction mechanism with MPro. The in vitro potency test shows that halicin inhibits the activity of MPro an IC50 of 181.7 â€‹nM. Native mass spectrometry and X-ray crystallography studies clearly indicate that the nitrothiazole fragment of halicin covalently binds to the catalytic cysteine C145 of MPro. Interaction and conformational changes inside the active site of MPro suggest a favorable nucleophilic aromatic substitution reaction mechanism between MPro C145 and halicin, explaining the high inhibition potency of halicin towards MPro.

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals.

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an α-ketoamide warhead, a P1 ß-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a ß-(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 MPro active site cysteine. The P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro 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 to inhibit ectopically expressed MPro in human 293T cells. In general, inhibitors 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 cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC50 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.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, 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 positions and the N-terminal protection group, we synthesized 18 tripeptidyl MPro inhibitors that contained also an aldehyde warhead and ß-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with MPro, their inhibition of MPro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC50 values that range from 4.8 to 650 nM. The determined in vitro enzymatic inhibition IC50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro MPro inhibition potency, the P3 position 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. In all structures, the MPro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to MPro. In MPro, large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit MPro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit MPro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of MPro.

A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds.

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 Nε-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.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 µM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T†Cells.

As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti-CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically reversible chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR-T activation during cancer treatment.

The P3 O - Tert -Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors.

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 ß -(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.

The N -Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors.

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 ß-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 ß-(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.